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Contributors
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
Kimberley A. Beaumont, Melanogenix Group, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia (85) Suzy Bianco, Department of Molecular and Cellular Pharmacology, Leonard M. Miller School of Medicine, University of Miami, Miami, Florida (33) Adrian J. L. Clark, Centre for Endocrinology, William Harvey Research Institute, Barts and The London, Queen Mary University of London, London, United Kingdom (155) Lucila L. K. Elias, Department of Physiology, School of Medicine of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil (155) Ana Claudia Latronico, Unidade de Endocrinologia do Desenvolvimento, Laborato´rio de Hormoˆnios e Gene´tica Molecular/LIM42 da Disciplina de Endocrinologia do Hospital das Clinicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sao Paulo 05403‐900, Brazil (33) Janis Lem, The Molecular Cardiology Research Institute, Tufts Medical Center; Program in Neuroscience; Program in Genetics, Program in Cell, Molecular and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine; and Department of Ophthalmology, Tufts Medical Center, Boston, Massachusetts 02111 (1) Yan Yan Liu, Melanogenix Group, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia (85) Katherine M. Malanson, The Molecular Cardiology Research Institute, Tufts Medical Center; and Program in Neuroscience, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts 02111 (1) Marco Martari, School of Medicine, Division of Endocrinology and Metabolism, Johns Hopkins University, Baltimore, Maryland 21287 (57) Roberto Salvatori, School of Medicine, Division of Endocrinology and Metabolism, Johns Hopkins University, Baltimore, Maryland 21287 (57) Leticia Ferreira Gontijo Silveira, Unidade de Endocrinologia do Desenvolvimento, Laborato´rio de Hormoˆnios e Gene´tica Molecular/LIM42 da Disciplina de Endocrinologia do Hospital das Clinicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sao Paulo 05403‐900, Brazil (33) Richard A. Sturm, Melanogenix Group, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia (85)
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Ya‐Xiong Tao, Department of Anatomy, Physiology, and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, Alabama 36849 (173) Milena Gurgel Teles, Unidade de Endocrinologia do Desenvolvimento, Laborato´rio de Hormoˆnios e Gene´tica Molecular/LIM42 da Disciplina de Endocrinologia do Hospital das Clinicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sao Paulo 05403‐900, Brazil (33)
Preface
G protein‐coupled receptors (GPCRs) comprise the largest family of membrane proteins with 800 members. They transduce signals from a diverse array of endogenous ligands, including ions, amino acids, nucleotides, lipids, peptides, and large glycoprotein hormones. They are also responsible for our sensing of exogenous stimuli including photons and odorants. GPCRs regulate almost every aspect of our physiological functions. It is estimated that 40–50% of currently used therapeutic drugs target GPCRs directly or indirectly. Because the current drugs target only a small portion of the GPCRs, opportunities for targeting the remaining GPCRs is enormous. Because GPCRs are such versatile regulators of physiological processes, it is not difficult to imagine that mutations in these genes will result in dysregulation of these physiological processes, therefore of pathophysiological significance. The first naturally occurring mutation in a GPCR was identified in rhodopsin (causing retinitis pigmentosa) in 1990. Since then, many GPCRs were found to be mutated in a plethora of diseases. Several volumes as well as numerous review articles were published on this topic. The current volumes are updates on this rapidly advancing field. Leading experts present their accounts on a diverse array of these GPCRs in health and disease. These include some of the earlier classical examples of diseased GPCRs such as rhodopsin and V2 vasopressin receptor to more recent additions including melanocortin‐4 receptor, GPR54, and GPR56. The studies of these naturally occurring mutations in GPCR genes have led to significant advances in our understanding of the physiology and pathophysiology of these GPCRs. Detailed understanding of the molecular defects are the basis of future personalized medicine. Some clinical trials have been done with impressive results. I hope the readers are excited to read the rapid progresses described in these two volumes. Indeed, these chapters are only selected examples of the field that combines thorough clinical observation, molecular genetics, pharmacology, biochemistry, cell biology, and epidemiology. I thank all the authors for taking time out of their busy schedules to write their outstanding contributions. I apologize for the occasional nagging reminding the due dates of their chapters. I cherish the opportunity to get to know these leading scientists by emails. I look forward to meeting them in person at scientific meetings.
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It has been a wonderful experience from the planning of the chapters and authors to seeing the chapters in print. For this, I am very grateful to Dr. P. Michael Conn, the Series Editor, for his trust, guidance, and friendship. I also thank the Editors at Elsevier, Ms. Lisa Tickner and Ms. Delsy Retchagar, for making these volumes a reality. It is a pleasure to work with them. Finally, special thanks to my wife, Zhen‐Fang, and my daughters Nancy, Rachel, and Lily for their understanding and love. YA-XIONG TAO Auburn, Alabama
Rhodopsin‐Mediated Retinitis Pigmentosa Katherine M. Malanson*,{ and Janis Lem*,{,z,} *The Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts 02111 {
Program in Neuroscience, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts 02111
z
Program in Genetics, Program in Cell, Molecular, and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts 02111 }
Department of Ophthalmology, Tufts Medical Center, Boston, Massachusetts 02111
I. Introduction .................................................................................. II. Rhodopsin Function ........................................................................ A. Retina Structure......................................................................... B. Rod Cell‐Specific Expression of Rhodopsin ...................................... C. Classification of Rhodopsin Mutations ............................................. III. Mechanisms of Rod Degeneration ...................................................... A. P23H, VPP: A Model for Misfolded and Mislocalized Rhodopsin Mutants .............................................. B. P347S: A Model for a Nonautonomous Pathway and Destabilized Rhodopsin Mutants..................................................................... C. K296E: A Model for Persistent Signaling and Alternative Signaling Pathways...................................................................... IV. Mechanisms of Cone Degeneration ..................................................... A. Trophic Factors.......................................................................... B. Toxic Factors ............................................................................. C. Metabolic Support ...................................................................... V. Treatments .................................................................................... A. Gene Therapy............................................................................ B. Vitamin A and Pharmacological Supplementation............................... C. Transplantation of Stem Cells or Retinal Sheets ................................. VI. Conclusions ................................................................................... References ....................................................................................
Progress in Molecular Biology and Translational Science, Vol. 88 DOI: 10.1016/S1877-1173(09)88001-0
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Copyright 2009, Elsevier Inc. All rights reserved. 1877-1173/09 $35.00
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Retinitis pigmentosa (RP) is a genetically and phenotypically heterogeneous group of diseases that cause blindness. Mutations within the rhodopsin gene account for approximately 25% of autosomal dominantly inherited RP cases. Therefore, understanding the mechanisms causing rhodopsin‐mediated RP has a significant health impact. To date, results from multiple labs indicate that rhodopsin‐mediated RP pathogenesis does not share a common mechanism of degeneration. There is strong evidence that multiple mechanisms are involved, including protein misfolding, mislocalization, release of toxic products, and aberrant signaling. Development of effective treatments requires investigation of the mechanism involved in the different rhodopsin mutations. This chapter focuses on the mechanisms by which rhodopsin mutations cause retinal degeneration, as well as potential therapeutic strategies to treat the disease.
I. Introduction Retinitis pigmentosa (RP) is a genetically heterogeneous group of diseases that produces phenotypes ranging from relatively mild night blindness (nyctalopia) to severe and complete blindness. Early in the disease process, rod photoreceptors (specialized neuronal cells adapted for exquisite light sensitivity) begin to die, causing night blindness. Patients have difficulty seeing in dim light, such as night driving, and adapting to changes in light intensity, such as entering a darkened movie theater. The disease progresses slowly with the continued loss of photoreceptors, resulting in loss of peripheral vision and producing ‘‘tunnel vision’’ where only central vision is retained. Additionally, many patients report seeing continued flashes of light (photopsia). In late stages of the disease, central vision is also lost, causing complete blindness. The key diagnostic test for RP is the electroretinogram (ERG), which measures rod and cone function. The ERG detects changes in rod function early in the disease process, often before patients are mentally aware of visual dysfunction. Because of the remarkable adaptive abilities of the human brain, as much as 90% of rod cells can be lost before a patient is aware of visual changes. Thus, by the time most patients seek medical attention, the disease is in advanced stages. The slow, progressive degeneration of rod photoreceptors inevitably leads to the loss of cone photoreceptors, which are responsible for high acuity, bright light vision. Cone photoreceptors are concentrated in the center of the retina in the ‘‘fovea.’’ The reason for the sequential degeneration is not fully understood, but it appears that cone survival is dependent upon the survival of rod photoreceptors.
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The prevalence of RP in the United States is one in 4,000 individuals, affecting approximately 100,000 people. Worldwide, RP affects approximately 1.5 million individuals. RP is genetically inherited as an X‐linked, autosomal recessive or autosomal dominant disease. The first mutation associated with human RP was identified in the gene encoding rhodopsin, the G‐protein coupled receptor (GPCR) of rod photoreceptor cells. Since then, mutations in 140 other genes have been mapped or identified involving genes with a variety of functions (for a summary, see RetNet at http://www.sph.uth.tmc.edu/ retnet/). Genetic screens of unrelated RP patient populations show that individual affected genes account only for a few percent of RP cases. Upwards of 40% of RP cases are of unknown genetic etiology. However, mutations in the rhodopsin gene account for approximately 25% of autosomal dominant retinitis pigmentosas (ADRPs), a significant proportion of RP patients. Thus, understanding the mechanisms by which rhodopsin mutations cause RP has a significant health impact. This review focuses on our current knowledge of rhodopsin mutants and their role in retinal degenerative disease. Because of space limitations, we are unable to discuss many studies and apologize to those investigators whose work is not included here. Our discussions within this chapter will focus on the genetics, biochemistry, and physiology of specific rhodopsin mutations that have been studied in greater detail.
II. Rhodopsin Function A. Retina Structure Rhodopsin protein is abundantly expressed in the retina, the light‐sensitive neurosensory tissue that lines the back of the eye. The protein is expressed in a tissue‐ and cell‐specific manner. Figure 1 is a schematic of a mammalian eye. Vision in mammals begins with the entry of light through the transparent cornea. Light passes through the anterior chamber of the eye, through the pupillary opening and is focused by the lens. A visual image is focused on the retina, the tissue comprised of photoreceptors and other neuronal and glial cells. The retina is a highly organized, stratified structure (Fig. 2). Thus, degenerative changes are easily recognized by loss of organization and stratification. The retina is bounded by retinal pigment epithelial (RPE) cells, which provide metabolic support to abutting photoreceptor cells. There are two major classifications of photoreceptor cells—the rods and the cones. Rods are responsible for dim‐light vision, whereas cones are responsible for color and high‐acuity vision in bright light. Photoreceptors have a polarized cell structure, with outer segments at one end of the cell and the synapse at the other. Rhodopsin is
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Sclera Choroid Pigment epithelium Retina Anterior chamber Cornea
Fovea
Vitreous chamber Lens
Iris Conjunctiva Optic nerve Blind spot
FIG. 1. Schematic of the mammalian eye. The mammalian eye is responsible for converting the light energy of the environment into a neural response that can be decoded by the brain. Light enters the eye via the transparent cornea, passes through the anterior chamber and lens until it falls on the retina, the specialized tissue at the back of the eye that contains the light‐sensitive photoreceptor cells.
found within the membranous discs of the outer segments, which stratify as a layer adjacent to the retinal pigment epithelium (RPE). The outer segments are joined to the inner segment by a modified cilium. The inner segment contains the mitochondria and endoplasmic reticulum (ER), where proteins are synthesized, then transported through the cilium into the outer segment. The nuclei of rod and cone photoreceptor cells are stratified in a separate ‘‘outer nuclear layer’’ (ONL) and can often be distinguished by the distinctive staining pattern of the nucleus, with rods having densely stained, rounded nuclei and cones having nuclei that stain heterogeneously. Photoreceptor cell synapses ramify in a single layer referred to as the ‘‘outer plexiform layer’’ (OPL) and synapse directly or indirectly to second‐order cells that localize within another strata called the ‘‘inner nuclear layer’’ (INL). INL cells include bipolar cells, horizontal cells, and amacrine cells. In addition, nuclei of the glial support cells are found in the INL. The bipolar, horizontal, and amacrine cell synapses ramify in the ‘‘inner plexiform layer,’’ where they synapse with cells of the innermost layer of the retina, the ganglion cells in the ‘‘ganglion cell layer’’ (GCL). Axons of the ganglion cells create the optic nerve through which visual information is sent to the brain.
RHODOPSIN‐MEDIATED RETINITIS PIGMENTOSA
Rod photoreceptor
5
Retinal pigmented epithelium Outer segments
Cone photoreceptor
Inner segments
Outer nuclear layer (ONL)
Horizontal cell Bipolar cell
Outer plexiform layer (OPL) Inner nuclear
Amacrine layer (INL) cell
Inner plexiform layer (IPL) Ganglion cell
Ganglion cell layer (GCL)
Light
FIG. 2. Retina structure. The retina is a highly organized tissue with cells organized into specific layers. Rod and cone photoreceptor outer segments contain membranous discs packed with rhodopsin. The nuclei for the photoreceptor cells are located within the outer nuclear layer (ONL). Because photoreceptor cells are lost during retinal degeneration, counting the number of nuclei in a column in the ONL is a measure of the extent of degeneration. Photoreceptor cells synapse onto second‐order neurons in the outer plexiform layer (OPL). The INL contains the bipolar, amacrine, and horizontal cells. The ganglion cells, whose axons form the optic nerve, are located within the ganglion cell layer (GCL). Scale bar = 25 micrometers.
B. Rod Cell‐Specific Expression of Rhodopsin Rhodopsin is the GPCR expressed specifically and exclusively in rod photoreceptor cells. Rhodopsin molecules are densely packed in photoreceptor outer segments, making rod photoreceptors sensitive to a single photon of light. Rhodopsin is the prototypical Class A receptor, possessing seven transmembrane domains. Unlike other GPCRs in which direct ligand binding activates the receptor, rhodopsin covalently binds its ligand, 11‐cis‐retinal. The 11‐cis‐ retinal is an inverse agonist, holding rhodopsin in the inactive state. A schematic of the phototransduction cascade is presented in Fig. 3. Activation by a single photon of light produces a conformational change, converting 11‐cis‐retinal to all‐trans‐retinal. The conformational change
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Rhodopsin
Light
R*
R
Plasma membrane
Arrestin
R
Photoreceptor disc
b Transducin
a GTP R*
g a*
GDP
g
5⬘ GMP
Rhodopsin kindase
b
a b*
g
cGMP
Na+ Ca2+
a* cGMP PDE
cGMP
cGMP
CNG channels
a b g g 4 Na+
NCKX1
Ca2+ K+
FIG. 3. Phototransduction cascade. Activation by a single photon of light produces a conformational change in rhodopsin by converting 11‐cis‐retinal to all‐trans‐retinal. This isomerization creates a conformational change in rhodopsin, which then binds transducin, the rod‐specific heterotrimeric G‐protein. The substitution of GDP by GTP on the transducin a‐subunit activates transducin. Activation of transducin causes the dissociation of transducin bg complex from the active GTP‐bound a‐subunit. The activated a‐subunit of transducin then activates cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) by binding the phosphodiesterase g‐subunit, removing its inhibitory effect on the catalytic cGMP phosphodiesterase a‐ and b‐subunits. Activated cGMP PDE hydrolyzes cGMP to 50 GMP, lowering the intracellular levels of cGMP. The decrease in cGMP concentrations results in the closure of the cyclic nucleotide gated (CNG) channels, blocking the inward flow of sodium and calcium. The decreased flow of sodium and calcium hyperpolarizes the membrane. This change in membrane potential is transmitted as a neural signal through the secondary neurons of the retina to the ganglion cells and, ultimately to the brain. Rhodopsin is inactivated by phosphorylation by rhodopsin kinase, which uncouples rhodopsin and transducin, followed by binding of visual arrestin to completely quench rhodopsin signaling activity.
activates rhodopsin (R*) so that it can bind the rod‐specific heterotrimeric G‐protein, transducin. Transducin is activated by the substitution of GDP by GTP on the transducin a‐subunit. This substitution causes the dissociation of the transducin bg‐complex from the active GTP‐bound a‐subunit. The activated transducin a‐subunit activates the cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) by binding the phosphodiesterase g‐subunits, removing their inhibitory effect on the catalytic cGMP phosphodiesterase
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a‐ and b‐subunits. Activated cGMP phosphodiesterase a‐ and b‐subunits hydrolyze cGMP to 50 GMP, lowering intracellular levels of cGMP, a secondary messenger of photoreceptor cells.1–3 Intracellular concentrations of cGMP regulate cyclic nucleotide gated (CNG) channels located within the plasma membrane of photoreceptor cells. In darkness, cGMP intracellular levels are elevated and bind to the CNG channels, holding them in the open configuration. Open CNG channels allow the entry of sodium and calcium into the photoreceptor outer segment. Rhodopsin activation results in the hydrolysis of cGMP, thus decreasing intracellular cGMP concentration. The reduction in cGMP concentration reduces the proportion of cGMP bound channels and increases the proportion of closed CNG channels. Closure of the CNG channels blocks the flow of sodium and calcium into the cell, effectively hyperpolarizing the plasma membrane. This change in membrane potential is transmitted as a neural signal through the secondary neurons of the retina to the ganglion cells and, ultimately to the brain. Restoration of light‐activated rhodopsin to a light‐sensitive state is well characterized. Inactivation of rhodopsin is a multistep process. First, rhodopsin kinase phosphorylates light‐activated rhodopsin, uncoupling rhodopsin and transducin. Next, visual arrestin binds phosphorylated rhodopsin, completely quenching rhodopsin signaling activity. Finally, all‐trans‐retinal is restored to its light‐sensitive conformation through the visual pathway, which takes place within RPE cells.4–6
C. Classification of Rhodopsin Mutations More than 100 different rhodopsin mutations have been identified in patients with RP. Most of these mutations are point mutations and a small number are deletions. Mutations are located throughout the protein in the intradiscal, transmembrane, and cytoplasmic domains (Fig. 4). There is poor correlation between disease severity and location of mutations. The mechanisms by which rhodopsin mutations cause RP have been the focus of much scientific research. To date, many different hypotheses have been proposed and tested, including impaired protein folding and mislocalization, release of toxic products and aberrant signaling. With over 100 different rhodopsin mutations identified, it is not surprising that a single mechanism that leads to rhodopsin‐mediated ADRP has not been found. Research suggests that rhodopsin mutations cause ADRP through multiple mechanisms, highlighting the importance of understanding the underlying mechanism of each mutation to develop effective therapies. Studies by Nathans and collaborators addressed the mechanism of rhodopsin mutant mediated RP by sorting rhodopsin mutants into two classes.7 Mutant rhodopsins expressed in a heterologous cell system were classified into two
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HOOC
P347
Cytoplasm Membrane K296 Intradiscal P23 H2N FIG. 4. Rhodopsin secondary structure and point mutations associated with ADRP. Secondary structure of rhodopsin with point mutations that have been associated with retinitis pigmentosa colored in black. Note that the mutations are within every domain of the protein. The positions of the three mutations discussed in further detail are labeled, P23, K296, and P347.
groups based on the mutant proteins’ expression levels, ability to regenerate spectrally active rhodopsin by association with 11‐cis‐retinal, and subcellular localization. Class 1 mutants expressed at levels comparable to that of wild‐type rhodopsin, regenerated with 11‐cis‐retinal to form light‐responsive rhodopsin, and localized properly to the plasma membrane. Class 1 mutations tended to be within the C‐terminus of rhodopsin. Class 2 mutants were more common than Class 1 mutants and were characterized by decreased expression compared to expression of wild‐type rhodopsin, inability to associate with 11‐cis‐retinal and mislocalization within heterologous cells. Class 2 mutations tended to occur in the intradiscal, transmembrane, and cytoplasmic domains of rhodopsin. The findings of this study suggested that Class 2 mutants might cause degeneration through decreased expression of the rhodopsin protein, inability to form spectrally active rhodopsin, and mislocalization of protein. However, since the Class 1 mutants appeared normal in all tests, the question of mechanism remained for this class of mutations. This classification system remains popular, although other groups have developed other subclasses based on added criteria. A modified classification system taking more recent data into account was proposed by Mendes et al.8 For example, expression of some Class 1 mutants in transgenic animals revealed that the C‐terminal mutations affected trafficking and were not targeted appropriately to the photoreceptor outer segment. The new classification system is not mutually exclusive. Mutant rhodopsins can fall within several categories. The system proposed that the new Class 1 mutants folded normally but mislocalized within the photoreceptor cells. Class 2 mutants misfolded, were retained within the ER, and did not associate
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with 11‐cis‐retinal. Class 3 mutants affected endocytosis. Class 4 mutants affected rod opsin stability and posttranslational modifications. Class 5 mutants had an increased activation rate for transducin, but no obvious folding defect. Class 6 mutants also had no obvious folding defect and were constitutively active in absence of chromophore and in the dark. Both classification systems group rhodopsin mutants that share common mechanisms of degeneration and therefore, might share common therapies. While groups of rhodopsin mutations may cause disease through similar mechanisms, no single pathogenic mechanism common to all rhodopsin mutations has been found that would allow for a single treatment for all rhodopsin-mediated cases of RP. The diversity of mechanisms causing rhodopsin‐mediated RP suggests the requirement for ‘‘personalized medicine.’’
III. Mechanisms of Rod Degeneration A. P23H, VPP: A Model for Misfolded and Mislocalized Rhodopsin Mutants One of the best studied rhodopsin mutations is a point mutation of proline at residue 23 (P23). P23 is located in the N‐terminus of rhodopsin, within the intradiscal space (Fig. 4). While other rhodopsin mutations are more prevalent in other continents, the P23H rhodopsin mutation is the most prevalent one in the United States, suggesting a founder effect. In vitro expression of the P23H mutant rhodopsin showed mislocalization of misfolded protein, making it a Class 2 mutant by the classification system of Sung et al. Protein misfolding and mislocalization have both been implicated as causes of other neurodegenerative diseases.9–11 Misfolded proteins can cause ER stress, activate the unfolded protein response (UPR), and result in the formation of protein aggregates within cells, all of which can lead to apoptosis. Recently, it was shown that mislocalized rhodopsin killed cells independent of light by activating normally inaccessible signaling pathways.12,13 The P23H rhodopsin mutant provides a model system for studying how protein misfolding and mislocalization cause cell death. 1. MISFOLDING Gene expression studies in a heterologous cell system suggested that P23H rhodopsin is misfolded and mislocalized.7 Protein expression levels were reduced and showed altered difference spectra and oligosaccharide profiles. The reduction in protein expression levels suggested the mutant protein was not stable and was either not formed initially or was degraded faster than wild‐type rhodopsin. To assess whether the P23H mutant produced a functional
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rhodopsin molecule (composed of the opsin apoprotein and the 11‐cis‐retinal chromophore ligand), the difference spectra were analyzed. Normal rhodopsin produces an absorbance spectrum that shows a characteristic shift when exposed to light. Misfolded rhodopsins unable to bind the chromophore ligand do not produce the characteristic light‐shifted absorbance spectrum. The difference spectrum is calculated by subtracting the absorbance spectra after photobleaching from the absorbance spectra before photobleaching. The P23H difference spectra were abnormal compared to wild‐type rhodopsin, indicating the P23H mutant rhodopsin does not combine with 11‐cis‐retinal to form spectrally active rhodopsin (Fig. 5). Misfolded P23H rhodopsin was also suggested by the lack of complex oligosaccharides, which are added during protein maturation in the Golgi complex. P23H mutant rhodopsin misfolding has been confirmed by additional in vitro studies. Liu et al. used circular dichroism (CD) spectral techniques to analyze misfolded and correctly folded rhodopsins (defined by their inability or ability, respectively, to bind retinal). The CD spectra indicated that the misfolded P23H rhodopsin had 50–70% less helical content than wild‐type rhodopsin and lacked the characteristic banding pattern typical of
0.075
WT
0.000
Absorbance
495 nm −0.075 0.050
P23H
0.000
−0.050 300
500 Wavelength (mm)
700
FIG. 5. P23H difference spectra. Photobleaching difference spectra of wild‐type rhodopsin and P23H rhodopsin mutant expressed in a heterologous cell system. The 495 nm absorbance peak observed in the wild‐type rhodopsin is not seen in P23H mutant rhodopsin, indicating the mutant protein is misfolded and not able to respond to light. Adapted with permission from Ref. 7.
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wild‐type rhodopsin, indicating that the mutant has a different tertiary structure.14 Finally, a trypsin digest of misfolded P23H mutant rhodopsin degraded the mutant protein into small fragments, whereas properly folded rhodopsin was relatively resistant to trypsin digestion. Both the CD spectra and trypsin digestion further supported the hypothesis that P23H mutant rhodopsin is misfolded. As a rule, misfolded proteins are ubiquitinated and targeted for degradation via the proteasome pathway. Illing et al. confirmed the involvement of the ubiquitin pathway in P23H mutant rhodopsin degradation.15 In the absence of proteasome inhibitors, the mutant rhodopsin was unstable and quickly targeted for degradation via ubiquitination. However, in the presence of proteasome inhibitors and dominant negative ubiquitin, P23H mutant rhodopsin was stabilized. These results strongly suggest that P23H mutant rhodopsin is unstable, ubiquitinated, and degraded via the proteasome pathway. Because rhodopsin molecules can form large oligomeric complexes, it was hypothesized that the presence of mutant rhodopsin could interfere with wild‐ type rhodopsin function. In fact, it was later confirmed that after expressing both P23H mutant rhodopsin and wild‐type rhodopsin in vitro, the presence of P23H mutant rhodopsin impaired delivery of wild‐type rhodopsin to the plasma membrane.16 Coexpression of mutant and wild‐type rhodopsins lead to an increase in proteasome‐mediated degradation and steady‐state ubiquitination of wild‐type rhodopsin, demonstrating that P23H mutant rhodopsin has a dominant negative effect. This study is especially critical because it was one of the first to identify how mutant rhodopsin can interfere with wild‐type rhodopsin activity. 2. MISLOCALIZATION Studies by Sung et al. suggested the P23H mutant rhodopsin also did not localize correctly.7 In the retina, rhodopsin is synthesized on the rough ER and transported via the Golgi apparatus to the plasma membrane. The rhodopsin‐ laden plasma membrane at the rod cilium invaginates and eventually pinches off to form disc membranes. In the heterologous cell system used in this study, wild‐type rhodopsin localized to the plasma membrane. In contrast, the P23H mutant rhodopsin localized intracellularly with a staining pattern indicating that it was trapped within the ER. P23H mutant rhodopsin was noticeably absent from the plasma membrane. A Drosophila model expressing Rh1P37H, which corresponds to human rhodopsin P23H, was studied to determine in vivo localization of the mutant rhodopsin.13 Data from these flies indicated that the mutant rhodopsin accumulated primarily in the ER with a small portion located in the rhabdomeres, the fly equivalent to rod outer segments. The study also indicated that P23H mutant rhodopsin was active when localized properly, although with a
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significantly reduced activity. The mislocalized mutant rhodopsin activated the stress‐specific mitogen‐activated protein kinases, p38 and JNK. Both p38 and JNK are known to control stress‐induced apoptosis. Activation of these kinases links mislocalized rhodopsin directly to the induction of apoptosis. Transgenic frogs have also been used to analyze P23H mutant rhodopsin localization in vivo. Frogs were created that overexpressed frog, bovine, human, and murine forms of P23H mutant rhodopsin.17,18 All of the transgenic frogs expressing P23H mutant rhodopsin underwent retinal degeneration. Analysis of the frogs indicated that the mutant rhodopsin was expressed at very low levels and that the mutant protein was retained within the ER. Interestingly, it was observed that light sensitive degeneration varied between the mutant rhodopsin species. While frogs expressing bovine P23H mutant rhodopsin had complete protection from degeneration when raised in the dark, frogs expressing human P23H mutant rhodopsin only had a partial rescue, and frogs expressing frog and murine P23H mutant rhodopsin had no protection from degeneration. In the frogs protected against degeneration, the mutant rhodopsin localized properly to the outer segments. The mutant rhodopsin was only able to localize properly after truncation of the N‐terminus. Truncation of the N‐terminus allowed the mutant protein to exit the ER and then transport to the outer segment. P23H mutant rhodopsin localization in vivo has also been analyzed using transgenic mice. While the initial characterization of the transgenic mice suggested that the mutant rhodopsin predominantly localized correctly to rod outer segments,19,20 later data suggested that the majority of the mutant rhodopsin was mislocalized21,22 and was retained within the inner segment and in the OPL early in the degeneration. Interestingly, the buildup of P23H mutant rhodopsin in the OPL also correlated with the accumulation of mislocalized phosphodiesterase and transducin to the OPL.21 In summary, several lines of evidence, including in vitro and in vivo data from Drosophila, frogs, and mice, suggest that P23H mutant rhodopsin mislocalizes. The mislocalization of the mutant rhodopsin molecule has been associated with the mislocalization of other signaling molecules, and may lead to degeneration via activation of p38 and JNK kinases.
B. P347S: A Model for a Nonautonomous Pathway and Destabilized Rhodopsin Mutants Seven different point mutations of the proline at residue 347 in rhodopsin have been linked to RP—P347S, P347L, P347A, P347G, P347C, P347R, and P347T. P347 is located in the C‐terminal cytoplasmic tail of rhodopsin (Fig. 4),
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an area suggested to be critical for proper protein trafficking and sorting. The last five residues of rhodopsin, of which P347 is one, are highly conserved among species, indicating their importance. In vitro data demonstrate that P347S mutant rhodopsin regenerates normally with 11‐cis‐retinal, has a normal response to light, activates transducin, is phosphorylated by rhodopsin kinase, can bind arrestin, and localizes properly.23 In summary, all of the in vitro data suggest that P347S mutant rhodopsin behaves the same as wild‐type rhodopsin, placing it within Sung et al.’s Class 1 rhodopsin mutants. 1. NONAUTONOMOUS DEGENERATION The creation of transgenic chimeric mice expressing P347S mutant rhodopsin in only a subset of photoreceptor cells demonstrated that degeneration was through a nonautonomous pathway.24 The retinas of the chimeric mice had populations of photoreceptor cells expressing only wild‐type rhodopsin, and populations of photoreceptor cells expressing both mutant and endogenous rhodopsin. Analysis of three chimeric mice demonstrated that the level of degeneration correlated to the percentage of cells expressing the P347S mutant rhodopsin. Furthermore, mice with a greater number of cells expressing only wild‐type rhodopsin had slower rates of retinal degeneration. Yet, photoreceptor cells expressing only wild‐type rhodopsin were also dying. The observation that photoreceptor cells expressing only wild‐type rhodopsin were degenerating indicated the involvement of a nonautonomous pathway in the degeneration. After demonstrating the involvement of a nonautonomous pathway, researchers hypothesized that photoreceptor cells expressing the mutant rhodopsin released a toxic factor that killed neighboring photoreceptor cells. If the photoreceptor cells expressing mutant rhodopsin released a toxic factor, then cells adjacent to those expressing the mutant rhodopsin should degenerate first. However, this localized degeneration was not observed. In fact, the photoreceptor cells of chimeric mice degenerated in a uniform fashion regardless of proximity to P347S mutant‐expressing cells. These results suggested that the release of a diffusible toxic factor from degenerating photoreceptor cells was not the cause of neighboring cell death.24 These observations do not rule out the involvement of a trophic factor in the degeneration. In summary, this study not only demonstrated the involvement of a nonautonomous pathway but also suggests that the degeneration was not dependent on the release of a toxic factor from degenerating cells. Later, we will further discuss the involvement of both the release of toxic factor and the necessity of a trophic factor in cone cell degeneration.
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2. CELL SORTING P347 is located within the last five residues of the C‐terminal tail of rhodopsin. These five residues are highly conserved across species, presumably due to their potential involvement in cell sorting and trafficking. Several studies have investigated whether protein mislocalization is involved in the degeneration caused by the P347S mutation in rhodopsin. As previously mentioned, when P347S mutant rhodopsin is expressed in a heterologous cell system, it localizes at levels similar to wild‐type rhodopsin at the plasma membrane, suggesting that P347S rhodopsin is able to localize correctly.7 However, mislocalization of mutant protein was predicted when a frog retinal cell‐free system was used to examine mutant rhodopsin trafficking.25 The study found that the last five residues of rhodopsin are critical for proper protein sorting. Synthetic peptides containing the last five residues of rhodopsin as competitive inhibitors prevented cell sorting of wild‐type rhodopsin. When the last five residues were deleted from the competitive peptide, wild‐type rhodopsin sorted to the membrane. Introduction of the P347S mutation into the peptide permitted wild‐type rhodopsin trafficking, suggesting that residue P347 plays a critical role in proper cell trafficking and sorting. Two lines of P347S transgenic mice were made in independent labs. The first line of mice created had normal retinal development until the third postnatal week, after which photoreceptor cells started dying.26 By 7 weeks of age, these P347S transgenic mice had only half of their ONL remaining. In this mouse model, while some mutant rhodopsin was properly targeted to the rod outer segments, some mutant rhodopsin was mislocalized in the inner segment.26 A second independent line of P347S transgenic mice was also made.23 There was a direct correlation between the severity of degeneration and the level of P347S mutant rhodopsin mRNA in these mice. These P347S transgenic mice had a much slower rate of retinal degeneration than those created by Chang et al.26 In these mice, the P347S mutant rhodopsin localized predominantly in the outer segments, but also accumulated in submicrometer‐sized extracellular vesicles near the junction of inner and outer segments. The accumulation of the mutant rhodopsin in these extracellular vesicles suggested that protein trafficking might play a contributory role in the retinal degeneration present in these mice, as a majority of the mutant rhodopsin localized properly to the rod outer segments. Together, these studies suggest that protein mislocalization may play a role, but does not fully account for the degeneration seen in the P347S model.
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3. RHODOPSIN STABILITY AND SIGNALING Abnormal signaling has been suggested to be involved in rhodopsin‐ mediated ADRP. The involvement of an abnormal signaling pathway has been investigated with P347S mutant rhodopsin mice. Using P347S rhodopsin mutant mice, Wiess et al., it was demonstrated that there were increased levels of cAMP in P347S retinas compared to wild‐type retinas.27 Since cAMP levels are not normally regulated through the canonical phototransduction pathway, increased levels of cAMP in rhodopsin mutant mice suggested the involvement of a noncanonical pathway. Together with the P347S mislocalization studies, these data suggest the mutant rhodopsin might be activating a secondary pathway that is not normally utilized when rhodopsin is localized properly to the outer segments. Previous work has shown that persistent photosignaling can cause retinal degeneration.28,29 Furthermore, degeneration caused by persistent photosignaling can be rescued by introducing a null mutation for the a‐subunit of transducin which prevents G‐protein‐mediated signaling. Since P347S mutant rhodopsin predominantly localizes to rod outer segments, regenerates with 11‐ cis‐retinal, and has a normal light response, it was proposed that P347S mutant rhodopsin caused degeneration through persistent photosignaling. We tested this hypothesis by placing P347S‐expressing animals onto an a‐transducin null background. The results of this experiment demonstrated that the presence of a‐transducin provides protection from degeneration, as removal of the a‐transducin increased the rate of degeneration.30 The results do not rule out the involvement of another pathway through which the mutant rhodopsin may signal preferentially in the absence of a‐transducin. The accelerated degeneration in the absence of a‐transducin also suggested a role for transducin in the stabilization of P347S mutant rhodopsin. Further results suggested that the P347S mutant rhodopsin was unstable and released all‐trans‐retinal at a faster rate than wild‐type rhodopsin.30 Free all‐trans‐ retinal is toxic to the cells and an increased rate of release could be toxic to the photoreceptor cells. To directly test the role of chromophore toxicity, we placed the P347S‐expressing mice onto an Rpe65 null genetic background. The Rpe65 enzyme is critical for the recycling of chromophore and in its absence cells cannot produce 11‐cis‐retinal. Without 11‐cis‐retinal, all‐trans‐retinal cannot be produced. Therefore, if the degeneration present in the P347S mutant is caused by increased release of all‐trans‐retinal, then placing the mice on the Rpe65 null background would provide protection. To our surprise, placing the mice on the Rpe65 null background did not slow the degeneration (Fig. 6). We believe that the Rpe65 null mutation did not rescue the degeneration because the P347S mutant rhodopsin may require 11‐cis‐retinal to
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12.0
*
ONL thickness
10.0 8.0
*
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P347S Tra Rpe65 n
− +/+ +/+ 6
− −/− +/+ 6
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+ −/− +/+ 6
+ −/− −/− 6
FIG. 6. P347S protein stability. The role of 11‐cis‐retinal in stabilizing P347S rhodopsin. The outer nuclear layer (ONL) thickness in retinas from 4‐month‐old P347S rhodopsin mutant mice in the presence or absence of transducin and Rpe65 are plotted. The absence of a‐transducin alone or a‐transducin and Rpe65 did not cause degeneration (compare three left columns). The presence of P347S rhodopsin caused degeneration (compare first and fourth columns). Loss of a‐transducin in P347S rhodopsin mice produced a statistically significant decrease in ONL thickness (compare fourth and fifth columns). The combined loss of a‐transducin and Rpe65 did not rescue degeneration (last column). Error bars show S.E.M. *P < 0.005.
properly fold and traffic to the outer segments. We are currently starting experiments to determine the necessity of chromophore in mutant rhodopsin localization. Taken together, these studies suggest that the mutant P347S rhodopsin has abnormal biochemistry that leads to the degeneration present in the mouse models. The mutant rhodopsin may activate a noncanonical signaling pathway within the inner segments, resulting in the increased levels of cAMP in the retina. The P347S mutant rhodopsin also is unstable and releases toxic all‐trans‐retinal faster than wild‐type rhodopsin.
C. K296E: A Model for Persistent Signaling and Alternative Signaling Pathways The lysine to glutamic acid substitution at residue 296 of rhodopsin is associated with human RP. The mutant is well studied at the biochemical level. K296 is located within the seventh transmembrane domain of rhodopsin (Fig. 4) and is the site of choromphore attachment. Additionally, K296 forms a
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salt bridge with E113 to restrict the motion of the protein, keeping it in its inactive conformation.31 The K296E mutation blocks both the formation of the Schiff base and the salt bridge. Without the formation of a Schiff base, the chromophore cannot attach, which prevents the formation of spectrally active rhodopsin. However, without the formation of the salt bridge, opsin cannot be locked into its inactive conformation. Not surprisingly, K296E is constitutively active in vitro. In a heterologous cell system, Robinson et al. found that K296E constitutively activated transducin independently of light.32 These results suggested that K296E mutant rhodopsin fits into a group of other rhodopsin mutations that cause retinal degeneration through persistent photosignaling. 1. PERSISTENT PHOTOSIGNALING The creation of a mouse expressing K296E mutant rhodopsin allowed investigators to study the mechanisms involved in retinal degeneration in vivo. Mice expressing K296E rhodopsin undergo progressive retinal degeneration. In vivo, the K296E mutant rhodopsin localized to rod outer segments, was phosphorylated, and bound arrestin.33 These results suggested that the mutant rhodopsin was in the inactive state and not constitutively active, contrary to in vitro results. Indeed, the degeneration associated with K296E was independent of persistent photosignaling.30,33 Several lines of evidence suggested that K296E mutant rhodopsin was not causing degeneration through light‐dependent signaling in vivo. First, removing all light stimuli by dark‐rearing K296E mutant mice did not protect from degeneration. Dark‐reared mutant mice had decreased ERG amplitudes,33 indicating progressive retinal degeneration despite the lack of light stimulus. Second, if degeneration was caused by persistent photosignaling, combining the K296E rhodopsin mutant with a mutation that blocks photosignaling should provide protection from the degeneration. Removing the a‐subunit of transducin has been shown to block photosignaling.34 Therefore, K296E rhodopsin mutant mice were placed onto an a‐subunit transducin null background and degeneration was analyzed. However, protection from degeneration was not observed when these two mutations were combined. Instead, K296E rhodopsin mutant mice had a faster rate of degeneration in the absence of transducin, suggesting that the presence of a‐transducin provides some degree of protection against degeneration. In summary, these results suggest that the K296E rhodopsin mutation is not causing degeneration through persistent photosignaling, and that the presence of the a‐transducin provides protection from degeneration for this rhodopsin mutation. The protective effect of transducin suggests that the K296E mutant rhodopsin may be signaling through a G‐protein‐independent pathway, which may lead to degeneration.
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2. RHODOPSIN–ARRESTIN COMPLEX FORMATION Evidence from Drosophila studies suggested that the formation of stable rhodopsin–arrestin complexes caused degeneration. In addition, preventing the formation of these complexes prevented degeneration.35–37 To analyze the involvement of rhodopsin–arrestin complex formation in K296E‐mediated degeneration, mice were placed onto an arrestin knockout background. The removal of arrestin prevented the formation of rhodopsin–arrestin complexes. If degeneration was dependent on the formation of these complexes, preventing their formation should provide protection from degeneration. Analysis of K296E, arrestin knockout mice demonstrated that the presence of rhodopsin– arrestin complexes contributed to, but was not the sole mechanism of degeneration.38 In conclusion, these studies demonstrated the potential involvement of abnormal protein complexes in retinal degeneration in rhodopsin‐mediated ADRP.
IV. Mechanisms of Cone Degeneration Perhaps the most puzzling question about RP is why rod cell death is inevitably followed by cone cell death. Although cone cells account for only 5% of photoreceptors, they are essential for high‐acuity daylight vision and their degeneration has devastating effects. Most of the RP disease gene alleles are rod cell specific, yet cone photoreceptors progressively die as well. However, the loss of rods is not concomitant with the loss of cones. In human RP patients, cone photoreceptor cells can survive for years in the absence of rods before dying. There are no known forms of RP where rods die and cones survive. Conversely, cone‐specific genetic mutations can result only in cone cell death, leaving rod cells intact. This fact has lead to the hypothesis that cones depend on rods for their survival. Three hypotheses on the mechanisms underlying cone cell death include: (1) the loss of rod‐derived trophic factor(s), (2) release of toxic factor(s) during rod cell death, and (3) metabolic support imbalance following rod cell death. In the following sections, we discuss evidence supporting each of these theories.
A. Trophic Factors This hypothesis suggests that healthy rod photoreceptor cells release a trophic factor(s) that cones require for survival. Mohand‐Said et al. demonstrated the existence of a diffusible trophic factor that supported cone cell survival in organ coculture experiments.39 Retinas carrying residual cone cells from retinally degenerated mice were cocultured with rod‐containing
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* 9
8
7
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Estimated rods total number (⫻10−3)
Estimated cones total number (⫻10−4)
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FIG. 7. Cone cell death mechanisms: involvement of rod‐derived trophic factor. Estimated total number (mean SD) of cones (A) and rods (B) in retinas from 5‐week‐old RP model mice cultured in DMEM alone (gray bars) or cocultured with retinas from 8‐day‐old wild‐type mice (black bars). Estimated number of cone cells increased when cultured with cells from rod‐ containing retinas. *P < 0.0001. Adapted with permission from Ref. 39.
retinas. The experiments showed that coculture with rods increased cone survival in the degenerating retina (Fig. 7). Since the cocultured retinas did not physically touch, the group concluded that the trophic factor was diffusible. The group also concluded that the trophic factor came from rod cells since control experiments with rodless retinas did not increase cone survival in the degenerating retina. Streichert et al. also demonstrated the existence of a rod‐ derived trophic factor which supported the survival of photoreceptor cells in a retinal degeneration mouse model.40 While this group did not specifically analyze the survival of cones, they did find that the rod‐derived trophic factor extended the life of rod photoreceptor cells in P23H rhodopsin mutant retinas. The survival‐promoting factor was diffusible, heat labile, and absent from RP retinas. The group tested a variety of trophic factors expressed in retinas, including basic fibroblast growth factor, brain‐derived neurotrophic factor, and glial cell‐derived neurotrophic factor, but no one factor provided the protection seen by coculture with normal rod cells. Two rod‐derived trophic factors were recently identified by expression cloning.41,42 The groups appropriately named the trophic factors rod‐derived cone viability factor (RdCVF) and rod‐derived cone viability factor‐2 (RdCVF2). Both were truncated thioredoxin‐like proteins expressed specifically in photoreceptor cells. They share similar gene and three‐dimensional protein structures, and both were shown to promote cone survival in vitro.
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Taken together, these studies suggest that healthy rod cells release a trophic factor that supports the viability of cone cells. Therefore, it follows that as rod cells die in RP, there is a decrease in this trophic factor, which in turn is responsible for cone photoreceptor cell death. However, the fact that cone cells are functional for years after rod cells have died in human RP patients argues against this theory.
B. Toxic Factors A second hypothesis on the mechanism of cone cell death in RP involves the release of toxic factors in the retina by dying rod cells or activated microglia. The toxic factors could be reactive oxygen species, excess glutamate, or products of apoptosis that accumulate in retinas lacking rod photoreceptor cells. Ripps proposed a mechanism whereby toxic factors released from degenerating rods cells travel through gap junctions and cause cone cell death.43 Possible toxic factors include glutamate and products of apoptosis. While the field generally agrees that the release of excess glutamate is unlikely to be the cause of cone cell death, the release of apoptotic byproducts may play a role in cone cell death in RP. In the central nervous system, activated microglia migrate to the damaged area and phagocytose cellular debris. Activated microglia also secrete molecules that can kill surrounding healthy neurons. These molecules include nitric oxide, reactive oxygen species, excitatory amino acids, proteases, and proinflammatory cytokines.44 Gupta et al. demonstrated the presence of activated microglia in degenerating human retinas using immunocytochemistry.45 In normal human retina, quiescent microglia were small, stellate cells associated with the inner retina blood vessels. However, in degenerating retinas, numerous activated microglia were present in the ONL in degenerating areas. The activated microglia were enlarged amoeboid cells that contained rhodopsin‐positive cytoplasm. While the group did not demonstrate that the microglia were secreting toxic substances that might kill neighboring photoreceptor cells, previous evidence from the central nervous system suggests that the release of toxic substances from microglia might play a role in RP. Reactive oxygen species may also increase following rod photoreceptor cell death. Rod photoreceptor cells are responsible for most of the oxygen consumption in the outer retina. Oxygen levels in the outer retina vary inversely with oxygen consumption. As the number of rod photoreceptors decreases so does the oxygen consumption, which increases the levels of oxygen in the outer retina.46,47 The increase in oxygen in the outer retina may lead to the creation of toxic reactive oxygen species which may be responsible for cone cell death.
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Shen et al. demonstrated the involvement of reactive oxygen species in cone photoreceptor cell loss.48 Using P347L mutant rhodopsin transgenic pig retinas, biomarkers of oxidative damage to lipids, proteins, and DNA were found within cone inner segments after the loss of rod photoreceptors. This study strongly suggests the involvement of oxidative stress in cone cell loss. The involvement of oxidative stress in retinal degeneration is also supported by pharmacological studies in which administration of antioxidants increased photoreceptor survival.49,50 Treatment with antioxidants promoted cone survival in three separate RP mouse models. Antioxidant administration increased cone cell density and rhodopsin mRNA levels, slowed the decline of function as measured by ERG and slowed the thinning of the ONL. These results strongly support the involvement of oxidative damage in cone photoreceptor cell loss. Taken together, these results support the idea that cone photoreceptor cells are lost following rod cell death due to the increase of toxic substances in the retina. These toxic substances may be released by dying rod photoreceptor cells or by activated microglia. The toxic substances may be a byproduct of rod cell death, as is the case for increased oxygen levels, resulting in oxidative damage.
C. Metabolic Support A third hypothesis for the mechanism of cone cell death following rod photoreceptor cell loss involves the requirement of rod photoreceptor cells for cone cell metabolic support.51 The hypothesis hinges on the idea that photoreceptor outer segments receive most of their metabolic support from the RPE. Since 95% of photoreceptors in the retina are rods, and there are approximately 20–30 photoreceptor outer segments in contact with each RPE cell, then only one or two cone photoreceptor outer segments contact a single RPE cell. This is not true in the macula of the human retina, an area that is exclusively cones. However, rodents lack a macula, which limits our abilities to completely analyze this third hypothesis. Cepko and collaborators compared four mouse models of RP, including the P23H/VPP mutant rhodopsin mouse.51 Gene expression arrays were used to identify common changes that occurred in all four models at the onset of cone death. The study identified a large number of genes that were involved in cellular metabolism, suggesting cones were suffering from a shortage of nutrients. The study also found that cones showed signs of autophagy, a process of self‐digestion. One group of genes identified included several components of the mammalian target of rapamycin (mTOR) pathway, which regulates cellular metabolism. To bypass this pathway, mice were treated with insulin. Mice receiving insulin had transiently prolonged cone survival compared to controls. The results of this study suggested that as rod photoreceptor cells died, the
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interactions between photoreceptor outer segments and the RPE are disrupted, which upset the flow of nutrients, including glucose, to the cone photoreceptor outer segments. While this study is very convincing, there are two pieces of evidence that suggest changes in metabolic support are not the only mechanism of cone cell death in RP. First, if lack of metabolic support is the sole mechanism causing cone photoreceptors to die, the macula, an area of the human retina containing only cone cells, should never be affected. While the cone photoreceptors in the macula live the longest in human RP, they too eventually die, suggesting that the disruption of outer segment–RPE interaction cannot be the sole mechanism of cone cell death. Second, in a rat model of RP, photoreceptors could be rescued from degeneration by the application of various growth factors even when a thick layer of debris was present that separated the photoreceptors from their source of nourishment.52 Therefore, while metabolic support does seem to play a role in the degeneration of cone photoreceptors, it seems unlikely that it is the sole mechanism of cone cell loss in RP.
V. Treatments There is currently no universally effective treatment for the different types of RP. While different mechanisms have been found to play a role in the degeneration associated with individual or groups of rhodopsin mutations, no common mechanism has been found that provides a therapeutic target for most or all cases of rhodopsin‐mediated ADRP. In the absence of a shared mechanism, many different therapies have been suggested and tested within labs, including gene therapy, transplantation with stem cells or retinal tissue, and pharmacological treatment with supplements to protect photoreceptor cells. This section will focus on some of the work that has been done with each of these potential therapies.
A. Gene Therapy Gene therapy is theoretically an ideal treatment for rhodopsin‐mediated ADRP. As previously discussed, mutations within rhodopsin cause degeneration through a toxic gain‐of‐function as opposed to a loss‐of‐function. Therefore, the goal of gene therapy for rhodopsin‐mediated ADRP is to decrease the toxic gain‐of‐function of the mutant rhodopsin. Decreasing the gain‐of‐function of the mutant rhodopsin has typically been achieved through inhibiting the expression of the mutant protein or increasing the ratio of wild‐type to mutant rhodopsin, as the rate of degeneration slows when the ratio of wild‐type to
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mutant rhodopsin is high. Decrease in the toxic gain‐of function has also involved targeting the apoptotic pathway to extend the life of photoreceptor cells. LaVail and colleagues demonstrated the potential of gene therapy for the treatment of rhodopsin‐mediated ADRP using recombinant adeno‐associated virus expressing hairpin and hammerhead ribozymes.53 Ribozymes are small RNAs that cleave mutant transcripts in an allele‐specific manner, leaving the wild‐type transcript intact. Rats expressing P23H mutant rhodopsin were injected with ribozymes at postnatal day 15 (P15), before the onset of degeneration, and at P30 or P45, when approximately 40–45% of the photoreceptors had degenerated. Rats injected at both the early and late time points with ribozymes targeting the mutant rhodopsin had increased ONL thickness compared to controls, indicating that the treatment increased the lifespan of photoreceptor cells (Fig. 8). Rats injected at P15 also had significantly increased ERG amplitudes compared to controls 6 months after treatment, indicating that the therapy not only increased the lifespan of photoreceptor
A
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FIG. 8. Gene therapy: the use of ribozymes to treat P23H rats. Measurements of the outer nuclear layer (ONL) thickness in P130 rats after late‐stage ribozyme injection at P30 and P45. ONL thickness from wild‐type (WT) nontransgenic rats (open squares) and the uninjected P23H transgenic (Tg) rats (filled squares) is plotted against age. P23H rats were injected subretinally with rAAV vectors carrying either hairpin (A) or hammerhead (B) ribozymes at either P30 (closed circles) or P45 (open triangles). For comparison, rats injected at P15 and analyzed at P130 are shown (filled triangle). Adapted with permission from Ref. 53.
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cells, but also preserved their function. It is important to note that the treatment had the same protective effect at all time points studied, suggesting that this therapy can be initiated in patients after the onset of degeneration. This study demonstrated that decreasing the expression of mutant rhodopsin could protect photoreceptors from degeneration and preserve function. Yet, many questions still remain on the feasibility for treatment in humans. One such question is the ability to design viruses that would specifically target mutant rhodopsins. With over 100 different mutations identified within rhodopsin that cause ADRP, having a specific therapy for each mutant is a large hurdle to overcome. One way to bypass the need for mutant‐specific therapy is to target a more generalized mechanism, such as apoptosis, the end stage found within rhodopsin‐mediated ADRP. Using an adenovirus expressing an apoptosis inhibitor, Leonard et al. demonstrated the potential use of a more generalized therapy.54 The adenovirus expressed the X‐linked inhibitor of apoptosis (XIAP) which inhibits caspase‐3, ‐7, and ‐9. The virus was injected into rats expressing either a Class 2 mutant rhodopsin, P23H, or a Class 1 mutant rhodopsin, S334ter at P14‐P17 before degeneration was evident. At 4 months, injection with virus preserved ONL thickness for both rhodopsin mutants. However, the ERG results suggested there was only functional protection for the Class 2, P23H mutant rhodopsin rats. ERG results at all time points tested demonstrated that the S334ter mutant rhodopsin rats had no change in ERG amplitudes compared to controls. However, the injected P23H mutant rhodopsin rats had increased ERG amplitudes compared to controls at all time points studied, with the greatest change over time. The ERG findings may downplay the positive results since the injection only treats approximately 20% of the retina, and the ERG measures a total retinal response, not isolated to the injected area. By expressing an inhibitor for apoptosis, this study demonstrated that gene therapy need not only focus on specific rhodopsin mutations, but could focus on a shared end pathway and still protect against degeneration. The difference in response to adenovirus injection of S334ter and P23H rhodopsin mutants suggests that the apoptotic pathways utilized may be different between the two mutants. Another group has tested the idea that suppression of both mutant and wild‐type rhodopsin using short hairpin RNA, followed by replacement with wild‐type rhodopsin that is immune to RNA degradation, could rescue degeneration.55–57 Suppression of both mutant and wild‐type rhodopsin also gets around the hurdle of needing to personalize therapy for each of the 100 different rhodopsin mutations. The group used a modified recombinant adeno‐associated virus that preferentially infected photoreceptor cells. While injection with the virus rescued the degeneration present in P347S mutant rhodopsin mice, the rescue was localized to areas receiving the injection and was transient, suggesting the need for multiple injections in a single patient.
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The investigators suggested replacement with wild‐type rhodopsin that is immune to degeneration by the RNA, but they have yet to demonstrate the feasibility of doing so after onset of degeneration. Still, the approach is promising for the development of generalized therapy for rhodopsin‐mediated ADRP. Many studies have illustrated the potential benefits of gene therapy in treatment of rhodopsin‐mediated ADRP. However, several questions still remain, including the timing of therapy and viral toxicity. One critical issue that needs to be determined with gene therapy is the timing of treatment. As each rhodopsin mutation has a different rate of photoreceptor cell death, the field will need to determine if and when there is a time point when rescue of photoreceptor degeneration is no longer possible. Patients typically do not visit the clinic until they have significant vision impairment, when a majority of their photoreceptor cells have died. Thus, it will be critical to determine if gene therapies are therapeutically effective after a majority of photoreceptor cells have died. Another complication of gene therapy is the adverse side effects, including fatal forms of severe combined immunodeficiency (SCID) syndrome, leukemia, and fatal inflammatory response that have been associated with the use of adenoviruses. Investigators are making great strides at creating viruses that will only infect photoreceptor cells and thus minimize off target effects. Only once the viruses are proven safe will gene therapy be a potential treatment for human RP patients. Another question for therapies focused on decreasing mutant rhodopsin expression is the level of suppression required to maintain long‐term effects. Most human patients with rhodopsin‐mediated RP have a mutation in one allele. The one allele mutation results in approximately 50% of the total rhodopsin being mutant protein. In comparison, animal models currently being studied express mutant rhodopsin in addition to endogenous wild‐type rhodopsin. In animal models, the mutant rhodopsin only accounts for 1%, at most, of the total rhodopsin. Yet, this 1% can lead to total blindness. The animal models suggest that therapies focusing on decreasing expression of mutant rhodopsin will need to completely or near completely eliminate the expression of the mutant rhodopsin to have a lasting therapeutic effect. At best, reducing levels of mutant rhodopsin will delay, but not arrest photoreceptor degeneration.
B. Vitamin A and Pharmacological Supplementation Ligand binding has long been shown to increase the stability of the apoprotein opsin.58 Therefore, it follows that supplementation with vitamin A, which is converted into 11‐cis‐retinal, may provide a therapeutic effect by stabilizing the mutant rhodopsin.
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Berson et al. demonstrated the beneficial effects of vitamin A supplementation for patients with RP.59,60 Vitamin A supplementation slowed the rate of degeneration, measured by ERG function, in patients with common forms of RP. Since the results of this study were averages of groups irrespective of genotype, the effectiveness of vitamin A supplementation for rhodopsin‐ mediated ADRP could not be determined. Follow‐up studies specifically addressed the effect of vitamin A supplementation on rhodopsin‐mediated RP. Vitamin A supplementation was shown to protect mice expressing a Class 2 rhodopsin mutation,61 which are classically thought to cause degeneration through protein misfolding. Mice expressing a Class 2 mutant rhodopsin that received vitamin A supplementation had preserved ERG amplitudes, longer inner and outer segments, as well as thicker ONLs compared to control. As a control for the experiment, mice expressing P347S mutant rhodopsin, a Class 1 mutation, which is thought not to misfold, were also analyzed. Mice expressing the Class 1 mutant rhodopsin did not receive the same beneficial effects following vitamin A supplementation. These results suggested that vitamin A supplementation may have beneficial effects for the degeneration caused by Class 2 rhodopsin mutants. In vitro work has also suggested that pharmacological supplementation to aid protein folding can protect against degeneration for Class 2 rhodopsin mutants. Pharmacological chaperones applied to cells expressing P23H mutant rhodopsin rescued some of the misfolding effects.62 The mutant rhodopsin in the presence of the chaperone had mature glycosylation patterns, was light sensitive, and localized to the plasma membrane.63 Retinal chromophore applied during opsin synthesis also increased P23H protein levels five‐ or sixfold, suggesting that in the absence of retinal, the opsin was targeted for protein degradation. In summary, in vitro and in vivo data support the beneficial therapeutic effect of vitamin A or a similar chaperone to aid in proper folding of mutant rhodopsin.
C. Transplantation of Stem Cells or Retinal Sheets Retinal transplantation has a long history, and it comes as no surprise that transplantation of either retina tissue or retinal stem cells has been investigated as a potential therapy for RP. Therapeutic studies originally focused on the use of retina tissue for transplantation. Later, it was found that integration with the host retina was vastly improved when retinal progenitor cells were transplanted as opposed to whole retina sheets. Retinal progenitor cells, also called retinal stem cells, are responsible for producing all the cells of the retina during development and are derived from fetal or newborn mice and rats, or human fetuses. Studies using these progenitor cells transplanted into either healthy or degenerating rat and
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mice retinas found that the cells migrate into retinal layers, develop morphological characteristics of retinal cell types, but do not fully integrate into the ONL and do not express photoreceptor cell‐specific genes.64–67 However, MacLaren et al. recently demonstrated that transplantation of newly born rod cells, harvested at the peak of rod genesis, integrated morphologically and made functional connections.68 Transplanted cells restored response to dim light in mice that were previously blind. However, using newly born rod cells for therapy in humans has the added complication that in humans these cells would need to be harvested from fetuses in the second trimester. There is hope that cultured cells with the same multipotency can be developed. Ghosh et al. grafted E48 pig retinas into a 6‐month‐old P347L transgenic pig when the ONL had thinned to less than one‐half of its original thickness.69 While the graft and host retina did not make neuronal connections, and the function of the host retina was initially reduced following transplantation, the graft did rescue rods and cones in the host retina from degeneration. The survival of host photoreceptor cells was not a local phenomenon, as host rod survival in eyes receiving a transplant was more pronounced at a distance from the graft. These results suggest that grafting a developing retina can provide protection from degeneration potentially through the release of trophic factors or other molecules that protect from degeneration. Together, these results suggest transplantation of either retinal stem cells or retinal sheets may provide protection from degeneration. However, several hurdles still exist before this therapy can be adequately used in humans, including the source of the stem cells and retinal tissue.
VI. Conclusions Results of studies from multiple independent labs indicate that rhodopsin‐ mediated ADRP pathogenesis does not utilize a common mechanism of degeneration. Molecular genetic studies and therapeutic testing reveal response differences for each of the rhodopsin mutants. There is strong evidence for multiple pathologic mechanisms, including protein misfolding, mislocalization, release of toxic products, and aberrant signaling. It is also clear that the mechanisms are not mutually exclusive, with each mechanism contributing differentially to disease pathogenesis. Yet, it is likely that there are other mechanisms that have not yet been discovered and have a major contribution to the degenerative process. In order to be highly effective, future therapies will need to take into account the various mechanisms that apply to different mutations. It is our hope that, with new information, we can group similar mutation mechanisms together that will share effective therapies for the successful treatment of inherited RP.
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Acknowledgments The authors thank Lisa M. Hynes for her expert assistance in preparing figures for this chapter. The authors are supported by grants from the US National Institutes of Health (EY12008, EY01745), the Massachusetts Lions Eye Research Fund, Research to Prevent Blindness and the Foundation Fighting Blindness.
References 1. Fesenko EE, Kolesnikov SS, Lyubarsky AL. Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment. Nature 1985;313:310–3. 2. Cobbs WH, Barkdoll AE, Pugh EN. Cyclic GMP increases photocurrent and light sensitivity of retinal cones. Nature 1985;317:64–6. 3. Haynes LW, Kay AR, Yau KW. Single cyclic GMP‐activated channel activity in excised patches of rod outer segment membrane. Nature 1986;321:66–70. 4. Ebrey T, Koutalos Y. Vertebrate photoreceptors. Prog Retin Eye Res 2001;20:49–94. 5. Pepperberg DR, Crouch RK. An illuminating new step in visual‐pigment regeneration. Lancet 2001;358:2098–9. 6. Kono M, Goletz PW, Crouch RK. 11‐cis‐ and all‐trans‐retinols can activate rod opsin: rational design of the visual cycle. Biochemistry 2008;47:7567–71. 7. Sung CH, Schneider BG, Agarwal N, Papermaster DS, Nathans J. Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci USA 1991;88:8840–4. 8. Mendes HF, van der Spuy J, Chapple JP, Cheetham ME. Mechanisms of cell death in rhodopsin retinitis pigmentosa: implications for therapy. Trends Mol Med 2005;11:177–85. 9. Williams AJ, Paulson HL. Polyglutamine neurodegeneration: protein misfolding revisited. Trends Neurosci 2008;31:521–8. 10. Soto C, Estrada LD. Protein misfolding and neurodegeneration. Arch Neurol 2008;65:184–9. 11. Muchowski PJ. Protein misfolding, amyloid formation, and neurodegeneration: a critical role for molecular chaperones? Neuron 2002;35:9–12. 12. Alfinito PD, Townes‐Anderson E. Activation of mislocalized opsin kills rod cells: a novel mechanism for rod cell death in retinal disease. Proc Natl Acad Sci USA 2002;99:5655–60. 13. Galy A, Roux MJ, Sahel JA, Leveillard T, Giangrande A. Rhodopsin maturation defects induce photoreceptor death by apoptosis: a fly model for RhodopsinPro23His human retinitis pigmentosa. Hum Mol Genet 2005;14:2547–57. 14. Liu X, Garriga P, Khorana HG. Structure and function in rhodopsin: correct folding and misfolding in two point mutants in the intradiscal domain of rhodopsin identified in retinitis pigmentosa. Proc Natl Acad Sci USA 1996;93:4554–9. 15. Illing ME, Rajan RS, Bence NF, Kopito RR. A rhodopsin mutant linked to autosomal dominant retinitis pigmentosa is prone to aggregate and interacts with the ubiquitin proteasome system. J Biol Chem 2002;277:34150–60. 16. Rajan RS, Kopito RR. Suppression of wild‐type rhodopsin maturation by mutants linked to autosomal dominant retinitis pigmentosa. J Biol Chem 2005;280:1284–91. 17. Tam BM, Moritz OL. Characterization of rhodopsin P23H‐induced retinal degeneration in a Xenopus laevis model of retinitis pigmentosa. Invest Ophthalmol Vis Sci 2006;47:3234–41. 18. Tam BM, Moritz OL. Dark rearing rescues P23H rhodopsin‐induced retinal degeneration in a transgenic Xenopus laevis model of retinitis pigmentosa: a chromophore‐dependent mechanism characterized by production of N‐terminally truncated mutant rhodopsin. J Neurosci 2007;27:9043–53.
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19. Wu TH, Ting TD, Okajima TI, Pepperberg DR, Ho YK, Ripps H, et al. Opsin localization and rhodopsin photochemistry in a transgenic mouse model of retinitis pigmentosa. Neuroscience 1998;87:709–17. 20. Olsson JE, Gordon JW, Pawlyk BS, Roof D, Hayes A, Molday RS, et al. Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa. Neuron 1992;9:815–30. 21. Roof DJ, Adamian M, Hayes A. Rhodopsin accumulation at abnormal sites in retinas of mice with a human P23H rhodopsin transgene. Invest Ophthalmol Vis Sci 1994;35:4049–62. 22. Frederick JM, Krasnoperova NV, Hoffmann K, Church‐Kopish J, Ruther K, Howes K, et al. Mutant rhodopsin transgene expression on a null background. Invest Ophthalmol Vis Sci 2001;42:826–33. 23. Li T, Snyder WK, Olsson JE, Dryja TP. Transgenic mice carrying the dominant rhodopsin mutation P347S: evidence for defective vectorial transport of rhodopsin to the outer segments. Proc Natl Acad Sci USA 1996;93:14176–81. 24. Huang PC, Gaitan AE, Hao Y, Petters RM, Wong F. Cellular interactions implicated in the mechanism of photoreceptor degeneration in transgenic mice expressing a mutant rhodopsin gene. Proc Natl Acad Sci USA 1993;90:8484–8. 25. Deretic D, Schmerl S, Hargrave PA, Arendt A, McDowell JH. Regulation of sorting and post‐ Golgi trafficking of rhodopsin by its C‐terminal sequence QVS(A)PA. Proc Natl Acad Sci USA 1998;95:10620–5. 26. Chang GQ, Hao Y, Wong F. Apoptosis: final common pathway of photoreceptor death in rd, rds, and rhodopsin mutant mice. Neuron 1993;11:595–605. 27. Weiss ER, Hao Y, Dickerson CD, Osawa S, Shi W, Zhang L, et al. Altered cAMP levels in retinas from transgenic mice expressing a rhodopsin mutant. Biochem Biophys Res Commun 1995;216:755–61. 28. Hao W, Wenzel A, Obin MS, Chen CK, Brill E, Krasnoperova NV, et al. Evidence for two apoptotic pathways in light‐induced retinal degeneration. Nat Genet 2002;32:254–60. 29. Woodruff ML, Wang Z, Chung HY, Redmond TM, Fain GL, Lem J. Spontaneous activity of opsin apoprotein is a cause of Leber congenital amaurosis. Nat Genet 2003;35:158–64. 30. Brill E, Malanson KM, Radu RA, Boukharov NV, Wang Z, Chung HY, et al. A novel form of transducin‐dependent retinal degeneration: accelerated retinal degeneration in the absence of rod transducin. Invest Ophthalmol Vis Sci 2007;48:5445–53. 31. Oprian DD. The ligand‐binding domain of rhodopsin and other G protein‐linked receptors. J Bioenerg Biomembr 1992;24:211–7. 32. Robinson PR, Cohen GB, Zhukovsky EA, Oprian DD. Constitutively active mutants of rhodopsin. Neuron 1992;9:719–25. 33. Li T, Franson WK, Gordon JW, Berson EL, Dryja TP. Constitutive activation of phototransduction by K296E opsin is not a cause of photoreceptor degeneration. Proc Natl Acad Sci USA 1995;92:3551–5. 34. Calvert PD, Krasnoperova NV, Lyubarsky AL, Isayama T, Nicolo M, Kosaras B, et al. Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha‐subunit. Proc Natl Acad Sci USA 2000;97:13913–8. 35. Iakhine R, Chorna‐Ornan I, Zars T, Elia N, Cheng Y, Selinger Z, et al. Novel dominant rhodopsin mutation triggers two mechanisms of retinal degeneration and photoreceptor desensitization. J Neurosci 2004;24:2516–26. 36. Alloway PG, Howard L, Dolph PJ. The formation of stable rhodopsin–arrestin complexes induces apoptosis and photoreceptor cell degeneration. Neuron 2000;28:129–38. 37. Kiselev A, Socolich M, Vinos J, Hardy RW, Zuker CS, Ranganathan R. A molecular pathway for light‐dependent photoreceptor apoptosis in Drosophila. Neuron 2000;28:139–52.
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38. Chen J, Shi G, Concepcion FA, Xie G, Oprian D. Stable rhodopsin/arrestin complex leads to retinal degeneration in a transgenic mouse model of autosomal dominant retinitis pigmentosa. J Neurosci 2006;26:11929–37. 39. Mohand‐Said S, Deudon‐Combe A, Hicks D, Simonutti M, Forster V, Fintz AC, et al. Normal retina releases a diffusible factor stimulating cone survival in the retinal degeneration mouse. Proc Natl Acad Sci USA 1998;95:8357–62. 40. Streichert LC, Birnbach CD, Reh TA. A diffusible factor from normal retinal cells promotes rod photoreceptor survival in an in vitro model of retinitis pigmentosa. J Neurobiol 1999;39:475–90. 41. Chalmel F, Leveillard T, Jaillard C, Lardenois A, Berdugo N, Morel E, et al. Rod‐derived cone viability factor‐2 is a novel bifunctional‐thioredoxin‐like protein with therapeutic potential. BMC Mol Biol 2007;8:74. 42. Leveillard T, Mohand‐Said S, Lorentz O, Hicks D, Fintz AC, Clerin E, et al. Identification and characterization of rod‐derived cone viability factor. Nat Genet 2004;36:755–9. 43. Ripps H. Cell death in retinitis pigmentosa: gap junctions and the ‘bystander’ effect. Exp Eye Res 2002;74:327–36. 44. Suzumura A, Takeuchi H, Zhang G, Kuno R, Mizuno T. Roles of glia‐derived cytokines on neuronal degeneration and regeneration. Ann N Y Acad Sci 2006;1088:219–29. 45. Gupta N, Brown KE, Milam AH. Activated microglia in human retinitis pigmentosa, late‐onset retinal degeneration, and age‐related macular degeneration. Exp Eye Res 2003;76:463–71. 46. Yu DY, Cringle SJ, Su EN, Yu PK. Intraretinal oxygen levels before and after photoreceptor loss in the RCS rat. Invest Ophthalmol Vis Sci 2000;41:3999–4006. 47. Yu DY, Cringle S, Valter K, Walsh N, Lee D, Stone J. Photoreceptor death, trophic factor expression, retinal oxygen status, and photoreceptor function in the P23H rat. Invest Ophthalmol Vis Sci 2004;45:2013–9. 48. Shen J, Yang X, Dong A, Petters RM, Peng YW, Wong F, et al. Oxidative damage is a potential cause of cone cell death in retinitis pigmentosa. J Cell Physiol 2005;203:457–64. 49. Komeima K, Rogers BS, Campochiaro PA. Antioxidants slow photoreceptor cell death in mouse models of retinitis pigmentosa. J Cell Physiol 2007;213:809–15. 50. Komeima K, Rogers BS, Lu L, Campochiaro PA. Antioxidants reduce cone cell death in a model of retinitis pigmentosa. Proc Natl Acad Sci USA 2006;103:11300–5. 51. Punzo C, Kornacker K, Cepko CL. Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa. Nat Neurosci 2009;12:44–52. 52. Steinberg RH. Survival factors in retinal degenerations. Curr Opin Neurobiol 1994;4:515–24. 53. LaVail MM, Yasumura D, Matthes MT, Drenser KA, Flannery JG, Lewin AS, et al. Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long‐term survival and late‐stage therapy. Proc Natl Acad Sci USA 2000;97:11488–93. 54. Leonard KC, Petrin D, Coupland SG, Baker AN, Leonard BC, LaCasse EC, et al. XIAP protection of photoreceptors in animal models of retinitis pigmentosa. PLoS ONE 2007;2: e314. 55. Chadderton N, Millington‐Ward S, Palfi A, O’Reilly M, Tuohy G, Humphries MM, et al. Improved retinal function in a mouse model of dominant retinitis pigmentosa following AAV‐delivered gene therapy. Mol Ther 2009;17(4), 593–9. 56. O’Reilly M, Millington‐Ward S, Palfi A, Chadderton N, Cronin T, McNally N, et al. A transgenic mouse model for gene therapy of rhodopsin‐linked retinitis pigmentosa. Vision Res 2008;48:386–91. 57. O’Reilly M, Palfi A, Chadderton N, Millington‐Ward S, Ader M, Cronin T, et al. RNA interference‐mediated suppression and replacement of human rhodopsin in vivo. Am J Hum Genet 2007;81:127–35. 58. Hubbard R. The thermal stability of rhodopsin and opsin. J Gen Physiol 1958;42:259–80.
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59. Berson EL, Rosner B, Sandberg MA, Hayes KC, Nicholson BW, Weigel‐DiFrano C, et al. Vitamin A supplementation for retinitis pigmentosa. Arch Ophthalmol 1993;111:1456–9. 60. Berson EL, Rosner B, Sandberg MA, Hayes KC, Nicholson BW, Weigel‐DiFranco C, et al. A randomized trial of vitamin A and vitamin E supplementation for retinitis pigmentosa. Arch Ophthalmol 1993;111:761–72. 61. Li T, Sandberg MA, Pawlyk BS, Rosner B, Hayes KC, Dryja TP, et al. Effect of vitamin A supplementation on rhodopsin mutants threonine‐17 ! methionine and proline‐347 ! serine in transgenic mice and in cell cultures. Proc Natl Acad Sci USA 1998;95:11933–8. 62. Noorwez SM, Kuksa V, Imanishi Y, Zhu L, Filipek S, Palczewski K, et al. Pharmacological chaperone‐mediated in vivo folding and stabilization of the P23H‐opsin mutant associated with autosomal dominant retinitis pigmentosa. J Biol Chem 2003;278:14442–50. 63. Noorwez SM, Malhotra R, McDowell JH, Smith KA, Krebs MP, Kaushal S. Retinoids assist the cellular folding of the autosomal dominant retinitis pigmentosa opsin mutant P23H. J Biol Chem 2004;279:16278–84. 64. Chacko DM, Rogers JA, Turner JE, Ahmad I. Survival and differentiation of cultured retinal progenitors transplanted in the subretinal space of the rat. Biochem Biophys Res Commun 2000;268:842–6. 65. Klassen HJ, Ng TF, Kurimoto Y, Kirov I, Shatos M, Coffey P, et al. Multipotent retinal progenitors express developmental markers, differentiate into retinal neurons, and preserve light‐mediated behavior. Invest Ophthalmol Vis Sci 2004;45:4167–73. 66. Coles BL, Angenieux B, Inoue T, Del Rio‐Tsonis K, Spence JR, McInnes RR, et al. Facile isolation and the characterization of human retinal stem cells. Proc Natl Acad Sci USA 2004;101:15772–7. 67. Canola K, Angenieux B, Tekaya M, Quiambao A, Naash MI, Munier FL, et al. Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate. Invest Ophthalmol Vis Sci 2007;48:446–54. 68. MacLaren RE, Pearson RA, MacNeil A, Douglas RH, Salt TE, Akimoto M, et al. Retinal repair by transplantation of photoreceptor precursors. Nature 2006;444:203–7. 69. Ghosh F, Engelsberg K, English RV, Petters RM. Long‐term neuroretinal full‐thickness transplants in a large animal model of severe retinitis pigmentosa. Graefes Arch Clin Exp Ophthalmol 2007;245:835–46.
Human Diseases Associated with GPR54 Mutations Milena Gurgel Teles,* Leticia Ferreira Gontijo Silveira,* Suzy Bianco,{ and Ana Claudia Latronico* *Unidade de Endocrinologia do Desenvolvimento, Laborato´rio de Hormoˆnios e Gene´tica Molecular/LIM42 da Disciplina de Endocrinologia do Hospital das Clinicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sao Paulo 05403-900, Brazil {
Department of Molecular and Cellular Pharmacology, Leonard M. Miller School of Medicine, University of Miami, Miami, Florida
I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII.
Introduction .................................................................................. Structure and Expression of GPR54 .................................................... The GPR54 Ligand ......................................................................... GPR54 and KISS1 Genes in Evolution................................................. Kisspeptin/GPR54 Signaling Pathway .................................................. KISS1/GPR54 and Cancer ................................................................ Neuroendocrine Studies................................................................... Expression Studies .......................................................................... gpr54 and kiss1 Knockout Mice ......................................................... Loss‐of‐Function Mutations of GPR54................................................. The Role of GPR54 in Central Precocious Puberty ................................. Conclusion.................................................................................... References ....................................................................................
34 35 37 38 39 40 41 43 44 44 49 51 51
G protein‐coupled receptor 54 (GPR54) was first described as an orphan receptor in the rat brain one decade ago. At that time, all we knew about this receptor was that it had a high homology with other G protein‐coupled receptors, like galanin receptors. Later, its endogenous ligand, kisspeptin, was identified and the kisspeptin‐GPR54 system became one of the most important excitatory neuroendocrine regulators of puberty initiation. Several loss‐of‐ function mutations in GPR54 gene were described to be associated with sporadic and familial normosmic isolated hypogonadotropic hypogonadism phenotype in humans. Consistent with this fact, knockout mice for gpr54/ recapitulated the human phenotype of the lack of reproductive maturation. On the other hand, a unique activating mutation (R386P) was recently described in Progress in Molecular Biology and Translational Science, Vol. 88 DOI: 10.1016/S1877-1173(09)88002-2
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Copyright 2009, Elsevier Inc. All rights reserved. 1877-1173/09 $35.00
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this receptor in a girl with central precocious puberty. This missense mutation located at carboxy‐terminal tail of the GPR54 leads to prolonged activation of intracellular signaling pathways in response to kisspeptin, suggesting an uncommon model of G protein‐coupled receptor activation. This chapter will describe the kisspeptin‐GPR54 complex physiology and its current role in human diseases.
I. Introduction Numerous hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli exert their effects on cells by binding to G protein‐coupled receptors.1 These receptors are seven‐transmembrane spanning domain receptors, and belong to the largest superfamily of proteins and regulate the function of virtually every cell.2 As pointed out by their name, these receptors possess an inherent ability of recruiting and regulating the activity of specific heterotrimeric G proteins. These highly specialized transducers are composed of three subunits, one alpha, one beta, and one gamma (a, b, g). They can modulate the activity of multiple signaling pathways leading to diverse biological responses.1 The binding of an endogenous ligand to its cognate G protein‐coupled receptor takes place on the cell surface. It may stabilize the receptor on its active conformation or induce a conformational change that activates the downstream signaling pathway regulated by that receptor. Once the receptor is on its active conformation, the a‐subunit is released from the heterotrimer originally bound to the receptor. The free a‐subunit, in turn, may regulate several signaling pathways inside the cell. These include the modulation of adenylyl cyclase or membrane‐bound phospholipases as well as the modulation of ion channels. G protein‐coupled receptors play a significant role in body homeostasis, helping keeping vital physiological processes, such as cardiac function, body temperature, blood pressure, brain function, and, of course, reproductive capacity under tight control. Therefore, it is not surprising that abnormal G protein‐coupled receptor signaling has been identified as the underlying cause of so many human diseases. Understanding the signaling pathways regulated by these receptors is the first step in the development of new diagnostic tools and treatment for the associated diseases. Distinct structural abnormalities of genes encoding G protein‐coupled receptors have been implicated in a number of endocrine disorders, including reproductive disorders. Loss‐of‐function mutations of the G protein‐coupled receptors are usually associated with hormone resistance conditions and, consequently with the phenotypes of complete or partial hormonal deficiency.3
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In contrast, the gain‐of‐function mutations affecting these receptors are associated with hormonal hyperfunction conditions. The abnormal receptor activation determined by these mutations, most of them are missense mutations in the transmembrane domain, generally occur in the absence of the ligand (constitutively activation) and rarely in the presence of the cognate or noncognate ligand, characterizing the nonconstitutively and promiscuous receptor activation models, respectively.3,4 One of the great mysteries of human biology is what initiates puberty. Definitely, we currently know that gonadotropin‐releasing hormone (GnRH) is a decapeptide central to the initiation of the reproductive hormone cascade. It is produced by hypothalamic neurons in a pulsatile manner and released into the hypophyseal portal circulation to stimulate the biosynthesis and secretion of the pituitary gonadotropins, luteinizing hormone (LH) and follicle‐ stimulating hormone (FSH). The pituitary gonadotropins stimulate gonadal function, including gametogenesis and steroid hormone synthesis. However, little is known about the precise mechanisms underlying the maturation and function of GnRH neurons and the regulation of GnRH secretion.5 The episodic release of GnRH seems to be modulated by excitatory and inhibitory signals transmitted by neurohormones and neurotransmitters acting at the level of the hypothalamus. The complex composed of a G protein‐coupled receptor, GPR54, and its endogenous ligand, kisspeptin, was recently described as a new excitatory neuroregulator system for the secretion of GnRH. In the last 6 years, several loss‐of‐function point mutations and deletions of the human GPR54 were identified in familial or sporadic isolated hypogonadotropic hypogonadism without olfactory abnormalities.6–11 In addition, a unique gain‐of‐function missense mutation in the carboxy‐terminal tail of the GPR54 was identified in a girl with idiopathic central precocious puberty.12 Taken together, these findings have established the essential role of the GPR54 as a genetic determinant and irrefutable gatekeeper of normal reproductive function.13
II. Structure and Expression of GPR54 GPR54 receptor is a member of the rhodopsin family of G protein‐coupled receptor superfamily. It was first cloned in 1999 as an orphan receptor in brain of rats.14 Based on the high‐sequence conservation of G protein‐coupled receptor transmembrane regions, Canadian researchers used the degenerate PCR strategy to discover this new receptor.14 The rat GPR54 contains an open reading frame of 1191 bp encoding for a protein of 396 amino acids and has 30–40% of homology to galanin receptors. Using in situ hybridization of rat brain sections, the distribution of GPR54 mRNA was found to be discretely localized in many areas.14 However, the highest levels of expression in rat brain
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were seen in hypothalamic and amygdaloid nuclei.14 A BLAST search with the rat GPR54 sequence revealed high identity with a human 3.5 Mb contig located in chromosome 19p13.3.14 The human GPR54 gene has five exons and four introns in a length of approximately 3.5 kb. It encodes a seven‐transmembrane receptor with 398 amino acids and has also a weak homology with the galanin receptors, although neither galanin nor galanin‐like peptide bind to GPR54 (Fig. 1).14–16 The human GPR54 receptor, also named AXOR12 and hOT7T175, is widely expressed in the brain, particularly in the hypothalamus, midbrain, pons, medulla, hippocampus, and amygdala and also in the pituitary, pancreas, placenta, and spinal cord.14–16 Lower level expression was found in the heart, muscle, kidney, liver, intestine, thymus, lung, and testis.14–16
NH2 R297L
L102P e1
e2
i2
i1
e3
C223R
R331stop 1001_1002insC
L148S IVS4/EX5_del155 i3
R386P
Stop399R COOH FIG. 1. Schematic representation of the G protein‐coupled receptor 54 (GPR54). It is a seven‐ transmembrane receptor, with three extracellular (e1, e2, and e3), and three intracellular (i1, i2, and i3) loops together with one extracellular amino‐terminal tail (–NH2) and one intracellular carboxy‐terminal tail (–COOH). The GPR54 inactivating mutations described so far in patients with normosmic hypogonadotropic hypogonadism are represented by black dots. The unique nonconstitutively activating mutation is indicated in a red dot.
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Notably, the human GPR54 gene contains a GC‐rich sequence. This sequence feature could be a complicate factor for gene amplification in human studies, requiring generally special PCR conditions for human DNA analysis.7 Alignment of the predicted amino acid sequences of GPR54 from mammalian and nonmammalian species reveals that the percentage of homology between GPR54 from primates and rodent sequences is greater than 80%.17 This high degree of sequence conservation for the receptor indicates that GPR54 is well conserved during evolution.17
III. The GPR54 Ligand The endogenous ligand of GPR54 receptor was unknown until 2001, when peptides with high affinity and agonist activity for the GPR54 were isolated from the placenta by three independent groups.15,16,18 These small peptides were all derived from the same precursor peptide, kisspeptin‐1, encoded by the KISS1 gene15,16,18 (Fig. 2). By that time, the KISS1 gene was already known as a metastasis suppressor gene.19 The KISS1 mRNA was originally isolated from a nonmetastatic human malignant melanoma cell line using a modified subtractive hybridization technique.19 The KISS1 gene was so named to combine the laboratory nomenclature for putative ‘‘suppressor sequences’’ with acknowledgment of the gene’s discovery in Hershey, Pennsylvania, a city known for its famous chocolate Signal peptide NH2 1
20
68
121
145 COOH
KP-145
15.4 kDa
KP-54, metastin
RF-NH2
5.9 kDa
KP-14
RF-NH2
1.7 kDa
KP-13
RF-NH2
1.6 kDa
KP-10
RF-NH2
1.3 kDa
FIG. 2. Products of the KISS1 gene: a 145‐amino acid propeptide called kisspeptin‐145 (KP‐145) or the KISS‐1 peptide. Shown are cleavage sites on the propeptide that lead to the production of the RF‐amidated kisspeptin 54, also known as metastin. Shorter peptides (such as kisspeptin‐10, ‐13, and ‐14) were identified by mass spectrometry with different weights. These peptides share a common C‐terminus and RF‐amidated motif with kisspeptin‐54.
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kisses.19 The KISS1 was mapped to the long arm of chromosome 1 (1q32) and comprises three exons, spanning a region of 6.36 kb in the human genome.20 The initial product of the KISS1 gene is a hydrophylic precursor peptide, kisspeptin‐1, containing a putative 19 amino acid signal sequence (predictive of secreted peptides), two potential dibasic cleavage sites, and one putative site for terminal cleavage and amidation.20 Kisspeptin‐1 is submitted to proteolytic processing followed by the transfer of a NH2 group to the carboxy‐terminal domain. The largest and more abundant cleavage product of kisspeptin‐1 is kisspeptin‐54 (68–121), with 54 amino acids, also known as metastin.21 Smaller cleavage products of 14, 13, and 10 residues have also been isolated from the human placenta and share with kisspeptin‐54 the highly conserved amidated carboxy‐terminal sequence of 10 amino acids (Fig. 2).15,16,18 All these fragments have high receptor‐binding affinity, which lies within the C‐terminal RF‐amide decapeptide.15,16,18 The absence of any of the 10 residues or of the C‐terminal NH2 group drastically decreases the receptor affinity.16 The shorter peptides, kisspeptin‐14, ‐13, and ‐10, seem to be the products of kisspeptin‐54 degradation, since no potential cleavage sites that might originate them have been detected.18,21 Kisspeptin‐54 was initially named metastin due to its capacity to suppress metastasis of malignant melanoma and breast cancer cell lines in vivo,19,22 but nowadays, the term kisspeptins has become more widely used to denominate this group of peptides with a C‐terminal Arg‐Phe‐NH2 motif, characteristic of the RF‐amide super family.23 The full‐length precursor peptide kisspeptin‐1 contains a PEST sequence (proline, glutamic acid, serine, and threonine rich sequence), which although upstream of the kisspeptin‐13 and kisspeptin‐10 sequences, is still present in the kisspptin‐54 sequence. This motif predisposes proteins for ubiquitination and proteosome degradation and suggests that cytosolic kisspeptin‐1 and kisspeptin‐54 would have a short half‐life.24
IV. GPR54 and KISS1 Genes in Evolution KiSS1 and GPR54 genes have been cloned in a variety of vertebrate species, mostly mammals.25 In mice, the longest cleavage product of the precursor kisspeptin‐1, corresponding to kisspeptin‐54 in humans, is 52 amino acids long. Although human and mouse kisspepetins share relatively low overall homology (52%), kisspeptin‐10 is highly conserved between mouse and human, varying by only one amino acid.26 Very recently, the cDNAs encoding gpr54 and kiss1 have been identified in different species of teleosts, including zebrafish, medaka, fugu, tilapia, fathead minnow, and gray mullet.27–34
GPR54 MUTATIONS AND HUMAN DISEASES
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Several nonmammalian species were shown to express a second isoform of KISS1, designated KISS2.25,35 In addition, two or three forms of the GPR54 genes have been identified in various vertebrates, including fish, amphibians, and a mammalian monotreme species, the platypus.25 The nonmammalian KISS1 gene was found to be the ortholog of the mammalian KiSS1 gene, while the KISS2 gene is a novel form, encoding a C‐terminally amidated decapeptide. Alignment of predicted amino acid sequences revealed that teleost kisspeptin‐1 sequences were poorly conserved in comparison to their mammalian homologs, with the exception of the region representing kisspeptin‐10, which is highly conserved, with 80–100% identity with the mammalian kisspeptin.31,33
V. Kisspeptin/GPR54 Signaling Pathway Initial studies on the characterization of GPR54 ligands showed that kisspeptin stimulation of heterologous cell models overexpressing the rat or human GPR54 resulted in a robust intracellular calcium release.15,16,18 These original studies demonstrated that activation of GPR54 receptor by kisspeptins results in coupling to the Gaq/11 pathway, with activation of phospholipase C (PLC), subsequent phosphatidylinositol biphosphate (PIP2) hydrolysis, and accumulation of inositol triphosphate (IP3) in the cells, leading to intracellular calcium mobilization and protein kinase C activation and arachidonic acid release15,16,18 (Fig. 3). Moreover, kisspeptin did not modify basal or forskolin‐ induced cAMP levels, thus supporting the notion that GPR54 does not couple to Gs and/or Gi/o subfamilies.16 Kisspeptins also induce the activation of additional signaling mechanisms involving the family of the mitogen‐activated protein kinases (MAPK).15,36 In CHO cells expressing human or rat GPR54, kisspeptin induced strong and sustained stimulation of phosphorylation of the MAP kinases extracellular signal regulated kinases, ERK1 and ERK2, and a weak stimulation of p38 MAPK phosphorylation, whereas no activation was observed for stress‐activated protein kinase/c‐Jun NH2‐terminal kinase (SAPK/JNK).15,36 Surprisingly, despite the involvement of the MAPK pathways in the control of cell cycle and proliferation, kisspeptin stimulation resulted in inhibition of cell proliferation.15 Castellano et al.37 showed that MAPK pathway activation is required for kisspeptin action in GnRH neurons. Blocking of the MAPK pathway abolished the kisspeptin‐induced GnRH secretion in hypothalamic fragments ex vivo. Nevertheless, the specific pattern of kinases activated by GPR54 stimulation seems to be dependent on the cellular context studied.38 Activation of ERK1/ 2 seems to be the most conserved kinase signal among the cell types studied, although its precise role in the antimetastatic and antimigratory actions of kisspeptin/GPR54 is still to be fully established.36
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Kisspeptin GnRH neuron GPR54 b
g
Gαq PIP2
PLCg
DAG PKC
Ca2+
IP3
RAF1
Ca2+ Ca2+ Ca2+
MEK
ER
ERK pERK FIG. 3. Intracellular signaling cascade of GPR54 receptor after stimulation by kisspeptin. Abbreviations used are: PLC, phospholipase C; PIP2, phosphatidylinositol; IP3, inositol triphosphate; PKC, protein kinase C; DAG, diacyl glycerol; RAF1 and MEK, serine/threonine‐specific kinases; ERK, extracellular‐regulated kinase; pERK, phosphorylated extracellular‐regulated kinase; ER, endoplasmic reticulum.
In addition, it was demonstrated that KISS1 overexpression in a fibrosarcoma HT‐1080 cell line, with unknown GPR54 expression, decreased matrix metalloproteinase 9 (MMP‐9) expression, but not of other MMPs.39 This effect, which seemed to be independent of GPR54 binding and MAPK activation, was presumably attributed to a reduction in NFkB binding to the MMP9 promoter.39 The MAPK independent suppression of MMP9 may suggest alternative signaling pathways to GPR54. However, it is unclear if a decrease in MMP9 can also be observed after GPR54/kisspeptin signaling.36,40
VI. KISS1/GPR54 and Cancer Northern blot analyses comparing mRNAs from a panel of human melanoma cells revealed that KISS1 mRNA expression occurred only in nonmetastatic melanoma cells.19 Transfection of a full‐length KISS1 cDNA into highly
GPR54 MUTATIONS AND HUMAN DISEASES
41
metastatic human melanoma cell lines (C8161) suppressed metastasis to the lung by more than 95% following intravenous or orthotopic injection in an expression‐dependent manner, suggesting that KISS1 expression was capable to suppress the metastatic potential of malignant melanoma cells.19,22 In vitro experiments have shown that nonmetastatic cell lines (CHO cells) overexpressing GPR54 exhibited a decrease in proliferation, motility, invasion, with concomitant increases in focal adhesions, and stress fiber formation, when exposed to exogenous kisspeptins.41 These characteristics are consistent with changes expected in cells which would have lower metastatic potential. Kisspeptin exposure has shown effects on migration in other cell lines with high endogenous or experimentally elevated levels of GPR54.40,41 Overexpression of KISS1 into a human metastatic breast cancer cell line MDA‐MB‐435 induced a suppression of metastases to the lung greater than 95% following orthotopic injection.22 In contrast, KISS1 expression had no effect on adhesion, motility, or invasion in MDA‐MB‐435 cells, although there was a significant decrease in soft agar colony formation.40,41 In line with this, kisspeptin severely reduced cell proliferation in MDA‐MB‐435S cells overexpressing GPR54.42 Interestingly, Nash et al.43 used C8161.9 human melanoma cells overexpressing KISS1 to demonstrate that kisspeptin secretion was required for multiple organ metastasis suppression and for maintenance of disseminated cells in a dormant state. This was independent of GPR54 expression in the C8161.9 cells, suggesting the existence of another kisspeptin receptor and/or paracrine signaling.43 In fact, the mechanism of metastasis suppression induced by kisspeptin is still uncertain. It is not entirely clear if kisspeptins binding to GPR54 is required for this function, since most studies were performed in cells that had experimentally elevated GPR54 expression, exposed to variable exogenous doses of kisspeptins, which could be either pharmacologic or physiologic.40 KISS1 mRNA expression have been evaluated in several tumor types, including melanoma, breast cancer, follicular, and papillary carcinoma of the thyroid, superficial, and invasive bladder neoplasms, esophageal squamous cell carcinoma, gastric cancer, and hepatocellular carcinoma.40 In general, loss or reduction of KISS1 expression was inversely correlated with tumor progression, metastatic potential, and survival.40
VII. Neuroendocrine Studies The finding that GPR54 inactivation causes hypogonadotropic hypogonadism in humans motivated a series of pharmacological and physiological studies, which confirmed the crucial role of the kisspeptin/GPR54 system in the hypothalamic–pituitary–gonadal axis activation.44
42
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Infusion of kisspeptin peptides by several administration routes, such as intracerebroventricular, subcutaneous, intraperitoneal, and intravenous, was capable to elicit powerful LH and FSH secretion in several mammalian species, including rodents,45–49 sheep,50 monkeys,51,52 and even humans.53 In mice, intracerebroventricular doses as low as 1 fmol evoke nearly maximal LH responses after 30 min.45 Interestingly, previous administration of a GnRH antagonist can abolish completely the kisspeptin effects on LH, indicating that its action is through the GnRH.45,51 Administration of kisspeptin to gpr54 knockout mice is not capable of stimulating LH release, suggesting that the stimulatory effects of this peptide are mediated through this receptor only.49 In addition, kisspeptin administration to immature animals was capable to induce precocious pubertal development.51,54 Intermittent intracerebroventricular kisspeptin administration to immature female rats for 7 days has been shown to induce precocious activation of the gonadotropic axis. Administration of 1 nmol of kisspeptin every 12 h between 26 and 31 days postpartum induced vaginal opening in 75% of females at the age of 31 days, an age‐point when none of the female animals injected with vehicle presented canalization of the vagina.54 The rats injected with intermittent kisspeptin also presented with enlarged uterus and increased serum levels of LH and estradiol.54 Likewise, in agonadal juvenile male rhesus monkeys, in which pituitary responsiveness to GnRH had been increased by a priming infusion of exogenous GnRH, intravenous infusions of human kisspeptin‐10 every hour, for 48 h, elicited a sustained train of endogenous hypothalamic GnRH discharges, as reflected by the pulsatile pattern of circulating LH concentrations.55 On the other hand, similarly to the GnRH/GnRH‐receptor system, it has been shown that continuous infusion of kisspeptin decreases LH levels in agonadal juvenile male monkeys, suggesting that kisspeptin secretion is probably pulsatile, and the continuous stimulation may induce desensitization of the receptor.56 Even though continuous kisspeptin infusion decreased LH levels, in day 4 of treatment, administration of NMDA, an excitatory amino acid analog, and GnRH were still able to elicit an LH surge, indicating that signaling capacity of the GnRH receptor as well as LH stores were intact.56 The neuroendocrine circuitries that serve as convergence points between the body weight and reproduction systems have been recently explored.57 Rare mutations in both the leptin gene and its receptor have been identified in patients with hypogonadotropic hypogonadism and morbid obesity.58 Interestingly, the administration of kisspeptin can correct the hypogonadism of leptin deficient animals, suggesting that the kisspeptin/GPR54 pathway may serve a converging function between the reproductive and nutritional systems.59
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VIII. Expression Studies Kisspeptin and GPR54 are widely expressed in human tissues.15,16,18 It was demonstrated by RT‐PCR that KISS1 mRNA is expressed mainly in the placenta, but also in the brain, including the hypothalamus, basal ganglia, pituitary, and peripheral organs, such as pancreas, testis and, in lower levels, in the liver and small intestine.15,16,18 GPR54 transcripts were particularly abundant in placenta, brain, pituitary, spinal cord, and pancreas, whereas lower levels were detected in other tissues, including lymphocytes, spleen, testis, and adipose tissue.15,16,18 In situ hybridization study of the mouse and the rat brains, showed that expression of KISS1 was highest in the arcuate (ARC) and anteroventral periventricular (AVPV) nuclei, known to send projections to the medial preoptic area, where there is an abundance of GnRH cell bodies.60 Moreover, it was demonstrated that GnRH neurons coexpress gpr54 transcripts.60 Hypothalamic kiss1 and gpr54 mRNA expression increases progressively across the pubertal development, reaching maximum levels at the beginning of puberty in rodents and primates.47,51 In male and female rodents, hypothalamic kiss1 mRNA expression increased dramatically after bilateral gonadectomy in adult animals and returned to the anterior levels after sexual steroids replacement as demonstrated by immunohistochemistry and in situ hybridization, suggesting an inhibitory effect for the sex steroids.47 In the AVPV, however, kiss1 expression was decreased after gonadectomy and increased after sex steroid replacement, in a positive feedback pattern.60,61 Moreover, whereas the overall distribution of kisspeptin immunoreactivity was very similar between males and females, the number of cell bodies expressing kisspeptin in the AVPV is over 10‐fold higher in the female.62 Interestingly, the AVPV is one of the only sexually dimorphic areas in the rodent brain that is larger in the female than the male.60 Virtually, all kisspeptin neurons in the ARC and the AVPV were shown to coexpress the estrogen receptor‐a (ER‐a), whereas only 25–30% of these neurons express the ER‐b. Furthermore, ER‐b knockout ovariectomized female mice respond to estradiol administration in the same way as the wild‐ type animals, showing that the estradiol effects are mediated by the ER‐a. In males, testosterone effects are mediated by the androgen receptor as well as the ER‐a, after testosterone aromatization in estradiol in the ARC. However, in the AVPV, testosterone‐induced kiss1 transcription seems to be mediated exclusively via ER‐a. The ARC is known to control the negative feedback regulation of GnRH and gonadotropin secretion, whereas the estrogen‐ induced kisspeptin increase in the AVPV seems to be involved in the positive feedback regulation which leads to the preovulatory LH surge in females.60,63
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In addition to the hypothalamic nuclei, it was recently demonstrated by immunohistochemistry that kisspeptin and GPR54 are coexpressed by the gonadotrophs, suggesting the possibility of an autocrine or paracrine action of kisspeptin in the pituitary.64 However, in vitro studies assessing the direct stimulatory effects of kisspeptins on gonadotropin secretion in the pituitary have given conflicting results.65 Gutierrez‐Pascual et al.66 demonstrated that kisspeptin‐10 induced a rise in free cytosolic calcium concentration in approximately 10% of male rat pituitary cells, including not only gonadotrophs, but also somatotrophs.
IX. gpr54 and kiss1 Knockout Mice gpr54 and kiss1 knockout mice have been generated by different groups of researchers.7,67–70 The phenotype of these animals is in general similar, with slight variations, which were attributed to the type of mutation carried by each line.70 Both the gpr54 and kiss1 knockout mice fail to undergo pubertal maturation and show poor development of the gonads, hypogonadotropic hypogonadism and infertility. Spermatogenesis and ovulation are severely impaired and mutant females do not show estrous cycling. Nevertheless, GnRH neurons were anatomically intact in these animals, with normal hypothalamic GnRH content, indicating that migration of GnRH neurons occurs normally. Apart from the reproductive system, no alterations have been described in other organs or systems. The gonads and the anterior pituitary retain functional responses to exogenous GnRH stimulation, consistent with the primary defect being a failure to secrete GnRH from the hypothalamus.70 When injected with kisspeptin, gpr54 knockout mice fail to increase gonadotropins, whereas kiss1 knockout mice respond with increased gonadotropin levels, suggesting that kisspeptins act directly and uniquely through GPR54 signaling to stimulate gonadotropin release.69 Taken together, these observations suggest that the primary defect in these animals is a failure to secrete GnRH from the hypothalamus and provide evidence that these molecules constitute an authentic receptor/ligand pair with no obvious redundancy or overlap with other signaling pathways.70
X. Loss‐of‐Function Mutations of GPR54 Congenital isolated hypogonadotropic hypogonadism (IHH) is defined as a deficiency of the pituitary secretion of LH and FSH, which results from defects in the synthesis, secretion, and action of GnRH, leading to impairment of pubertal maturation and reproductive function.71 It is characterized by low levels of sex steroids in the presence of low or inappropriately normal LH and
GPR54 MUTATIONS AND HUMAN DISEASES
45
FSH serum levels, with no anatomical lesion in the hypothalamo‐pituitary tract and no other associated pituitary hormone deficiency.72 It is called Kallmann syndrome when associated to anosmia.73 IHH is an infrequent and heterogeneous disorder, which can be sporadic or inherited as, autosomal recessive, dominant, or an X‐linked trait. IHH is usually diagnosed in the second or third decade of life, presenting with delayed pubertal development, primary amenorrhea, or infertility.72 The presence of microphallus and cryptorchidism at birth or in younger age may disclose the diagnosis before puberty.73 An increasing number of genes has been described to be involved in the pathogenesis of IHH with normal sense of smell, including defects in GnRH receptor (GnRHR), in fibroblast growth factor receptor‐1 (FGFR1), in prokineticin (PROK2) and neurokinin B (TAC3), and their respective receptors (PROKR2 and TACR).74–77 In 2003, using the linkage analysis methodology, two different groups first described GPR54 loss‐of‐function mutations in patients with normosmic IHH.7,8 de Roux et al.8 described one consanguineous family with five affected members that harbored a 155 bp deletion in homozygous state within the transition of intron 4 and exon 5 of the GPR54 gene. The unaffected members were heterozygous or homozygous for the wild‐type sequence.8 This deletion removes the G protein binding domain which is located on the third intracellular loop of GPR54. Therefore, even if present on the cell surface and able to bind kisspeptin, receptors carrying this deletion are unable to couple to G proteins and thus incapable of signaling.8 Seminara et al.7 reported one novel homozygous mutation in six members with normosmic IHH of a large consanguineous Saudi Arabian family. The four males and two females with IHH harbored a missense mutation in homozygous state within exon 3 of GPR54 gene. The nucleotide thymine was changed by a cytosine resulting in the substitution of a leucine by a serine at position 148 (L148S). This amino acid change takes place in the second intracellular loop of GPR54 receptor, near the DRW/Y motif. This motif has been shown to regulate the activation and inactivation of a number of G protein‐coupled receptors. The critical role of the DRW/Y motif is highlighted by several descriptions of mutations in and around this motif that result in constitutive activation or inactivation of the mutated receptors.78 In fact, functional studies indicate that inositol phosphate production by the L148S GPR54 mutant is severely impaired.7 Interestingly, the mutation of an analogous leucine on the a1‐ adrenergic receptor has been recently reported to equally impair receptor function, indicating that leucine at this position is required for adequate receptor activation.79 On the other hand, expression levels and trafficking of L148S GPR54 mutant to the cell surface have been reported to be similar to those of the wild‐type GPR54. Using tagged receptors, Wacker et al.79 demonstrated that immunoprecipitation of L148S GPR54 mutant is comparable to
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that of the wild‐type GPR54, indicating that this mutation does not affect receptor expression. Likewise, confocal imaging and cell sorting studies show that trafficking of the L148S GPR54 mutant to the plasma membrane and its cellular localization are not altered.79 Two other mutations (R331X and X399R) in compound heterozygous state were identified in an Afro‐American patient, but in this case, with sporadic IHH in Seminara’s study.7 The first mutation was an insertion of a stop codon at position 331 on the beginning of the intracellular carboxyl tail, whereas the second mutation was the substitution of the expected stop codon of GPR54 by an arginine. The signaling capacity of both mutants individually transfected into COS‐7 cells as measured by kisspeptin‐stimulated inositol phosphate production in transfected cells was reported to be profoundly impaired.7 The introduction of the stop codon at position 331 would, at best, produce a truncated GPR54 receptor missing the entire intracellular carboxyl tail. Such deletion is expected to severely disrupt receptor signaling. On the other hand, the replacement of the normal stop codon (X399R) is predicted to affect stability and may result in protein misfolding followed by redirection of this abnormal GPR54 to destruction. Interestingly, all the affected patients described by Seminara et al.7 had at least a partial response to acute GnRH stimulation test. In fact, the Afro‐American patient with sporadic normosmic IHH due to compound heterozygous mutations exhibited a leftward‐shifted dose response curve when compared to other patients with IHH without GPR54 mutations.7 Since then, four other inactivating GPR54 mutations were described in patients with normosmic IHH (Table I). Semple et al.11 described one sporadic case of normosmic IHH harboring a compound heterozygous mutation in GPR54. The boy had micropenis and undescended testes at birth and undetectable serum gonadotropins at 2 months of age. The first variant was a transition c.667T > C in exon 4, leading in a substitution of a cysteine near the cytoplasmic end of the fifth transmembrane helix for arginine (C223R), while the second mutation was a transversion in exon 5, c.891 G > T, resulting to the substitution of an arginine in the third extracellular loop for leucine (R297L). The proband’s mother was heterozygous for the R297L mutation, and experienced menarche at 11 years of age. His father is believed to have normal reproductive function but was not available for testing.11 There was no family history of hypogonadism. Cysteine at the position 223 is evolutionarily conserved from fish to humans, suggesting physiological relevance. The importance of this cysteine for GPR54 function was reiterated by the lack of calcium signaling of GPR54 receptors carrying this mutation. On the other hand, the arginine on position 297 is not highly conserved; and calcium signaling by this GPR54 mutant was only mildly affected.11 Nevertheless, this mild effect was enough to result in hypogonadism when combined with the C223R amino acid GPR54 substitution.11
TABLE I HUMAN GPR54 INACTIVATING MUTATIONS ASSOCIATED WITH NORMOSMIC HYPOGONADOTROPIC HYPOGONADISM Case
Mutation (cDNA)
Mutation (protein)
Patient’s status
Familial/sporadic
Affected members
References
1
IVS4‐13‐142del155
Truncated protein
Homozygous
Familial
4M/1F*
8
2
c.443T > C
L148S
Homozygous
Familial
4M
7
3
c.991C > T/c.1195T > A
R331X/X399R
Compound heterozygous
Sporadic
M
7
4
c.667T > C/c.891G > T
C223R/R297L
Compound heterozygous
Sporadic
M
11
5
c.1101_1002insC
Frameshift
Homozygous
Sporadic
M
6
6
c.305T > C
L102P
Homozygous
Familial
1M
9
7
c.305T > C
L102P
Homozygous
Familial
2M
9
8
IVS2‐4‐2delGC insACCGGCT
Truncated protein
Homozygous
Familial
2M
10
*M ¼ male; F ¼ female.
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The first frameshift mutation in GPR54 was described by Lanfranco et al.6 It was a homozygous insertion of a cytosine after nucleotide position 1001 (1001_1002insC). This insertion resulted in a frameshift in the open reading frame with elongation of the GPR54 protein from 398 to 441 amino acids.6 His parents were cousins of second degree and had German descendency. There was no report of family history of hypogonadism or infertility. He suffered from delayed puberty, bilateral undescended testes, and presented with mild hypospadia. He had low levels of LH and FSH together with low testosterone levels. His gonadotropin response to GnRH acute stimulation test was pubertal. After a 2‐year GnRH pulsatile treatment he fathered a healthy male child.6 More recently, a novel GPR54 missense mutation was described by Tenenbaum‐Rakover et al.9 A substitution in homozygous state of a cytosine for thymidine 305 resulted in the change of a proline by a leucine at position 102 (L102P) of the first extracellular loop. This mutation was described in two families with three and four affected members with normosmic IHH, respectively.9 Affected patients were all born from consanguineous parents. In vitro studies of the L102P mutant showed that despite displaying normal affinity for kisspeptin, maximal binding to this ligand was shown to be approximately 50% decreased when compared to the wild‐type GPR54, suggesting that membrane levels of the L102P GPR54 mutant are reduced by half. In addition, kisspeptin‐ stimulated signaling (as measured by inositol phosphate accumulation) of the L148S GPR54 mutant was absent.9 Figure 1 shows the localization of the inactivating mutations reported in the coding region of the GPR54 receptor. Recently, we described one novel homozygous GPR54 gene mutation in two Brazilian siblings with normosmic IHH. These two siblings carrying the new mutation in GPR54 gene were born to apparently nonconsanguineuos parents. They presented with micropenis and had no secondary sexual characteristics at the age of 14 and 18 years. Both had low testosterone levels (16.5 and 24 ng/dL) and prepubertal levels of basal gonadotropins.10 This new variant was an insertion/deletion (indel) mutation characterized by the deletion of three nucleotides (GCA) at position 2 to 4, and insertion of seven nucleotides (ACCGGCT) in the 30 splice acceptor site of intron 2 of GPR54 gene.10 The nucleotide changes were absent in a control population of 120 ethnically matched controls. Their mother carried the same mutation in the heterozygous state and reported normal pubertal development. Their father is believed to have had normal reproductive maturation and function, but was not available for testing. This complex indel mutation removed the 30 splice acceptor site of intron 2. Computational splice site prediction confirms that the substitution of the GCA nucleotides by ACCGGCT in the GPR54 mutated patients could result in aberrant intron retention or exon 3 skipping due to disruption of the constitutive site and the use of cryptic splice acceptor sites. Both events would result in truncated receptors.10 Ongoing in vitro studies will confirm this prediction.
GPR54 MUTATIONS AND HUMAN DISEASES
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GPR54 mutations are an infrequent cause of IHH, accounting for less than 5% of all reported normosmic IHH cases. This prevalence is significantly higher in familial cases. GPR54 alterations were identified in 3 out of 180 sporadic IHH patients (1.6%) and 5 of 24 familial cases (20.8%). Patients with inactivating GPR54 mutations present with delayed puberty and apparently no associated condition. Cryptorchidism and micropenis are described in some cases, suggesting that the kisspetin–GPR54 system plays an important role in the testosterone production during late fetal and early neonatal sexual development.6–11 The acute response to GnRH acute stimulation test was variable among patients with GPR54 mutations and ranged from totally blunted to normal.6,7,9 A 9‐year follow‐up of the affected patient with the L102P mutation revealed progressive changes in pituitary response.9 The LH and FSH levels after GnRH stimulus progressively increased across the follow‐ up from prepubertal to almost pubertal levels around 21 years of age. This finding suggests that the L102P mutation leads to a more quantitative than qualitative defect of gonadotropic axis activation.9 Notably, treatment with chronic pulsatile GnRH therapy leaded patients to increase sperm maturation and reach fertility.6,7,9 In addition, one female patient with homozygous mutations in GPR54 had multiple conceptions, uncomplicated pregnancies and deliveries of healthy children, and lactation for several months postpartum.80
XI. The Role of GPR54 in Central Precocious Puberty Precocious puberty is defined as the development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys, and can be divided in two major groups according to the etiology: gonadotropin‐ dependent precocious puberty, also known as central or true precocious puberty and gonadotropin‐independent precocious puberty, also called peripheral precocious puberty.81 Central precocious puberty (CPP) results from premature activation of hypothalamic GnRH secreting neurons and leads to the development of secondary sexual characteristics, acceleration in linear growth, and progressive bone age advancement.81,82 CPP has noteworthy female gender predominance and over 90% of cases are considered idiopathic.83,84 In contrast, up to 75% of boys with central precocious puberty have an underlying central nervous system abnormality, such as hypothalamic hamartomas, especially in children younger than 4 years old.83,85,86 Although some gene defects were related to peripheral precocious puberty,87–89 little is known about the molecular basis of CPP without hypothalamic lesions. Only recently, mutations in the KiSS1/GPR54 complex, lately known as the gatekeepers of puberty, have been implicated in the pathogenesis of CPP.12 As inactivating GPR54 mutations were described in patients with
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normosmic IHH a question was raised: is the opposite also true? Recently, the first activating GPR54 mutation was identified in a patient with CPP after 53 children (50 girls and 3 boys) had been screened for GPR54 mutations.12 She had slow progressive thelarche from birth, suggesting an early, persistent, and slightly increased estrogen secretion. Isolated thelarche was ruled out on the basis of progressive secondary sexual development and accelerated growth and skeletal maturation. At 7 years of age, she presented with breast development acceleration and pubic hair was noted. In vitro studies demonstrated that the R386P mutation, located in the carboxy‐terminal tail of the GPR54 receptor, determined a prolonged activation of intracellular signaling pathway in response to kisspeptin, resulting in significantly higher accumulation of two second messengers of GPR54 signaling cascade: inositol phosphate (Fig. 4A) and phosphorylated extracellular‐ A
(% of maximum)
100 80
p < 0.05
60
Arg386Pro GPR
40
Wild-type GPR5
20 0 3
6
9
12 15 18
h
C
Wild-type GPR54 pERK
Total ERK 0
5
10
15
30
60
Arg386pro GPR54 pERK Total ERK
pERK: total ERK (% of maximum)
0
B
100 Arg386Pro GPR54
80 60
p < 0.001 Wild-type GPR54
40 20 0 0
20
40
60
min 0
5
10
15
30
60
min
FIG. 4. (A) Inositol phosphate (IP) levels after stimulation with kisspeptin‐10. The IP accumulation was significantly higher in cells transfected with R386P GPR54 mutant at 18 h. (B, C) Phosphorylated ERK (pERK) levels after stimulation with kisspeptin are significantly higher in cells transfected with the R386P GPR54 mutant at 60 min. Both experiments show a decrease in desensitization of the mutant receptor characterizing a nonconstitutively activating mutation. Adapted from Ref. 12.
GPR54 MUTATIONS AND HUMAN DISEASES
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regulated kinase (Fig. 4B and C). These findings indicate a significant reduction in the rate of desensitization of the mutant GPR54. This mechanism would result in an increased, prolonged cellular response, and hence the release of an increased‐amplitude pulse of GnRH in response to kisspeptin stimulation. In vitro affinity of mutant GPR54 for its ligand and intracellular signaling capacity under basal conditions were not altered, indicating that the R386P is a nonconstitutively activating GPR54 mutation. In addition, this finding revealed a unique mechanism that requires the presence of ligand for activation.12 The prolonged responsiveness to ligand stimulation and the location of this substitution indicate that desensitization of R386P GPR54 is altered, which is in agreement with the fact that the carboxyl‐terminal tail of G protein‐ coupled receptors is the main target of intracellular proteins that regulate receptor activity and half‐life. In fact, a constitutive activation of GPR54 might be expected to disrupt pulsatile GnRH release and cause delayed puberty, since continuous GnRH secretion lead to receptor desensitization. Indeed, continuous infusion of kisspeptin has been shown to decrease LH levels in agonadal juvenile male monkeys.56
XII. Conclusion GPR54 and its endogenous ligand, kisspeptin, are key regulators of the reproductive cascade. The discovery of loss‐of‐function and gain‐of‐function mutations in GPR54 in patients with idiopathic hypogonadotropic hypogonadism and central precocious puberty, respectively, has uncovered the fundamental role of the GPR54 pathway in the physiologic regulation of puberty and reproduction. Expression and pharmacologic studies establish the role of the GPR54 pathway in the stimulation of GnRH neurons during puberty. In the future, modulators of GPR54 activity and the super agonists of kisspeptin may prove valuable in clinical application in the fields of reproductive medicine in both animals and humans.
References 1. Cabrera‐Vera TM, Vanhauwe J, Thomas TO, Medkova M, Preininger A, Mazzoni MR, et al. Insights into G protein structure, function, and regulation. Endocr Rev 2003;24:765–81. 2. Gether U. Uncovering molecular mechanisms involved in activation of G protein‐coupled receptors. Endocr Rev 2000;21:90–113. 3. Lania AG, Mantovani G, Spada A. Mechanisms of disease: mutations of G proteins and G‐protein‐coupled receptors in endocrine diseases. Nat Clin Pract Endocrinol Metab 2006;2:681–93.
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4. Feng YH, Miura S, Husain A, Karnik SS. Mechanism of constitutive activation of the AT1 receptor: influence of the size of the agonist switch binding residue Asn(111). Biochemistry 1998;37:15791–8. 5. Kuohung W, Kaiser UB. GPR54 and KiSS‐1: role in the regulation of puberty and reproduction. Rev Endocr Metab Disord 2006;7:257–63. 6. Lanfranco F, Gromoll J, von Eckardstein S, Herding EM, Nieschlag E, Simoni M. Role of sequence variations of the GnRH receptor and G protein‐coupled receptor 54 gene in male idiopathic hypogonadotropic hypogonadism. Eur J Endocrinol 2005;153:845–52. 7. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno Jr JS, Shagoury JK, et al. The GPR54 gene as a regulator of puberty. N Engl J Med 2003;349:1614–27. 8. de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E. Hypogonadotropic hypogonadism due to loss of function of the KiSS1‐derived peptide receptor GPR54. Proc Natl Acad Sci USA 2003;100:10972–6. 9. Tenenbaum‐Rakover Y, Commenges‐Ducos M, Iovane A, Aumas C, Admoni O, de Roux N. Neuroendocrine phenotype analysis in five patients with isolated hypogonadotropic hypogonadism due to a L102P inactivating mutation of GPR54. J Clin Endocrinol Metab 2007;92:1137–44. 10. Teles MG, Trarbach E, Guerra G, Costa EMF, Baptista MTM, Castro M, et al. In: 89th Endocrine Society Annual Meeting, Toronto. 2007. 11. Semple RK, Achermann JC, Ellery J, Farooqi IS, Karet FE, Stanhope RG, et al. Two novel missense mutations in G protein‐coupled receptor 54 in a patient with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 2005;90:1849–55. 12. Teles MG, Bianco SD, Brito VN, Trarbach EB, Kuohung W, Xu S, et al. A GPR54‐activating mutation in a patient with central precocious puberty. N Engl J Med 2008;358:709–15. 13. Seminara SB, Crowley Jr WF. Kisspeptin and GPR54: discovery of a novel pathway in reproduction. J Neuroendocrinol 2008;20:727–31. 14. Lee DK, Nguyen T, O’Neill GP, Cheng R, Liu Y, Howard AD, et al. Discovery of a receptor related to the galanin receptors. FEBS Lett 1999;446:103–7. 15. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, et al. The metastasis suppressor gene KiSS‐1 encodes kisspeptins, the natural ligands of the orphan G protein‐coupled receptor GPR54. J Biol Chem 2001;276:34631–6. 16. Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, et al. AXOR12, a novel human G protein‐coupled receptor, activated by the peptide KiSS‐1. J Biol Chem 2001;276:28969–75. 17. Roa J, Tena‐Sempere M. KiSS‐1 system and reproduction: comparative aspects and roles in the control of female gonadotropic axis in mammals. Gen Comp Endocrinol 2007;153:132–40. 18. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, et al. Metastasis suppressor gene KiSS‐1 encodes peptide ligand of a G‐protein‐coupled receptor. Nature 2001;411:613–7. 19. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, et al. KiSS‐1, a novel human malignant melanoma metastasis‐suppressor gene. J Natl Cancer Inst 1996;88:1731–7. 20. West A, Vojta PJ, Welch DR, Weissman BE. Chromosome localization and genomic structure of the KiSS‐1 metastasis suppressor gene (KISS1). Genomics 1998;54:145–8. 21. Bilban M, Ghaffari‐Tabrizi N, Hintermann E, Bauer S, Molzer S, Zoratti C, et al. Kisspeptin‐10, a KiSS‐1/metastin‐derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 2004;117:1319–28. 22. Lee JH, Welch DR. Suppression of metastasis in human breast carcinoma MDA‐MB‐435 cells after transfection with the metastasis suppressor gene, KiSS‐1. Cancer Res 1997;57:2384–7. 23. Roa J, Aguilar E, Dieguez C, Pinilla L, Tena‐Sempere M. New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function. Front Neuroendocrinol 2008;29:48–69.
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24. Harms JF, Welch DR, Miele ME. KISS1 metastasis suppression and emergent pathways. Clin Exp Metastasis 2003;20:11–8. 25. Lee YR, Tsunekawa K, Moon MJ, Um HN, Hwang JI, Osugi T, et al. Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates. Endocrinology 2009;150:2837–46. 26. Stafford LJ, Xia C, Ma W, Cai Y, Liu M. Identification and characterization of mouse metastasis‐ suppressor KiSS1 and its G‐protein‐coupled receptor. Cancer Res 2002;62:5399–404. 27. Parhar IS, Ogawa S, Sakuma Y. Laser‐captured single digoxigenin‐labeled neurons of gonadotropin‐releasing hormone types reveal a novel G protein‐coupled receptor (Gpr54) during maturation in cichlid fish. Endocrinology 2004;145:3613–8. 28. Mohamed JS, Benninghoff AD, Holt GJ, Khan IA. Developmental expression of the G protein‐coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia. J Mol Endocrinol 2007;38:235–44. 29. Nocillado JN, Levavi‐Sivan B, Carrick F, Elizur A. Temporal expression of G‐protein‐coupled receptor 54 (GPR54), gonadotropin‐releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus. Gen Comp Endocrinol 2007;150:278–87. 30. Biran J, Ben‐Dor S, Levavi‐Sivan B. Molecular identification and functional characterization of the kisspeptin/kisspeptin receptor system in lower vertebrates. Biol Reprod 2008;79:776–86. 31. Kanda S, Akazome Y, Matsunaga T, Yamamoto N, Yamada S, Tsukamura H, et al. Identification of KiSS‐1 product kisspeptin and steroid‐sensitive sexually dimorphic kisspeptin neurons in medaka (oryzias latipes). Endocrinology 2008;149:2467–76. 32. Martinez‐Chavez CC, Minghetti M, Migaud H. GPR54 and rGnRH I gene expression during the onset of puberty in Nile tilapia. Gen Comp Endocrinol 2008;156:224–33. 33. van Aerle R, Kille P, Lange A, Tyler CR. Evidence for the existence of a functional Kiss1/Kiss1 receptor pathway in fish. Peptides 2008;29:57–64. 34. Kitahashi T, Takahashi M, Yamada Y, Oghiso Y, Yokohira M, Imaida K, et al. Occurrence of mutations in the epidermal growth factor receptor gene in X‐ray‐induced rat lung tumors. Cancer Sci 2008;99:241–5. 35. Kitahashi T, Ogawa S, Parhar IS. Cloning and expression of kiss2 in the zebrafish and medaka. Endocrinology 2009;150:821–31. 36. Castano JP, Martinez‐Fuentes AJ, Gutierrez‐Pascual E, Vaudry H, Tena‐Sempere M, Malagon MM. Intracellular signaling pathways activated by kisspeptins through GPR54: do multiple signals underlie function diversity? Peptides 2009;30:10–5. 37. Castellano JM, Navarro VM, Fernandez‐Fernandez R, Castano JP, Malagon MM, Aguilar E, et al. Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin‐releasing hormone system of the rat. Mol Cell Endocrinol 2006;257–258:75–83. 38. Castano JP, Martinez‐Fuentes AJ, Gutierrez‐Pascual E, Vaudry H, Tena‐Sempere M, Malagon MM. Intracellular signaling pathways activated by kisspeptins through GPR54: do multiple signals underlie function diversity? Peptides 2008;30:10–5. 39. Yan C, Wang H, Boyd DD. KiSS‐1 represses 92‐kDa type IV collagenase expression by down‐ regulating NF‐kappa B binding to the promoter as a consequence of Ikappa Balpha‐induced block of p65/p50 nuclear translocation. J Biol Chem 2001;276:1164–72. 40. Nash KT, Welch DR. The KISS1 metastasis suppressor: mechanistic insights and clinical utility. Front Biosci 2006;11:647–59. 41. Hori A, Honda S, Asada M, Ohtaki T, Oda K, Watanabe T, et al. Metastin suppresses the motility and growth of CHO cells transfected with its receptor. Biochem Biophys Res Commun 2001;286:958–63. 42. Becker JA, Mirjolet JF, Bernard J, Burgeon E, Simons MJ, Vassart G, et al. Activation of GPR54 promotes cell cycle arrest and apoptosis of human tumor cells through a specific transcriptional program not shared by other Gq‐coupled receptors. Biochem Biophys Res Commun 2005;326:677–86.
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43. Nash KT, Phadke PA, Navenot JM, Hurst DR, Accavitti‐Loper MA, Sztul E, et al. Requirement of KISS1 secretion for multiple organ metastasis suppression and maintenance of tumor dormancy. J Natl Cancer Inst 2007;99:309–21. 44. Tena‐Sempere M. The roles of kisspeptins and G protein‐coupled receptor‐54 in pubertal development. Curr Opin Pediatr 2006;18:442–7. 45. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, et al. A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 2004;145:4073–7. 46. Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T. Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 2004;320:383–8. 47. Navarro VM, Castellano JM, Fernandez‐Fernandez R, Barreiro ML, Roa J, Sanchez‐ Criado JE, et al. Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS‐1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone‐releasing activity of KiSS‐1 peptide. Endocrinology 2004;145:4565–74. 48. Thompson EL, Patterson M, Murphy KG, Smith KL, Dhillo WS, Todd JF, et al. Central and peripheral administration of kisspeptin‐10 stimulates the hypothalamic‐pituitary‐gonadal axis. J Neuroendocrinol 2004;16:850–8. 49. Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, et al. Kisspeptin directly stimulates gonadotropin‐releasing hormone release via G protein‐coupled receptor 54. Proc Natl Acad Sci USA 2005;102:1761–6. 50. Smith JT, Clay CM, Caraty A, Clarke IJ. KiSS‐1 messenger ribonucleic acid expression in the hypothalamus of the ewe is regulated by sex steroids and season. Endocrinology 2007;148:1150–7. 51. Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM. Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 2005;102:2129–34. 52. Plant TM. The role of KISS‐1 in the regulation of puberty in higher primates. Eur J Endocrinol 2006;155(Suppl. 1):S11–6. 53. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, et al. Kisspeptin‐54 stimulates the hypothalamic‐pituitary gonadal axis in human males. J Clin Endocrinol Metab 2005;90:6609–15. 54. Navarro VM, Fernandez‐Fernandez R, Castellano JM, Roa J, Mayen A, Barreiro ML, et al. Advanced vaginal opening and precocious activation of the reproductive axis by KiSS‐1 peptide, the endogenous ligand of GPR54. J Physiol 2004;561:379–86. 55. Plant TM, Ramaswamy S, Dipietro MJ. Repetitive activation of hypothalamic G protein‐ coupled receptor 54 with intravenous pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits a sustained train of gonadotropin‐releasing hormone discharges. Endocrinology 2006;147:1007–13. 56. Seminara SB, Dipietro MJ, Ramaswamy S, Crowley Jr WF, Plant TM. Continuous human metastin 45–54 infusion desensitizes G protein‐coupled receptor 54‐induced gonadotropin‐ releasing hormone release monitored indirectly in the juvenile male Rhesus monkey (Macaca mulatta): a finding with therapeutic implications. Endocrinology 2006;147:2122–6. 57. Cerrato F, Seminara SB. Human genetics of GPR54. Rev Endocr Metab Disord 2007;8:47–55. 58. Clement K, Vaisse C, Lahlou N, Cabrol S, Pelloux V, Cassuto D, et al. A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 1998;392:398–401. 59. Fernandez‐Fernandez R, Martini AC, Navarro VM, Castellano JM, Dieguez C, Aguilar E, et al. Novel signals for the integration of energy balance and reproduction. Mol Cell Endocrinol 2006;254–255:127–32.
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60. Smith JT, Clarke IJ. Kisspeptin expression in the brain: catalyst for the initiation of puberty. Rev Endocr Metab Disord 2007;8:1–9. 61. Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA. Regulation of Kiss1 gene expression in the brain of the female mouse. Endocrinology 2005;146:3686–92. 62. Clarkson J, Herbison AE. Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin‐releasing hormone neurons. Endocrinology 2006;147:5817–25. 63. Gottsch ML, Clifton DK, Steiner RA. Kisspepeptin‐GPR54 signaling in the neuroendocrine reproductive axis. Mol Cell Endocrinol 2006;254–255:91–6. 64. Richard N, Galmiche G, Corvaisier S, Caraty A, Kottler ML. KiSS‐1 and GPR54 genes are co‐expressed in rat gonadotrophs and differentially regulated in vivo by oestradiol and gonadotrophin‐releasing hormone. J Neuroendocrinol 2008;20:381–93. 65. Richard N, Corvaisier S, Camacho E, Kottler ML. KiSS‐1 and GPR54 at the pituitary level: overview and recent insights. Peptides 2008;30:123–9. 66. Gutierrez‐Pascual E, Martinez‐Fuentes AJ, Pinilla L, Tena‐Sempere M, Malagon MM, Castano JP. Direct pituitary effects of kisspeptin: activation of gonadotrophs and somatotrophs and stimulation of luteinising hormone and growth hormone secretion. J Neuroendocrinol 2007;19:521–30. 67. Funes S, Hedrick JA, Vassileva G, Markowitz L, Abbondanzo S, Golovko A, et al. The KiSS‐1 receptor GPR54 is essential for the development of the murine reproductive system. Biochem Biophys Res Commun 2003;312:1357–63. 68. d’Anglemont de Tassigny X, Fagg LA, Dixon JP, Day K, Leitch HG, Hendrick AG, et al. Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proc Natl Acad Sci USA 2007;104:10714–9. 69. Lapatto R, Pallais JC, Zhang D, Chan YM, Mahan A, Cerrato F, et al. Kiss1/ mice exhibit more variable hypogonadism than Gpr54/ mice. Endocrinology 2007;148:4927–36. 70. Colledge WH. Transgenic mouse models to study Gpr54/kisspeptin physiology. Peptides 2008;30:34–41. 71. Seminara SB, Hayes FJ, Crowley Jr WF. Gonadotropin‐releasing hormone deficiency in the human (idiopathic hypogonadotropic hypogonadism and Kallmann’s syndrome): pathophysiological and genetic considerations. Endocr Rev 1998;19:521–39. 72. Seminara SB, Oliveira LM, Beranova M, Hayes FJ, Crowley Jr WF. Genetics of hypogonadotropic hypogonadism. J Endocrinol Invest 2000;23:560–5. 73. Quinton R, Duke VM, Robertson A, Kirk JM, Matfin G, de Zoysa PA, et al. Idiopathic gonadotrophin deficiency: genetic questions addressed through phenotypic characterization. Clin Endocrinol (Oxf) 2001;55:163–74. 74. Beranova M, Oliveira LM, Bedecarrats GY, Schipani E, Vallejo M, Ammini AC, et al. Prevalence, phenotypic spectrum, and modes of inheritance of gonadotropin‐releasing hormone receptor mutations in idiopathic hypogonadotropic hypogonadism. J Clin Endocrinol Metab 2001;86:1580–8. 75. Topaloglu AK, Lu ZL, Farooqi IS, Mungan NO, Yuksel B, O’Rahilly S, et al. Molecular genetic analysis of normosmic hypogonadotropic hypogonadism in a Turkish population: identification and detailed functional characterization of a novel mutation in the gonadotropin‐ releasing hormone receptor gene. Neuroendocrinology 2006;84:301–8. 76. Pitteloud N, Acierno Jr JS, Meysing A, Eliseenkova AV, Ma J, Ibrahimi OA, et al. Mutations in fibroblast growth factor receptor 1 cause both Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism. Proc Natl Acad Sci USA 2006;103:6281–6. 77. Topaloglu AK, Reimann F, Guclu M, Yalin AS, Kotan LD, Porter KM, et al. TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction. Nat Genet 2009;41:354–8.
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78. Alewijnse AE, Timmerman H, Jacobs EH, Smit MJ, Roovers E, Cotecchia S, et al. The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H(2) receptor. Mol Pharmacol 2000;57:890–8. 79. Wacker JL, Feller DB, Tang XB, Defino MC, Namkung Y, Lyssand JS, et al. Disease‐causing mutation in GPR54 reveals the importance of the second intaracellular loop for class A G‐protein‐coupled receptor function. J Biol Chem 2008;283:31068–78. 80. Pallais JC, Bo‐Abbas Y, Pitteloud N, Crowley Jr WF, Seminara SB. Neuroendocrine, gonadal, placental, and obstetric phenotypes in patients with IHH and mutations in the G‐protein coupled receptor, GPR54. Mol Cell Endocrinol 2006;254–255:70–7. 81. Grumbach MM. The neuroendocrinology of human puberty revisited. Horm Res 2002;57 (Suppl. 2):2–14. 82. Brito VN, Latronico AC, Arnhold IJ, Mendonca BB. Update on the etiology, diagnosis and therapeutic management of sexual precocity. Arq Bras Endocrinol Metabol 2008;52:18–31. 83. Kakarla N, Bradshaw KD. Disorders of pubertal development: precocious puberty. Semin Reprod Med 2003;21:339–51. 84. Palmert MR, Boepple PA. Variation in the timing of puberty: clinical spectrum and genetic investigation. J Clin Endocrinol Metab 2001;86:2364–8. 85. Pescovitz OH, Comite F, Hench K, Barnes K, McNemar A, Foster C, et al. The NIH experience with precocious puberty: diagnostic subgroups and response to short‐term luteinizing hormone releasing hormone analogue therapy. J Pediatr 1986;108:47–54. 86. Partsch CJ, Sippell WG. Pathogenesis and epidemiology of precocious puberty. Effects of exogenous oestrogens. Hum Reprod Update 2001;7:292–302. 87. Weinstein LS, Shenker A, Gejman PV, Merino MJ, Friedman E, Spiegel AM. Activating mutations of the stimulatory G protein in the McCune‐Albright syndrome. N Engl J Med 1991;325:1688–95. 88. Shenker A, Weinstein LS, Moran A, Pescovitz OH, Charest NJ, Boney CM, et al. Severe endocrine and nonendocrine manifestations of the McCune‐Albright syndrome associated with activating mutations of stimulatory G protein GS. J Pediatr 1993;123:509–18. 89. Shenker A, Laue L, Kosugi S, Merendino Jr JJ, Minegishi T, Cutler Jr GB. A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 1993;365:652–4.
Diseases Associated with Growth Hormone‐Releasing Hormone Receptor (GHRHR) Mutations Marco Martari and Roberto Salvatori School of Medicine, Division of Endocrinology and Metabolism, Johns Hopkins University, Baltimore, Maryland 21287
I. Introduction .................................................................................. II. GHRHR Biology............................................................................. A. GHRHR and Its Ligand............................................................... B. Signal Transduction..................................................................... C. GHRHR Structure ..................................................................... D. GHRHR Organization and Regulation............................................. E. GHRHR Splice Variants............................................................... III. GHRHR Mutations and Polymorphisms................................................ A. GHRHR Mutations..................................................................... B. GHRHR Polymorphisms .............................................................. IV. Diseases Associated with GHRHR Mutations ......................................... A. Clinical Characteristics of Homozygous Individuals ............................ B. Clinical Characteristics of Heterozygous Carriers ............................... V. Conclusions ................................................................................... References ....................................................................................
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The growth hormone (GH)‐releasing hormone (GHRH) receptor (GHRHR) belongs to the G protein‐coupled receptors family. It is expressed almost exclusively in the anterior pituitary, where it is necessary for somatotroph cells proliferation and for GH synthesis and secretion. Mutations in the human GHRHR gene (GHRHR) can impair ligand binding and signal transduction, and have been estimated to cause about 10% of autosomal recessive familial isolated growth hormone deficiency (IGHD). Mutations reported to date include five splice donor site mutations, two microdeletions, two nonsense mutations, seven missense mutations, and one mutation in the promoter. These mutations have an autosomal recessive mode of inheritance, and heterozygous individuals do not show signs of IGHD, although the presence of an intermediate phenotype has been hypothesized. Conversely, patients with biallelic mutations have low serum insulin‐like growth factor‐1 and GH levels (with absent or reduced GH response to exogenous stimuli),
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resulting—if not treated—in proportionate dwarfism. This chapter reviews the biology of the GHRHR, the mutations that affect its gene and their effects in homozygous and heterozygous individuals.
I. Introduction Growth hormone (GH) is a single chain 191‐amino acid peptide hormone that promotes longitudinal growth and is involved in several metabolic pathways. GH increases bone mineralization, lipolysis, and protein synthesis. It controls glucose metabolism in the liver, and influences blood pressure and cardiac muscle mass and functions. GH also plays a role in the immune and reproductive systems.1 It acts both directly and indirectly through the actions of insulin‐like growth factor‐1 (IGF‐1, formerly also known as Somatomedin‐ C), which is produced both locally and in the liver, from where it is released in the general circulation.2 GH is produced, stored, and secreted by the somatotroph cells of the anterior pituitary under the regulatory control of two hypothalamic factors: one stimulatory, GH‐releasing hormone (GHRH), and one inhibitory, somatostatin (SRIF)3,4 (Fig. 1). GHRH and SRIF act on different G protein‐coupled receptors (GPCRs) expressed on the somatotroph cell membrane. They induce an increase (GHRH) or decrease (SRIF) in intracellular cyclic adenosine monophosphate (cAMP),5 with resulting activation or inhibition of the intracellular cascade that leads to GH release.6 Isolated growth hormone deficiency (IGHD) causes somatic growth failure to different extents, from mild to severe, according to its degree.7 GH is not needed during fetal development, and therefore affected children have normal body size at birth, but growth retardation is usually evident after the first year of life. The occurrence of IGHD is estimated to be between 1/4000 and 1/10,000 live births.8–10 Structural defects and acquired damages in the hypothalamic/ pituitary region have for a long time been considered the principal causes of IGHD. Nevertheless, only 12% of IGHD patients show clear anatomical abnormalities in that region by magnetic resonance imaging (MRI),11 while about 5–30% of IGHD patients exhibit some sort of familial occurrence.12 These findings suggest that genetic and functional factors should also be considered as these contribute a major percentage in causing IGHD. On the other hand, it must be noted that by using age‐adjusted pituitary measurements, MRI studies have recently shown that a certain degree of anterior pituitary hypoplasia (APH) is present in a significant number of IGHD individuals.13,14 However, when APH is associated with pituitary stalk agenesis (PSA) and ectopia of the posterior pituitary (PPE), IGHD children are more likely to develop additional pituitary hormone deficiency.15,16
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Hypothalamus (−) GHRH
(+) SRIF (−)
(+) (−)
Pituitary
GH
Indirect effects
Direct effects
Liver
Muscle/bone/heart adipose tissue (+)
IGF-1
GH receptor IGF-1 receptor GHRH receptor FIG. 1. Outline of the hypothalamus/pituitary axis, showing direct and indirect (IGF‐1‐mediated) effects of GH on peripheral tissues.
Familial IGHD has been classified into four different categories, according to pattern of inheritance, clinical traits, and effects of exogenous GH replacement therapy: Type IA: Autosomal recessive. GH levels are often undetectable, and GH
therapy generally induces the development of anti‐GH antibodies. Type IB: Autosomal recessive. It is the most common form of familial
IGHD; GH levels are low but often detectable and usually GH therapy does not induce anti‐GH antibodies. Type II: Autosomal dominant. GH levels are low but detectable and anti‐GH antibodies do not develop with GH therapy. Type III: X‐linked. It is a very rare form that presents different clinical findings among different families.
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Mutations in the human GH gene (GH1) can cause types IA, IB, and II phenotypes.17 While the prevalence of GH1 mutations is very high in type IA (66.7%), it is only 1.7% in IB patients.18 Because of the important role of GHRH in GH secretion, IGHD could be due to genetic alterations that affect the production, secretion, or action of GHRH. While it has been shown that abnormalities in the GHRH gene do not cause IGHD,19 numerous mutations in the human GHRH receptor (GHRHR) gene (GHRHR) have been reported in the last decade, with a prevalence of about 10% in IGHD IB.20 The purpose of this article is to review structure and functions of the GHRHR, summarize known GHRHR mutations, and describe the clinical features of homozygous affected individuals and heterozygous carriers.
II. GHRHR Biology A. GHRHR and Its Ligand GHRH is synthesized in the hypothalamus as a 107/108 amino acid precursor (prepro‐GHRH) (the 108 isoform contain an extra serine in the carboxy‐terminal region), which is later processed to give two peptides [GHRH(1–44)‐NH2 and GHRH(1–40)‐OH] with similar functions and potency in stimulating GH release.21 Only the first 29 residues are required to exhibit full biological activity.22 GHRH has a plasma half‐life of 7–50 min23–25 and it is degraded to inactive forms by dipeptidyl‐peptidase type IV and trypsin‐like endopeptidases.24,26 Internalization into target cells is another mechanism of GHRH inactivation that has been demonstrated in vitro.21,27 GHRH stimulates somatotroph cell growth28 and GH release.29 These actions are mediated by high affinity, low‐capacity receptors.22,30,31 GHRHR is widely expressed in the anterior pituitary gland and partial occupancy causes maximal GH response (only 10–20% of the receptors need to be occupied for maximal GH release).32 GHRHR mRNA has also been identified in the renal medulla33 and in other tissues, such as testes, placenta, and gastrointestinal tract, where it may play a role in paracrine and/or autocrine control.34,35 GHRHR has also been detected in the hypothalamus,36 where GHRH probably functions as a neurotransmitter.34 Splice variants (SVs) of GHRHR have been found in several normal and neoplastic tissues.37 GHRHR expression changes during fetal development and throughout the life. In rat pituitary, GHRHR first appears on embryonic day 19 (E19)38–40 and reaches a peak during E20 with no subsequent increase.41 Receptor expression declines after birth and surges again during puberty. A final decrease in GHRHR expression is seen with aging.42,43
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B. Signal Transduction The interaction between the receptor and its ligand induces activation of adenylate cyclase, which increases cAMP concentration, resulting in an increase in Ca2þ ions, and consequential GH release into the systemic circulation. The increase in cAMP results in activation of the protein kinase A pathway, determining cell proliferation, and GH and GHRHR synthesis.5,44 Figure 2 provides an outline of the actions that follow the interaction between GHRHR and its ligand. Although the adenylate cyclase–protein kinase A pathway is the principal transduction mechanism that mediates GHRH actions on somatotropic cells, other mechanisms, such as the inositol phosphate–diacylglycerol–protein kinase C system,45,46 the Ca2þ–calmodulin system,47,48 and the arachidonic acid–eicosanoid system49,50 are also believed to play a minor role. Uninterrupted or recurring stimulation of GHRHR in the pituitary leads to attenuation of GH release.51 Receptor desensitization is the main mechanism responsible for GH response attenuation and it involves the uncoupling of the
GHRH GHRHR Stimulation of GH1 and GHRHR transcription
GSa
Adenylate cyclase Protein kinase A cAMP
Ca2+
ATP
GH release
Ca2+ FIG. 2. Schematic representation of the cascade of events caused by the interaction between GHRHR and its ligand. Adenylate cyclase activation leads to increase in cAMP concentration, provoking influx of extracellular Ca2þ and release of GH. Activation of the protein kinase A pathway leads to GH and GHRHR transcription.
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G protein that activates the adenylate cyclase.52 This mechanism is common in this class of receptors and it involves a two‐step process that starts with receptor phosphorylation by a G protein‐coupled receptor kinase (GRK). The phosphorylation marks the receptor for beta arrestin binding, which leads to receptor silencing.53 Accordingly, in vitro studies showed that human GHRHR lacking intracellular phosphorylation sites (similarly to the ovine GHRHR) is more sensitive to GHRH stimulation if compared to the full‐length receptor.54 However, desensitized somatotroph cells can still release GH in response to other secretagogues [stress, arginine, GH‐releasing peptide (GHRP), TRH, and hypoglycemia]21 by using signal‐specific GH pools.55,56 GHRH desensitization can be prevented by SRIF, which restores GH response in GHRH‐desensitized cells.57 In addition, desensitization is not complete, as demonstrated by the fact that patients with GHRH‐secreting tumors present persistently elevated serum GH levels.58,59
C. GHRHR Structure The GHRHR belongs to family B group III (B‐III) of the GPCR superfamily.34 Other receptors in this family include receptors for vasoactive intestinal peptide (VIP), secretin, gastric inhibitory peptide, glucagon, glucagon‐like peptide‐1 (GLP‐1), and pituitary adenylate cyclase‐activating peptide.60,61 Structurally, the GHRHR is a 423‐amino acid transmembrane (TM) receptor coupled to a Ga subunit protein and it is made of an N‐terminal extracellular domain, seven hydrophobic a‐helices (which are interconnected by small loops and form the TM portion of the receptor), and a C‐terminal intracellular domain.34,60 The N‐terminal domain displays a glycosylation site at position 50, while a cleavage site is present at position 22 for signal peptide removal during protein postsynthetic modifications. The C‐terminal domain exhibits numerous phosphorylation sites likely meant to interact with GRK/beta arrestin. Several highly conserved sequences have been identified in all the mammalian GHRHRs (human, mouse, rat, bovine, and porcine) cloned to date54,62–68 and in the teleost fish.69 Figure 3 compares the primary structures of the GHRHR from different species and highlights the consensus areas. The N‐terminal portion of the receptor alone does not exhibit high affinity for GHRH but low‐affinity interactions can be detected.34 Mutant receptors lacking the extracellular domain or most of the TM domains appear to be unable to bind GHRH.70 Studies using a chimeric receptor, where the extracellular domains of VIP receptor was linked to the rest of the GHRHR, showed that the chimera was still able to bind GHRH. This demonstrates that the N‐terminus, though important in GHRH binding, does not determine ligand specificity.70 Ligand specificity is instead provided by the TM portion of the
DISEASES ASSOCIATED WITH
GHRHR MUTATIONS
Human Mouse Rat Cow Sheep Pig Pufferfish
1 1 1 1 1 1 1
MDRRM----W MDGLM----W MDSLL----W MDSRV----W MGSRV----W MDSGV----W McclerqekF
GAHVFCVLSP ATRILCLLSL ATWVLCLLNL GACVLCLLGP GACVLCLLGP AACIFCLLSS TVLIFCLHLW
■ LPTVLGHMHP CGVTLGHLHL WGVALGHLHL LPIVLGHVHP LPIVLGHVHP LPVALGHVHP HTPTAQAIHP
ECDFITQLRE ECDFITQLRD ECDFITQLRD ECDVITQLRE ECDVITQLRE ECDFITQLRE DCAIISAHQR
DESACLQA-DELACLQA-DELACLQA-DEQACLQA-DEQACLQA-DERTCLQA-AQEMCKQTrr
□ AEEMPNTTLG AEGTNNTSLG AEGTNNSSMG AEGMPNSTLG AEGMPNSTLG ADRMANSSSG SEAQNQTNQS
CPATWDGLLC CPGTWDGLLC CPGTWDGLLC CPRIWDGLLC CPRIWDGLLC CPRTWDGLLC CTTLWDNIRC
WPTAGSGEWV WPPTGSGQWV WPPTGSGQWV WPTAGSGEWV WPMAGSGEWV WPTAGPGEWV WPWAEVGQVV
Human Mouse Rat Cow Sheep Pig Pufferfish
85 85 85 85 85 85 91
FSSESGAVKR FGSDTGFVKR FGSDPGAVKR FSSEPGAVKR FSLEPGAVKR FSSEPGALKR FSSNQGFVYR
DCTITGWSEP DCTITGWSNP DCTITGWSDP DCTIAGWSEP DCTIAGWSEP DCTTTGWSEP NCTADGWSEL
FPPYPVACPV FPPYPVACPV FPPYPVACPV FPPYPEACPV FPPYPEACPV FPPYPEACPV YPPYQRACAV
PLELLAE-EE PLELLTK-EK PLELLTE-EK PLELLTE-EK PLELLTE-EK PLELLTD-EK rndsepesET
TM1 ** ********** SYFSTVKIIY TVGHSISIVA SYFSTVKIIY TTGHSISIVA SYFSTVKIIY TTGHSISIVA SYFSAVRIIY TMGHSVSAAA SYFSAVRIVY TMGHSVSAAA SYFSTVRIVY TTGHSVSAVA SYLATFRQIY TVGYATSLIT
********** LFVAITILVA LCVAIAILVA LCVAIAILVA LLVAIIILVA LLVAIIILVA LFVAIAILVA LITAIVVFTA
TM2 * * ********** LRRLHCPRNY VHTQLFTTFI LRRLHCPRNY IHTQLFATFI LRRLHCPRNY IHTQLFATFI LRRLHCPRNY IHTQLFITFI LRRLHCPRNY IHTQLFTTFI LRRLHCPRNY IHSQLFATFI FRKFRCTRNY IHVNLFSSFI
Human Mouse Rat Cow Sheep Pig Pufferfish
174 174 174 174 174 174 181
********** LKAGRVFLKD LKASAVFLKD LKASAVFLKD LKAAAVFLKD LKAAAVFLKD LKAGAVFLKD LRASAVFIKD
**** AALFHSDDTD AAIFQGDSTD AAVFQGDSTD ATLFHQENTD ATLFHRENMD AALFHSENTD TVLFADESLD
HCSFS--TVL HCSMS--TVL HCSMS--TIL HCSFSTVTVL HCSFSTV--L HCSFSTV--L HCSMSTT--A
TM3 ******** CKVSVAASHF CKVSVAISHL CKVSVAVSHF CKVSVATSHF CKASVTASHF CKVSVATSHF CKSAVAFFQF
****************** ATMTNFSWLL AEAVYLNCLL ATMTNFSWLL AEAVYLSCLL ATMTNFSWLL AEAVYLSCLL ATMTNFSWLL AEAVYLTCLL ATMTNFSWLL AEAVYLTCLL ATMTNFSWLL AEAVYLTCLL SILANYFWLL VEGMYLQTLL
TM4 * ********** ASTSPSSRRA FWWLVLAGWG ASTSPRSKPA FWWLVLAGWG ASTSPRSKPA FWWLVLAGWG VSTLPSTRRV FWWLVLAAWG ASTLPSTRRV FWWLVLAAWG ASTSPSTRRA FWWLVLAGWG ALTFVSQRKY FWWYILIGWG
********** LPVLFTGTWV LPVLCTGTWV LPVLCTGTWV LPLLFTGMWV LPLLFTSMWV LPLLFTGTWV LPSAVLVLWV
Human Mouse Rat Cow Sheep Pig Pufferfish
262 262 262 262 262 262 269
** SCKLAFEDIA GCKHSFEDTE GCKLAFEDTA GCKLAFEDVA GCKLAFEDVA GCKLAFEDVA LTRFIYDNRS
CWDLDDTSPY CWDLDNSSPC CWDLDDSSPY CWDLDDSSPY CWDLDDSSPY CWDLDDSSPY CWDDTDNVAI
TM5 ********** WWIIKGPIVL WWIIKGPIVL WWIIKGPIVL WWIIKGPIVL WWIIKGPIVL WWIIKGPIVL WWIIKGPITV
********** SVGVNFGLFL SVGVNFGLFL SVGVNFGLFL SVGVNFGLFL SVGVNFGLFL SVGVNFGLFL SLLVNILIFI
*** NIIRILVRKL NIICILLRKL NIICILLRKL NIIRILLRKL NIIRILLRKL NIIRILLRKL NVIRILVQKL
EP-AQGSLHT EP-AQGGLHT GP-AQGGLHT EP-TQGSLHT EP-TQGSLHT EP-AQGSLHT KssAMAGNHD
TM6 * ********** QSQYWRLSKS TLFLIPLFGI RAQYWRLSKS TLLLIPLFGI RAQYWRLSKS TLLLIPLFGI QHQYWRLSKS TLLLIPLFGI QPQYWRLSKS TLLLIPLFGI QPQYWRLSKS TLLLIPLFGI TGHYMRLAKS TLFLIPLFGM
******** HYIIFNFLPD HYIIFNFLPD HYIIFNFLPD HYVIFNFLPD HYVIFNFLPD HYVIFNFLPD HYTVFAFLPE
Human Mouse Rat Cow Sheep Pig Pufferfish
351 351 351 351 351 351 359
NAGLGIRLPL SAGLDIRVPL SAGLGIRLPL SAGLDIRLPL SAGLDIRLPL SAGLGIRLPL NTGVTARLYI
TM7 ********* ELGLGSFQGF ELGLGSFQGF ELGLGSFQGF ELGLGSFQGF ELGLGSFQGF ELGLGSFQGF ELGLGSFQGF
********** IVAILYCFLN IVAVLYCFLN VVAVLYCFLN IVAILYCFLN IVAILYCFLN IVAILYCFLN VVALLYCFMN
QEVRTEISRQEVRTEISRQEVRTEISRQEVRTEISRQEVRTEISRQEVRTEISRGEVQTELRRw
------------------------------------------------------lrkccnqnhp
------------------------------------------------------tqakrsitqv
-----KWHGH -----KWYGH -----KWYGH -----RWHGH -----RWHGH -----RWHGH tsrarEWFGQ
$$ $ $ RAKWTTPSRS CTEWTTPPRS CTEWTTPPRS HIKWTTPSHS HIKX-----HAKWAKPSRS ----------
Human Mouse Rat Cow Sheep Pig Pufferfish
415 415 415 415 415 415 449
$$ AAKVLTSMCX RLKVLTSECX RVKVLTSECX RVKVTvpvps ---------RAKVSacsra ----------
---------------------------cpqagepfig ---------gssrprahgd ----------
---------------------------thsgeX------------typglevpgq ----------
-----------------------------------wlclfltX --------
Total length 423 423 423 439 407 451 438
63
■ □ * X $ YWR
DPELLPAWRT DPELLPARRT DPELLPARRT DLELLPARVT DPELLPARRT DPELLPAWRT PPssanissv
TLPCPDFFSH SLPCPEFFSH SLPCPEFFSH SLPCPAFFSH SLPCPAFFSH TLPCPAFFSH NVSCAEVLQD
signal peptide cleavage site glycosylation site predicted transmembrane domain stop codon phosphorylation site consensus sequence
FIG. 3. The alignment of amino acid sequences of human, mouse, rat, cow, sheep, pig, and pufferfish GHRHRs shows its highly conserved primary structure. Sequences were retrieved from the EntrezProtein database and the alignment was performed according to Morgenstern.169
receptor, and specifically by the extracellular loops connecting the seven TM domains.34,70 Removal of the extracellular portion of the GHRHR not only decreases ligand binding, but it also negatively influences the transport of the receptor to the cell surface, with resulting protein accumulation in intracellular compartments.70 In vitro studies using mutated GHRHR presenting naturally occurring missense mutations showed the inability of the mutant receptors to respond to GHRH stimulation, while immunoprecipitation and immunofluorescence experiments showed that the mutations neither caused protein degradation nor affected the transport to the membrane.71
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D. GHRHR Organization and Regulation The GHRHR has been independently located on chromosome 7p14 by in situ hybridization72 and on chromosome 7p15 by polymerase chain reaction.73 It includes 13 exons and it spans about 15 kb, including a coding sequence of about 1.3 kb.34 Exons 1–4 encode for the extracellular domain, exons 5–12 for the TM region, and exon 13 encodes for the intracellular domain.34,74 GHRHR expression regulation in the pituitary by the transcription factor Pit‐1 (Pou1f1 according to the current official nomenclature) has been demonstrated for both humans75 and rats.76 Indeed, transgenic mice with a null mutation in the Pit‐1 transcription factor do not express GHRHR.77 The importance of Pit‐1 regulation for GHRHR expression has also been confirmed by the observation of a patient with a mutation in the Pit‐1 binding site of the GHRHR promoter region resulting in IGHD.78 Pit‐1 is not the only transcription factor that acts on the GHRHR promoter. The first 2000 bases of the 50 flanking region of the GHRHR display other regulatory sequences, including binding sites for the enhancer activator protein 1 (AP‐1) and AP‐4, the consensus sequences for the nuclear factor 1 (NF‐1) and a binding site for the upstream stimulatory factor (USF).79 Binding sites for estrogen receptor element (ERE), glucocorticoid receptor element (GRE), NF‐kB, and cAMP‐ response element binding protein (CREB) transcription factor are also present, allowing gene regulation by transcription factors that are induced by exogenous stimuli.75,79 Glucocorticoids stimulate GHRHR transcription in vivo and in vitro80–83 and they appear to be essential for somatotroph cell differentiation and GH production in the pituitary.84 In humans and rodents, GH secretion displays a characteristic sexual dimorphism. A similar pattern is seen in GHRHR expression in rats. A clear increase in GHRHR transcription that coincides with the onset of puberty has been reported in rats, with female expression levels only about 15% those of males,38,85 likely due to the inhibitory effect of estrogen on GHRHR promoter.79 Conversely, androgens have been shown to enhance GHRH expression in the arcuate nucleus of the hypothalamus.86 Thyroid hormones also influence GHRHR expression. Indeed, the decrease in GHRHR expression caused by thyroidectomy can be reversed by thyroxine (T4) replacement therapy.87 Although in humans no thyroid hormone receptor response element was found in the promoter region of GHRHR, this could be present outside of the areas studied to date.6,79 Indeed, triiodothyronine (T3) causes a dose‐dependent increase in GHRHR mRNA in rat primary pituitary cells82 and T3 responsive elements were later found in the 50 flanking region of the rat GHRHR gene.88 In a recent study, T3 has been shown to have little effect on GHRHR expression in rat cells derived from a mammotrophic
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pituitary tumor, suggesting that the acquisition of mechanisms responsible for T3‐regulation of GHRHR transcription could be involved in the process of functional development of GH cells.89 The GHRHR appears to be also regulated by GHRH itself.90–92 A putative CREB binding site that could mediate this effect is present in the promoter region and in vitro studies demonstrated regulation of the GHRHR promoter by forskolin.93
E. GHRHR Splice Variants Several SVs of the GHRHR have been reported in normal pituitary and pituitary tumors in mice,68,77 rats,65 and humans,94,95 and they appear to be more abundant in pituitary tumors. The first human SV discovered encodes a truncated GHRHR lacking the last two TM domains and it is functionally inactive. However, if in vitro coexpressed with the wild‐type receptor, it showed a dominant negative modulatory effect on GHRH‐induced signaling,96 inhibiting GHRH binding.97 Alternatively spliced GHRHR variants (named SV1, SV2, SV3, and SV4)98–100 have been identified and correspond to GHRHR mRNA species not found in pituitary tissues.98 SV1 begins at an alternative start codon on intron 3, which causes the 128 amino acids of the extracellular domain of the protein to be replaced by 26 novel residues. The other SVs present truncations at different levels of the TM portion of the protein.60,98 These four SVs have been detected in renal cell carcinomas,99 breast cancer lines,101 prostate cancers,100 gastroenteropancreatic carcinomas,102 bone tumors,103 in xenografts of human non‐Hodgkin’s lymphomas, pancreatic cancer, glioblastoma, and small cell lung carcinomas. Interestingly, SVs have also been found in human nonmalignant prostate, liver, lung, and kidney but their role in disease and normal cellular activity is still unclear.37 A biological role of these splicing variants is difficult to reconcile with the important role of the extracellular domain in ligand binding. The attention to SVs is made more relevant by the observation that competitive GHRH antagonists can inhibit growth and proliferation of tumors and tumor cell lines.104 While the in vivo consequences of the GHRH antagonists could be explained by the downregulation of the GH‐IGF‐1 axis,105,106 the in vitro antiproliferative effects imply a role of GHRH as autocrine/paracrine growth stimulating factor35 with mitogenic actions on somatotroph cells.28 Nevertheless, because the full‐length GHRHR has not been detected in cell lines responsive to GHRH antagonists, it is still unclear if the in vitro actions are the result of binding to such variants or to a different receptor.98
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III. GHRHR Mutations and Polymorphisms A. GHRHR Mutations The first naturally occurring GHRHR gene mutation was reported in rodents. Dwarf mice were found at the Jackson Laboratories in the 1970s. The dwarf phenotype, inherited as an autosomal recessive character, was later associated with isolated lack of GH.107 Because of its size, the mouse was named little (lit/lit), and it soon became a popular model for IGHD studies. GHRH was found to be ineffective in the little mouse108,109 and, after the cloning and characterization of the GHRHR gene,66,67 two groups independently discovered a missense mutation (D60G) in an area of the GHRHR gene encoding for the receptor extracellular domain.110,111 This single base change (A ! G) in codon 60 causes aspartate to be replaced by glycine (D60G). Aspartate in position 60 belongs to a group of about 10 amino acids (mostly cysteine) that are highly conserved in the GHRHR of different species (mouse, rat, human) (Fig. 3). Additionally, this aspartate residue is conserved in other receptors that belong to the same subfamily of GPCRs, such as the receptors for secretin, VIP, glucagon, and GLP‐1.110,112 Cells expressing the receptor containing the lit mutation do not respond to GHRH stimulation, but respond to agents that directly increase cAMP.110 Size and quantity of GHRHR mRNA transcript, protein stability, glycosylation, cellular distribution, and transport to the cell surface are not affected by the mutation,110,113 which causes inability of the receptor to bind GHRH.113 Similar consequences are observed if the same aspartate residue is mutated in VIP114 and glucagon115 receptors, confirming the importance of the extracellular domain in this class of receptors for ligand binding.70 The first human analog of the little mouse was found a few years later, when a GHRHR mutation was discovered in two IGHD Indian Muslim cousins living in New York and born from consanguineous parents.116 The affected children were homozygous for a G ! T mutation on exon 3, which introduces a stop codon at residue 72 (E72X) (residue 50 if counting after the removal of the 22‐amino acid signal peptide). In contrast with the GHRHR of the little mouse (synthesized and expressed on the cell surface), this mutation results in a receptor that lacks most of the functional domains and it is possibly not even translated due to nonsense‐mediated mRNA decay.117 Interestingly, the same mutation (E72X) was later reported in a Pakistani kindred (‘‘Dwarfs of Sindh,’’ from the name of the southernmost province of Pakistan), with 18 young people affected by IGHD,118,119 and in two Sri Lankan siblings.120 Although the three kindreds originated from the Indian subcontinent, they were not geographically or culturally related. Nevertheless, genetic analysis suggested that the three kindreds may be very distantly related and share a common ancestor (‘‘founder effect’’).121
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The E72X mutation has been later reported in a study from western India,122 where 78% of IGHD IB subjects (the majority not born from consanguineous unions) carried this mutation, making it the most prevalent GHRHR mutation in the Indian subcontinent. The reason of its spreading in this particular region (whether it confers a survival advantage) has not been clarified, but this mutation has never been reported in other regions of the world. We identified a large kindred with more than 100 individuals (approximately 74 currently alive) affected by autosomal recessive IGHD who live in the rural county of Itabaianinha (in the northeastern Brazilian state of Sergipe).123 All the affected individuals have a homozygous GHRHR mutation (IVS1 þ 1 G ! A), which alters the highly conserved guanine–thymine dinucleotide of the donor splice site of intron 1,124,125 likely resulting in incomplete removal of the intron sequence during mRNA processing. We had predicted that this mutation would cause retention of part of the intronic sequence and insertion of a stop codon (TGA) 213 bases downstream of the junction between exon 1 and intron 1.123 Very recently, a mutation in the same splice site (IVS1 þ 2 T ! C) has indeed confirmed this hypothesis.126 The Itabaianinha kindred is the largest population affected by a GHRHR mutation, and the largest population with IGHD described to date. Haplotype analysis suggested a ‘‘founder effect.’’ The high percentage of consanguineous unions in a very limited geographical area contributed to the spread of this mutation (with a calculated prevalence of one case of dwarfism per 304 inhabitants).123 The same mutation has been reported in other individuals, but always inside Brazil.127 Both for the Itabaianinha kindred and the ‘‘dwarfs of Sindh,’’ the exact timing of onset of the mutations and their origin are still not determined.128 Several other mutations in the GHRHR have been reported over the last few years. They include seven missense mutations,78,129–131,131a one nonsense mutation,132 two microdeletions,130,133 and four splice donor site mutations.126,132,134,135 One mutation in the promoter has been reported, affecting one of the Pit‐1 binding sites.78 A summary of GHRHR mutations described to date is given in Table I, together with information regarding location on the gene, geographical provenience, and inheritance pattern. Mutations are also graphically presented in Fig. 4. With the exception of L144H, each mutation is specific for a geographical area. The L144H mutation, affecting the first TM domain, was found in families from three different geographical areas (Europe, South, and North America).129,136 This mutation occurs in a CpG dinucleotide, an area of increased mutagenicity because C undergoes spontaneous deamination.137 Linkage studies excluded the possibility of a ‘‘founder effect’’ for this type of mutation,136 suggesting that this may be a ‘‘hot spot’’ for mutations.
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TABLE I SUMMARY OF ALL GHRHR MUTATIONS KNOWN TO DATE Mutation
Location
Type
Provenience
Inheritance
References
124 A ! C
Promoter
Pit‐1 binding
Portugal
CHa
78
IVS1þ1 G ! A
Intron 1
Splice donor
Brazil
HMZb
123
IVS1þ2 T ! G
Intron 1
Splice donor
Morocco
HMZ
126
Q43X
Exon 2
Nonsense
Canada
CH
132
E72X
Exon 2
Nonsense
India/Pakistan
HMZ
118–120
IVS3þ1 G ! A
Intron 3
Splice donor
Canada
CH
132
H137L
Exon 5
Missense
Canada
CH
130
L144H
Exon 5
Missense
Spain/Brazil/USA
CH and HMZ
129
A176V
Exon 6
Missense
Pakistan
HMZ
131
A222E
Exon 7
Missense
Pakistan
HMZ
129
F242C
Exon 7
Missense
USA
CH
129
IVS7þ1 G ! C
Intron 7
Splice donor
Morocco
HMZ
135
K329E
Exon 11
Missense
Portugal
CH
78
R357C
Exon 11
Missense
Israel
HMZ
131a
Del 1121–1124
Exon 11
Deletion
Japan
HMZ
133
Del 1140–1144
Exon 11
Deletion
Canada
CH
130
IVS12þ2 T ! A
Intron 12
Splice donor
Pakistan
HMZ
134
Mutations are listed according to their location in the gene and with information regarding type of mutation, geographical provenience, and type of inheritance. a Compound heterozygous. b Homozygous.
All the seven missense mutations reported to date involve the substitution of highly conserved amino acids that belong to one of the TM domains or connecting loops. Single amino acid substitutions result in loss of signal transduction and unresponsiveness to GHRH stimulation. In vitro studies performed on six of the seven missense mutations showed that the mutant receptors are expressed on the cell surface, but unable to bind GHRH.71 The two microdeletions involve a five‐base deletion (Del 1140–1144)130 and a four‐base deletion (Del 1121–1124);133 both in exon 11. Del 1140–1144 starts at codon 365 (corresponding to the seventh TM domain of the protein) and causes the coding sequence to shift out of frame, introducing a premature stop codon 67 bp after the beginning of the deletion. By the same mechanism, Del 1121–1124 (starting at codon 358) is predicted to cause a premature termination of GHRHR synthesis. In addition to the E72X, another nonsense mutation (Q43X) has been reported, which introduces a stop codon at the end of exon 2 resulting in a severely truncated protein.132
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IVS1 + 1 TÆA
Q43X
NH2
E72X
R357C
IVS3 + 1 GÆA Extracellular
IVS7 + 1 GÆT
H137L H137L
Cell membrane
Del 1121−1124 Del 1140−1144
A222E A176V
K329E
L144H
F242C
Intracellular
IVS12 + 2 TÆA HOOC
Splice donor mutation
Missense mutation
Nonsense mutation
Deletion
Exon boundaries
Glycosylation site
FIG. 4. Graphic representation of GHRHR protein structure and localization of the mutations reported to date in the coding sequence and splice sites. The mutation in the promoter region is not represented here.
After the Itabaianinha mutation, other splice site mutations were described. One involving the splice donor site at the beginning of intron 3 (IVS3 þ 1 G ! A),132 a second one involving the splice donor site at the beginning of intron 7 (IVS7 þ 1 G ! T),135 a third one involving the splice donor site in intron 12 (IV12 þ 2 T ! A),134 and a fourth one in intron 1 (IVS1 þ 2 T ! G).126 This last mutation is only one base downstream of the mutation found in the Itabaianinha kindred.123 The mechanism that leads to nonfunctional GHRHR was demonstrated through the creation of a minigene containing the mutated gene which results in a larger than normal splicing product due to the use of a cryptic donor splice site 50 of the normal site. In case of translation, this abnormal splicing product would generate a frameshift at the end of exon 1 (codon 19) that introduces 71 novel amino acids before inserting a stop codon that prematurely terminates the protein.126 As mentioned earlier, the only reported mutation in the promoter region of the GHRHR has been found in one of the Pit‐1 binding sites.78 This mutation involves an A ! C transversion at position 124 of the promoter region. In vitro studies showed that the mutant promoter has a much lower transcriptional activity compared to the normal promoter, resulting from severely reduced ability to bind the Pit‐1 transcription factor.78 Accordingly, a previous study where a 3‐bp artificial mutation was introduced in this area (129 to123,
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TATGCAA ! TGTACGA), including the A nucleotide mutated in the patient described by us, demonstrated that both Pit‐1 binding and promoter activity were markedly reduced.75 All GHRHR mutations are inherited as autosomal recessive traits, with possibly the only exception of Del 1121–1124. This mutation displays a ‘‘dominant negative’’ effect in vitro that should account for an autosomal dominant inheritance.60 Nevertheless, the homozygous patient was severely GH deficient and much shorter than the heterozygous parents, making the in vivo results of this dominant effect questionable.133 Although many of these mutations occur in inbred kindreds, where the homozygous state is caused by parental consanguinity, it is important to notice that GHRHR mutations are not limited to these kinds of family. Over the years, we have found four families in which the affected subjects were compound heterozygotes for two distinct mutations.78,129,132 This observation supports the hypothesis that faulty GHRHR alleles may be rather prevalent in the general population.
B. GHRHR Polymorphisms Polymorphisms in the GHRHR have been reported but no role in IGHD disease has been established.78,127 On the other hand, some polymorphisms (A57T and M422T) have been associated with an increased response to GHRH through an augmented cAMP production.138,139 Although the presence of constitutively active GHRHR mutations could in theory be considered crucial for the development of GH‐secreting pituitary tumors, several studies reported no increased risks of developing acromegaly in patients affected by those polymorphisms when compared to the general population, and analysis of tissues from GH‐secreting adenomas failed to confirm this hypothesis.139–141 Very recently, two haplotypes of the GHRHR have been reported to account for 1.8% of the variation in adult height after adjusting for sex, age and population affinity, making it the strongest genetic contributor to normal human variation in height so far identified.141a
IV. Diseases Associated with GHRHR Mutations A. Clinical Characteristics of Homozygous Individuals The common feature of subjects with biallelic GHRHR mutations is marked GH deficiency. Because of different GH measurement assays, different stimuli used during GH stimulation tests, and the multigene influence on stature, it is difficult to compare the degree of IGHD and of growth failure caused by the individual mutations. Although it is theoretically possible that
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less disruptive mutations may cause a milder degree of IGHD, this has never been demonstrated. Furthermore, most IGHD children in developed countries receive GH replacement therapy after the diagnosis is made. GH replacement therapy has been successfully used for the treatment of short stature of many children with GHRHR mutations. They respond very well to the therapy, showing a brisk increase in longitudinal growth and they reach their target adult height if treatment is started in timely fashion.78,129,131,135 In some instances where IGHD diagnosis was made late, GH has been associated with gonadotropin‐releasing hormone (GnRH) analog therapy in order to delay the onset of puberty and allow more time for the beneficial consequences of exogenous GH on longitudinal growth.142,143 However, the two largest kindreds (Sindh and Itabaianinha) have many adult IGHD subjects who have not been treated with GH, allowing an analysis of lifetime lack of GH due to such mutations. Affected subjects exhibit similar phenotypic features with proportionate short stature, poorly developed muscle mass, and excessive abdominal fat accumulation. Small developmental defects have been noted in some patients, especially ear deformities.119 In addition, they have a characteristic high‐pitched voice, due to the lack of GH effect on laryngeal development.144,145 The onset of puberty is usually delayed without affecting fertility. IGHD women of Itabaianinha tend to have a lower number of children (2.0 vs. an average of 5.5), probably due to delayed age of first intercourse and to the necessity of performing caesarean sections because of cephalic/pelvic disproportions, but have normal menopause timing and symptomatology. Their hormonal profiles are similar to unaffected women, apart from decreased serum prolactin (PRL) level.146 Low‐PRL concentration has also been reported in some individuals with the E72X mutation, suggesting a possible interference of the GHRHR mutation with PRL secretion, as PRL gene transcription is cAMP‐activated and cAMP concentration is increased by GHRHR activation (Ref. 120 and references therein). Nevertheless, the Itabaianinha IGHD women are able to nurse their children. Adult height for adult Itabaianinha IGHD males (18 years and older) is 123.3 7.62 cm compared to 163.0 4.53 cm for the normal‐appearing male subjects from the same area. For adult IGHD females, it is 118.8 8.53 cm compared to 150.8 7.31 cm of normal females.147 Adult height for Sindh IGHD males is 130.3 10.6 cm compared to 172.8 6.0 cm for randomly selected men from the same area and for adult IGHD females it is 113.5 0.7 cm.119 Patients from the Itabaianinha kindred have very low serum GH, IGF‐1, and IGF‐1 binding protein‐3 (IGFBP‐3) levels. IGHD status is confirmed by lack of significant GH response to stimulation test (clonidine, GHRH, insulin‐induced hypoglycemia, or l‐Dopa).
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Dwarfism appeared to have emerged recently in the Sindh region, with the oldest affected person being 28 years old at the time of the first report, in 1997. Conversely, in the Itabaianinha area, dwarfism emerged in the more remote past, with the first documented affected person being born in 1877. Both kindreds have a high frequency of consanguineous marriages and have been geographically isolated. In IGHD individuals from Itabaianinha, the size of interior organs, assessed using ultrasounds, has revealed reduced absolute size of liver, spleen, kidney, prostate, thyroid, and uterus if compared to unaffected controls, while ovaries and pancreas were similar in size.148,149 Once corrected for body surface area, prostate and ovaries show normal size, spleen, thyroid, and uterus remain smaller, while kidney, pancreas, and liver are actually larger than in control subjects.148 Hence, the growth of these last three organs appears to depend less from an intact GH/IGF‐1 axis than the longitudinal bone growth. On the other hand, the smaller uterus may also be caused by the decreased number of pregnancies.146 In addition to reduced thyroid volume (possibly due to reduced trophic effect of circulating IGF‐1 on thyroid cells), IGHD individuals also present low serum T3 and high serum‐free T4 levels149 likely caused by absence of the stimulatory effect of GH on 50 ‐deiodinase, the enzyme responsible for converting T4 into T3.150 MRI of the hypothalamic–pituitary region shows presence of APH in affected individuals both from the Itabaianinha and Sindh’s kindreds.120,151,152 APH is also reported in affected individual in most (but not all) other kindreds. Because large studies have never been done in very young children ( 40.0) or patients with severe obesity (BMI between 35.0 and 39.9) and severe medical complications of obesity. To identify more pharmacological therapeutic options, a better understanding of the regulation of energy homeostasis is essential. Obesity is caused by an imbalance of energy intake and energy expenditure. Genetic, environmental, and social factors contribute to the pathogenesis of obesity. Abundance of energy dense foods and sedentary life styles of the modern society are major environmental contributors. The role of social networks in obesity pathogenesis is suggested by an interesting recent study.6 However, multiple lines of studies, including twin and adoption studies, showed unequivocally that there is an important genetic component in body weight regulation (reviewed in Ref. 7). This chapter summarizes the studies on one of the genetic factors that causes monogenic obesity and contributes to common obesity, the melanocortin‐4 receptor (MC4R). Interestingly, some alleles of the MC4R confer protection from obesity.
II. The Melanocortin System The melanocortin system is an ancient system conserved from teleosts to mammals. It consists of four agonists, two antagonists, and five receptors. The four agonists, including ‐, ‐, and ‐melanocyte‐stimulating hormone (MSH),
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and adrenocorticotropin (ACTH), are derived from tissue‐specific posttranslational processing of the preprohormone proopiomelanocortin (POMC).8 The five receptors are called melanocortin receptors 1–5 (MC1R–MC5R) based on the sequence of their cloning. They are all members of Family A G protein‐coupled receptors (GPCRs) consisting of seven transmembrane domains (TMs) connected by alternating intracellular and extracellular loops, with the N‐terminus extracellular, and the C‐terminus intracellular. All five MCRs are coupled primarily to the stimulatory G protein Gs, therefore receptor activation results in increased cyclic adenosine monophosphate (cAMP) production in the cell. The two endogenous antagonists of the MCRs are agouti and agouti‐related peptide (AgRP). Agouti is an antagonist for MC1R. Binding of agouti to the MC1R decreases synthesis of eumelanin, the black/brown pigment, resulting in lighter coat color in the animals. AgRP, a peptide of 132 amino acids (in humans), antagonizes the actions of the MC3R and MC4R.9 It is expressed in the hypothalamus. AgRP was recently shown to act as an inverse agonist in that it decreases the basal activity of the MC4R.10,11 POMC has been cloned from fishes, frogs, reptiles, birds, and mammals. Human POMC consists of 267 amino acids, which can be cleaved into several peptides, including the melanocortins ‐, ‐, and ‐MSH, and ACTH, as well as ‐endorphin. There are two enzymes involved in the cleavage, prohormone convertases 1 and 2 (PC1 and PC2). The cleavage is tissue specific. In the anterior pituitary gland, POMC is processed into ACTH and other peptides by PC1. In the arcuate nucleus of the hypothalamus, POMC is further processed into ‐, ‐, and ‐MSH by the combined actions of PC1 and PC2. Cone and colleagues cloned the first two members of the MCRs, MC1R, and MC2R.12 The MC1R is the classical MSH receptor expressed in the skin that regulates pigmentation. The MC2R is the classical ACTH receptor expressed in the adrenal cortex that regulates adrenal steroidogenesis and cell proliferation. Subsequently, several groups reported the cloning of additional members of this family, including the MC3R,13,14 the MC4R,15 and the MC5R.16 The functions of these new MCRs were unknown when they were first cloned. Gene knockout techniques were instrumental in elucidating the functions of these MCRs. It was shown that knockout of the MC3R results in a unique metabolic syndrome. The knockout mice had normal food intake and body weight; however, the fat mass is increased significantly with decreased lean mass.17,18 Knockout of the MC4R resulted in increased food intake and decreased energy expenditure therefore obesity.19 Knockout of the MC5R revealed its involvement in regulating exocrine gland secretions.20
III. The Leptin‐Regulated Melanocortin Circuit With the groundbreaking discovery of leptin,21 a protein hormone produced by the adipocyte, tremendous progress has been made in elucidating the neural pathways regulating energy homeostasis during the last 15 years.
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The leptin‐regulated melanocortin circuit was found to be essential for energy homeostasis.22 In this circuit, leptin, after crossing the blood–brain barrier, binds to its receptors in two subsets of neurons in the arcuate nucleus. One subset of neurons coexpresses neuropeptide Y and AgRP, while the other subset of neurons coexpresses POMC and cocaine and amphetamine‐regulated transcript.23 Studies in mice and humans showed that genetic disruption of multiple components in this circuit cause obesity (Fig. 1). The expression levels of POMC and AgRP are modulated by the energy homeostasis of the animals. For example, fasting reduces POMC mRNA, although the decrease is small, about 24%.24,25 In another study, hypothalamic POMC mRNA was decreased >60% after a 2‐day fast.26 The response of POMC mRNA to overfeeding seems to be larger, with arcuate POMC increased to 185% in rats overfed for 10 days.27 AgRP gene responds more robustly to changes in energy balance. For example, in mice, AgRP was upregulated 13‐fold by a 2‐day fast.28 First order neurons (arcuate)
Second order neurons
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(+) BBB OBRb POMC CART
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FIG. 1. The leptin‐regulated melanocortin circuit. Leptin produced by adipocyte binds to two subsets of neurons that express leptin receptor (OBRb) in the arcuate nucleus. ‐MSH derived from POMC activates the MC4R, whereas AgRP inhibits the MC4R. Activation of the MC4R decreases food intake and increases energy expenditure resulting in a negative energy balance. The molecules highlighted in red, including leptin, leptin receptor, POMC, PC1 (or carboxypeptidase E, CPE in mice), and the MC4R, have been shown to cause obesity in mice and humans. Modified from Ref. 23.
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Ob/ob and db/db mice are well‐known examples of mice that have defective leptin (ob/ob) or leptin receptor (db/db) genes.21,29 Mice lacking the POMC‐ derived peptides suffer from obesity in addition to defective adrenal development (due to lack of MC2R signaling) and altered pigmentation (due to lack of MC1R signaling),30 recapitulating the phenotype of POMC‐deficient patients. Fat/fat mice are obese and hyperglycemic due to mutation in carboxypeptidase E gene resulting in inactive enzyme.31 Inefficient processing of POMC results in obesity, whereas inefficient processing of proinsulin results in defective insulin production and hyperglycemia. Human genetic studies showed that mutations in several components of this circuit, including leptin,32,33 leptin receptor,34 POMC,35 PC1,36 and MC4R, all result in monogenic obesity. Essentially human genetic studies replicated the rodent genetic studies. A total of 12 individuals have been shown to have homozygous mutations in leptin (LEP) gene. These patients have severe early‐onset obesity and undetectable serum leptin levels.37 Heterozygous LEP individuals have increased body weight. Treatment with exogenous leptin daily successfully corrected all the phenotypes, including reducing food intake, inducing onset of puberty and development of secondary sexual characteristics when treated to adolescent children, restoring impaired T cell‐mediated immunity, representing an outstanding example of personalized medicine resulting from genetic investigations.38 Several mutations in leptin receptor (LEPR) gene, including frameshift, missense, and nonsense mutations, have been reported.34,39 Individuals with homozygous or compound heterozygous mutations develop severe obesity, whereas individuals with heterozygous mutations have increased body weight. Serum leptin levels are appropriate for the increased body fat. Functional studies of the missense mutant leptin receptors showed that they indeed have impaired functions due to misfolding or impaired cell‐surface expression and signaling.40 Krude and colleagues were the first to report naturally occurring mutations in POMC gene from patients with severe early‐onset obesity.35 They originally described two patients. Patient 1 was found to be a compound heterozygote for two mutations that interfere with appropriate synthesis of ACTH and ‐MSH. Patient 2 was homozygous for a mutation that abolishes POMC translation. They subsequently reported three additional patients with POMC mutations.41 Mutations in the POMC gene in humans result in severe early‐onset obesity in addition to adrenal insufficiency (lack of MC2R signaling) and red hair and pale skin (lack of MC1R signaling), similar to Pomc gene knockout mice. A Turkish patient with POMC deficiency had brown hair with red roots.42 Hypocortisolemia leads to hypoglycemia and increased susceptibility to infection. In one case, it led to neonatal death.37 Individuals heterozygous for POMC mutations have increased risk for developing obesity.
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Two recent studies suggested that ‐MSH is also an important regulator of human energy homeostasis.43,44 Mutations in the coding region for ‐MSH in the POMC gene were found to be associated with early‐onset obesity. In vitro studies showed that mutated ‐MSH had impaired ability to bind and activate the MC4R. These data are consistent with a series of studies supporting the involvement of ‐MSH in regulating energy homeostasis (reviewed in Ref. 45). These experiments showed that ‐MSH has higher affinity for MC4R than ‐MSH; ‐MSH can activate MC4R in vivo; ‐MSH is present in hypothalamic nuclei that regulate feeding and its concentrations are modulated by nutritional status (the concentrations of ‐MSH are not); and importantly, ‐MSH is as potent as ‐MSH at inhibiting food intake in fasted animals. These results lead Harrold and Williams to conclude that ‐MSH is more convincing than ‐ MSH as the endogenous ligand that activates MC4R to inhibit food intake,45 although it cannot be excluded that ‐MSH is also involved. Two patients with compound heterozygous mutations and one patient with homozygous mutations in PC1 (PCSKI) gene have been identified so far.36,37 These patients have severe early‐onset obesity due to defect in POMC processing. PC1 is also involved in processing other substrates, including proinsulin, proglucagon, and progondotropin‐releasing hormone. Defect in processing of proinsluin results in postprandial hypoglycemia and increased ratio of plasma proinsulin/insulin ratio. Defect in proglucagon and progastrin processing might contribute to the severe neonatal small‐intestinal absorptive abnormality.46 Defect in progonadotropin‐releasing hormone processing results in hypogonadotropic hypogonadism, since gonadotropin‐releasing hormone is essential for stimulation of gonadotropin secretion. Gonadotropins then stimulate gonadal functions. Taken together, these studies demonstrated that the leptin‐regulated melanocortin circuit is indispensable for maintaining energy homeostasis in both rodents and humans.
IV. Melanocortin‐4 Receptor and Energy Homeostasis Multiple lines of investigations, including anatomical, genetic, and pharmacological studies, demonstrated convincingly the critical importance of the MC4R in regulating food intake and energy homeostasis. Using in situ hybridization, Mc4r was localized in numerous nuclei in the rat brain, including the cortex, thalamus, hypothalamus, and brainstem.47 Administration of the MC3/4R agonist melanotan II (a cyclic heptapeptide) into brain ventricles of rodents suppressed food intake and decreased body weight,48,49 while the MC3/4R antagonist SHU 9119 stimulated feeding and reversed the suppressive effects of melanotan II on food intake.48 Since the
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antagonist itself stimulated food intake, it was suggested that the arcuate POMC neurons exert a tonic inhibitory effect on feeding by releasing desacetyl‐‐MSH that then activates the MC4R (and potentially MC3R too). In addition to the arcuate and paraventricular nuclei as the possible sites of action for these melanocortin analogs, the brainstem, which has the highest expression of MC4R in the brain,47 may also be involved in the control of feeding.50 The agouti (Ay) mice, which overexpress agouti ectopically in many tissues including the hypothalamus, in addition to the phenotype of yellow coat color, are also obese, because agouti is a specific high‐affinity antagonist of the MC4R.51 Mouse agouti is not an antagonist for the MC3R or MC5R. Overexpression of AgRP also results in obesity.9,52 AgRP expression is elevated in the hypothalamus of ob/ob and db/db mice,9,53 again supporting a role for AgRP and MC4R in the regulation of feeding. Finally, Mc4r knockout mice provided the proof that MC4R is a key receptor in regulating energy homeostasis. The knockout mice had increased food intake and body weight, increased linear growth, and hyperinsulinemia.19 Even the heterozygous mice had bodyweight higher than the wild‐type (WT) littermates. The Mc4r knockout mice do not respond to the MC4R agonist melanotan II in food intake54 or energy expenditure.55 An elegant study using Cre‐lox technology to reactivate the Mc4r in paraventricular nucleus and a subpopulation of amygdala neurons showed that the MC4R in these neurons control food intake whereas other neurons expressing Mc4r control energy expenditure.56 There are also evidences suggesting that MC4Rs expressed outside of the hypothalamus are involved in regulating energy homeostasis. For example, among the central nervous system, MC4R mRNA is expressed at the highest level in the dorsal motor nucleus of the vagus.47 Intracerebroventricular administration of MC4R agonists into the fourth ventricle decreases food intake whereas the antagonists increase food intake.50,57
V. Naturally Occurring Mutations in MC4R Gene and Human Obesity A. Identification of MC4R Mutations In 1998, shortly after the Mc4r knockout study was published, two groups independently reported that frameshift mutations in the MC4R gene were associated with severe early‐onset obesity.58,59 Since then, numerous groups sought to identify novel MC4R mutations. More than 150 mutations have been identified so far in various patient cohorts.60–99 These mutations include at least 122 missense mutations, 2 inframe deletion mutations, 7 nonsense mutations, and dozens of frameshift mutations (Fig. 2) (the frameshift mutations are not
Y35C/X
G32E
S30F
R18C/H/L
L23R
S4F
D37V
M NV terminal
S Y G K G L S E S A N S H L R Y S S R N W L H L S T H M G R H T S N D G
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I121T
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Y80X
M79I P78L H76R
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D D C
I D S I125K S II D126Y F A G181D S127L V L I
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I137T I W174C
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I301T Y302F R310K E308K
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N240S A A303T G Q G238D
M
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M
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A I F201L V T L A175T F261S W T C C V F M A A F202L F W V F M200V T M V I C V253I L G I S
F Y
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Del170 I I A L S I I170V I M A A S G D V L D146N I169S Y V R V166I Y F R V T H I K R165Q/W T150I L
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Y S
T
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E T
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E
I
I C C Y
I316S P I317T/V L
G
T I
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I226T
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Y R S S L D C L G Cterminal
Y332C/H
C326R
L325F
FIG. 2. Naturally occurring mutations of the MC4R identified from various patient cohorts. Shown are the missense, nonsense, and inframe deletion mutations. Frameshift mutations are not included in the figure. The two polymorphisms that confer protection from obesity are indicated with red filling and letters. See the text for references.
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listed in Fig. 2). From Fig. 2, it is very clear that these mutations are scattered throughout the MC4R. A total of at least 103 residues were mutated, representing 31% of the receptor. Still, with each new screening report, novel mutations continue to be identified. Since the level of MC4R gene expression is important for regulating food intake (haploinsufficiency causes obesity), several studies tried to identify mutations or variants in the regulatory sequences, especially promoter region, in obese subjects. Although several variants were identified,80,100,101 none of them segregates with obesity phenotype, therefore their relevance to the pathogenesis of obesity is unknown at present. A polymorphism in the promoter of the MC4R gene is related to physical activity phenotype, potentially related to obesity.102 Therefore data from these experiments suggested that defect in MC4R gene transcription is likely not a major cause of severe early‐onset obesity. Recently, genome‐wide association scans showed that single nucleotide polymorphisms (SNPs) near MC4R (SNP rs17782313, rs12970134, and rs17700663) are associated with increased risk for obesity with increased intakes of total energy and dietary fat.103–105 These polymorphisms are located 109–188 kb downstream of MC4R.104 Although the effects of these SNPs are believed to be mediated by the MC4R, the exact mechanism is not clear at present. One hypothesis is that the transcriptional control of the MC4R is affected.
B. Prevalence of MC4R Mutations Earlier studies by Farooqi and colleagues showed that up to 6% of early‐ onset morbidly obese patients in some cohorts harbor MC4R mutations.71 The patients in this cohort are unique in that they had early‐onset (before 10 years of age) and severe (BMI SDS of greater than 4) obesity. Subsequently prevalence was calculated from a number of large scale screening studies of cohorts from different ethnic backgrounds, and different age (children, adolescents, and adults), as well as degree of obesity. Not surprisingly, lower prevalence was found in these studies. For example, Hinney and colleagues reported a prevalence of 1.9% pathogenic MC4R mutations in extremely obese German children and adolescents.73 In another smaller study of early‐onset severely obese German children, one pathogenic mutation was identified from 51 children; therefore the prevalence is also 1.96%.98 In a study of Finnish patients consisting of 56 children and 252 adults with severe obesity, only one pathogenic mutation was found from a child,78 therefore the prevalence in the children cohort is 1.8%, and only 0.32% in the whole cohort. In a large study of obese Europeans recently published, 1.72% of obese individuals have MC4R mutations compared with 0.15% in nonobese individuals.95
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An earlier study of obese North Americans raised doubt about MC4R mutations in severely obese patients.68 In Pima Indians, a Native American population with very high prevalence of obesity, 14 out of 300 severely obese patients had MC4R mutations, amounting to a prevalence of 4.6%.80 Recently, in a cohort of North American adults consisting of 889 severely obese and 932 lean controls, the prevalence of rare MC4R variants is 2.25% in the obese group and 0.64% in the lean group.106 This prevalence is similar to the Europeans. In Asians, the prevalence is generally lower. A small study of 50 severely obese Japanese did not identify any mutation in the MC4R except for the V103I polymorphism.107 Lam and colleagues screened 227 obese southern Chinese (from Hong Kong) and identified three missense MC4R variants.86 However, functional studies showed that all three variants function normally. The available genetic data from pedigree analysis also suggest that these variants are not the cause of obesity in these probands.86 In another study of 200 obese children from mainland China, three MC4R mutants were identified with a prevalence of 1.5%.87 Screening of 288 obese Chinese from Shanghai identified only one functionally relevant MC4R mutation, with a prevalence of 0.3%.108 In a screening of 227 children and adolescents from Singapore, three subjects had MC4R mutations, with a prevalence of 1.3%.94 It should be mentioned that some of the studies that investigated prevalence did not perform functional studies on the mutations identified therefore the estimate of the prevalence can be an overestimate. If the mutations do not impair function in vitro, the involvement of these mutations in the pathogenesis of obesity is questionable. Although the prevalence varies in different studies, there is no doubt that mutations in the MC4R are the most common monogenic form of early‐onset severe obesity and significant genetic cause for adult obesity.
C. Clinical Phenotypes of Patients Harboring MC4R Mutations Of course the most important phenotype of MC4R mutation carriers is their obesity. When they were offered a buffet meal, they have increased caloric intake. In addition to obesity, MC4R mutation carriers were also reported to have increase longitudinal growth.71 The effect of MC4R mutation on BMI is more pronounced in females than in males. It was estimated that MC4R mutations carriers, compared with MC4R WT relatives, are 9.5 and 4 kg/m2 higher in middle‐aged women and men, respectively.95,109 The exact mechanism for this MC4R genotype–sex interaction is not clear at present. Sex steroids might be contributing factors.
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Earlier studies showed that MC4R mutations do not affect energy expenditure,71 different from mouse genetic studies.56,110 Very recently, Baier and colleagues showed that Pima Indians with two pathogenic MC4R mutations (R165Q and a frameshift mutation with A insertion at nucleotide 100) had decreased energy expenditure.96 Recently, Farooqi and colleagues showed that there is a negative association between MC4R mutations and blood pressure.111 The prevalence of hypertension is significantly lower in subjects with MC4R mutations than in control subjects with no MC4R mutations. Insulin levels cannot account for the differences in blood pressure between the two groups. These results suggest that hypertension might be a side effect for treating obesity using MC4R agonists. Interestingly, several studies suggested that some MC4R polymorphisms might confer resistance to obesity. V103I is a common polymorphism with the minor allele appearing at a frequency of 2–4%. Rosmond et al. first reported lower abdominal obesity in heterozygotes for V103I compared with homozygotes for V103.112 Hebebrand and colleagues then expanded this observation to a larger cohort and meta‐analysis, confirming the negative association of the isoleucine allele with obesity rate.113 A subsequent meta‐analysis of several studies consisting of 29,563 individuals, 10,975 cases and 18,588 controls, showed that for the V103I polymorphism, the isoleucine allele had an 18% lower risk of obesity compared with the valine allele.114 Functional studies revealed no differences between the V103 and I103 MC4R in constitutive activity and ligand‐induced signaling as well as binding to the agonists.60,63,113,115 However, a recent study showed that I103 is less sensitive to AgRP inhibition.116 AgRP is an orexigenic signal; decreased response to AgRP might result in weaker orexigenic signal conferring lower risk of obesity. Taken together, these studies suggest that the V103I MC4R polymorphism contribute to pathogenesis of polygenic common obesity. Froguel and colleagues showed that another common polymorphism, I251L, confers stronger protection from obesity.117 This variant is negatively associated with childhood and adult severe obesity, with odds ratio ranging from 0.25 to 0.76, averaging 0.52. The reason for the protective effect of I251L is suggested to be due to its higher constitutive activity.116 Together with V103I (average odds ratio 0.80), these two variants may provide 2% protection of obesity, similar to the roughly 2% cause of obesity by pathogenic loss‐of‐function mutations in the MC4R.117 Therefore the MC4R is a double‐edged sword that can either cause obesity or protect from obesity, highlighting yet again the critical importance of MC4R in regulating human energy homeostasis. Although MC4R mutations cause hyperphagia, it was not clear whether they were associated with eating disorders. Most of the studies that reported MC4R mutations did not document the eating behavior of the patients
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harboring MC4R mutations. Hebebrand and colleagues reported a few cases of binge eating patients with functionally relevant MC4R mutations.118,119 Branson et al. suggested that all obese patients with MC4R mutations had binge eating disorder.120 However, 75% of their subjects had MC4R polymorphisms V103I, T112M, and I251L. These variants have been shown by multiple independent groups to be functioning normally.60,62,63 We further showed that the three novel variants they identified, T11A, F51L, and M200V, as well as T112M, have normal functions.121 These results raise serious doubts about the conclusion reached in the Branson et al. article. Additional clinical studies demonstrated that indeed MC4R mutations are not associated with binge eating disorder.84,122
D. Functional Characterizations of the MC4R Mutants Identification of a MC4R variant from an obese patient does not necessarily prove that the mutation is the cause of obesity in the patient. Additional data, including cosegregation of the mutation with obesity in the family and absence of the variant in the ethnically matched controls, provide additional support for a causative role of the mutation in causing obesity.123 In addition, functional characterization of the mutant receptor in vitro is indispensable to prove a causative role. Several assays can be used to study whether the mutant receptor is functioning normally. Because MC4R activate Gs after ligand binding, resulting in increased intracellular cAMP levels, signaling assays by either direct measurement of cAMP levels or indirect measurement of increased reporter gene expression driving by increased cAMP levels have been used to measure mutant MC4R signaling. These assays will reveal whether mutant receptors have either decreased or absent signaling in response to agonist stimulation. Ligand‐binding experiments can be used to measure whether these mutant receptors can bind to the ligand normally therefore differentiating whether the impaired signaling is due to defect in ligand binding or due to defect in signaling per se. For mutants that are defective in ligand binding, further experiments measuring cell‐surface expression, either by immunocytochemistry or flow cytometry, are needed to differentiate whether the defects in binding is due to defective cell‐surface expression or due to defect in ligand binding per se. For these experiments, it is preferable to use stably transfected cells. Transient transfection can over‐load the cell’s quality control system, resulting in an artificial intracellular retention of the receptors (see Ref. 124 for example). If no cell‐surface expression is observed, permeabilization will reveal whether intracellular staining can be observed, demonstrating whether the protein is produced but retained intracellularly or the mutant receptor expression level is impacted.
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In our own previous study using the strategy described above, we characterized 10 naturally occurring mutants, D37V, P48S, S58C, N62S, P78L, G98R, Y157S, I170V, C271Y, and N274S.125 For signaling assay, we measured cAMP levels in cells transfected with MC4R and stimulated with the widely used superpotent analog of ‐MSH, [Nle4,d‐Phe7]‐‐MSH (NDP‐MSH). We showed that cells transfected with WT MC4R increased intracellular cAMP levels in a dose‐dependent manner, with an EC50 of 0.24 nM. Some mutant receptors, such as N62S, P78L, Y157S, and C271Y, have either decreased or absent signaling in response to NDP‐MSH stimulation. Ligand‐binding experiments showed that these mutants also have decreased or absent binding to iodinated NDP‐MSH. To investigate whether these mutant receptors are expressed on the cell surface, confocal microscopy studies were performed on cells stably trasnfected with MC4Rs. We showed that WT MC4R is expressed on the cell surface, although some intracellular expression is also observed. The mutant receptors that have decreased or absent binding were shown to have decreased or absent cell‐surface expression. When these cells were permeabilized, intracellular staining was observed suggesting that mutant receptors were expressed but retained intracellularly.125 From previous extensive studies in other mutated GPCRs that cause human disease, intracellular retention is the most common defect for loss‐of‐function phenotype. Prominent examples include rhodopsin mutations in retinitis pigmentosa,126,127 the V2 vasopressin receptor mutations in nephrogenic diabetes insipidus (reviewed in Ref. 128), the endothelin B receptor mutations in Hirschsprung’s disease,129 the calcium‐sensing receptor mutations in familial hypocalciuric hypercalcemia or neonatal severe hyperparathyroidism,130 the gonadotropin‐releasing hormone receptor mutations in idiopathic hypogonadotropic hypogonadism,131 the lutropin and follitropin receptors mutations in reproductive disorders (reviewed in Ref. 132) (extensively reviewed in Ref. 133). Most of the patients harboring MC4R mutations are heterozygous. Therefore obesity due to MC4R mutations can be caused either by haploinsufficiency or dominant negative activity exerted by the mutant receptor. Cotransfection studies showed that most of the mutants do not have dominant negative activity.62,72,115 The only mutations that have been shown to have dominant negative activities is the D90N mutation in which the most conserved Asp in TM2 was mutated to Asn76 and S136F.134 Therefore, obesity due to MC4R deficiency is likely caused by haploinsufficiency, similar to the Mc4r knockout mice. Biebermann and colleagues, using fluorescence resonance energy transfer, demonstrated that these two MC4R mutants heterodimerize with WT MC4R and suggested that this is why they have dominant negative activities.76,134 However, it is not clear why the other intracellularly retained MC4R mutants do not exert dominant negative activity, especially since all the cotransfection
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studies were done in transient transfections. Numerous studies in other GPCRs have shown that in transient transfections, intracellularly retained mutant receptors decrease cell‐surface expression of cotransfected WT receptors through heterodimerization (see Refs. 124, 135–137 for example). Dimerization of GPCRs is thought to occur universally in almost all GPCRs,138 including MCRs,139 and plays an important role in GPCR biosynthesis and maturation.138 Recently, we showed that with Y302F mutant, although no dominant negative activity was observed when the superpotent ligand NDP‐MSH was used, dominant negative activity was observed when the endogenous ligand ‐MSH was used,98 suggesting that caution is warranted when nonendogenous ligands are used. A further caution is that with signal amplification, assays measuring activities downstream of cAMP may also mask subtle dominant negative activity. Based on the facts that heterozygous MC4R mutation carriers are obese and the penetrance of MC4R mutations in causing obesity is not 100%, O’Rahilly and colleagues concluded that the mode of inheritance in MC4R deficiency is codominance with modulation of expressivity and penetrance of the phenotype.7
E. Molecular Classification of the MC4R Mutants To catalog the numerous MC4R mutations, we were the first to propose a classification scheme for MC4R mutations.125 This scheme is based on the life cycle of the receptor and modeled after the classification of mutations in low‐ density lipoprotein receptor and cystic fibrosis transmembrane conductance regulator140,141 (Fig. 3): Class I. Null mutations. Due to defective protein synthesis and/or accelerated protein degradation, receptor protein levels are decreased. Nonsense mutants W16X,69 Y35X,61,119 and L64X68 might belong to this class, although expression studies are needed to verify this prediction. We recently showed that R7C, C84R, and W174C belong to this class.142 Flow cytometry showed that they have decreased total expression levels. C84R and W174C are also defective in intracellular trafficking; their cell‐surface expression had more dramatic decreases.142 Class II. Intracellularly retained mutants. The mutant receptors are produced but are retained intracellualrly, most likely in the endoplasmic reticulum due to misfolding being detected by the cell’s quality control system. This class comprises the largest set of MC4R mutations reported to date, including the frameshift mutations DCTCT at codon 211,115 the TGAT insertion at codon 244,115 and D750–751GA,79 S58C,70,125 N62S,72,125 P78L,70,125,143 N97D,72
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FIG. 3. Molecular classification of naturally occurring MC4R mutations in early‐onset severe obesity. See texts for detailed description. Reprinted with permission from Ref. 148. Copyright Elsevier, 2005.
G98R,125 I102S,70 L106P,72 I125K,72 S127L,142 S136P,97 R165Q,143 R165W,70,143 L250Q,70 Y287X,72 C271Y,72,125 C271R,74 P299H,70 Y302F,98 I316S,72 I317T.70,144 Class III. Binding defective mutants. These mutant MC4Rs are expressed on the cell surface, but are defective in ligand binding per se, with either decreased binding capacity and/or affinity, resulting in impairments in hormone‐stimulated signaling. These mutants include I137T,60 N97D, L106P, I125K, I316S,72 and D88–92.75 I102S and I102T have partial defect in NDP‐ MSH binding.121 Since AgRP is the natural antagonist of the MC4R, if the mutants are more sensitive to inhibition by AgRP, the mutants are functionally defective. It was shown that I316S has altered relative affinities for ‐MSH and AgRP.72 V103I is less sensitive than WT MC4R to AgRP inhibition.116 Therefore, these mutants can be classified as a subclass within this class. Class IV. Signaling defective mutants. These mutant MC4Rs are expressed on the cell surface, bind ligand with normal affinity, but are defective in agonist‐stimulated signaling with decreased efficacy and/or potency. Mutants D90N,76 S136F,91,97,142 I137T,60 A175T, and V253I72 may belong to this class.
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Recently, Vaisse and colleagues suggested that decreased constitutive activities of some MC4R mutants might be the cause of the obesity in the patients harboring these mutant MC4Rs.145 Of nine mutants identified from patients with binge eating disorder and nonobese or obese subjects that we studied, five had decreased constitutive activities whereas the other four had normal basal activities.121 All three variants identified from obese Chinese subjects had normal constitutive activities.86 Further studies are needed to demonstrate unequivocally the physiological significance of the basal activity of MC4R. We suggest that mutants with defective basal signaling are also Class IV mutants. Class V. Variants with unknown defect. These variants behave similarly as the WT MC4R in heterologous expression systems in the parameters studied. These variants have normal cell‐surface expression, ligand binding, and signaling (basal and ligand stimulated). Whether and how these variants cause energy imbalance and therefore obesity is unclear. Since our initial proposal of the classification scheme described above, several other groups proposed modified or alternative classification schemes. For example, MacKenzie suggested that Class III and Class IV mutants could be combined.146 Vaisse and colleagues suggested that MC4R mutants can be classified as Class 1, intracellularly retained, Class 2, expressed on the cell surface but defective in both basal and ligand‐induced signaling (Class 2A), basal signaling (Class 2B) or ligand‐induced signaling (Class 2C).147 With this scheme, mutants with defective protein levels or ligand binding or variants with normal functions are not included. Farooqi and colleagues suggested another classification scheme: Class I mutants have defective expression, normal binding, and normal activation; Class II mutants have defective expression, normal binding, and defective activation; Class III mutants have defective expression, defective binding, and defective signaling; and Class IV mutants have normal expression and binding but impaired signaling.97 The reason for this proposal might be due to an imperfect replication of in vivo situation with the current heterologous expression systems used in all the laboratories performing functional studies of the naturally occurring mutants, namely the presence of spare receptors.148 Frequently, even though the expression levels are decreased, maximal signaling is not decreased. Heterozygous mutations of the MC4R cause obesity by haploinsufficiency as well as the intermediate phenotype of heterozygous Mc4r knockout mice would argue against the presence of spare receptors in the hypothalamus. Therefore there is likely no Class I mutants in vivo. The reason for observing Class II mutants is likely a technical one. The ligand‐ binding experiments performed in most laboratories are not saturation binding due to the high cost of radioligand. Therefore the so‐called maximal binding is an estimate. Farooqi’s Class III mutants correspond to our Class II mutants,
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and her Class IV mutants correspond to our Class IV mutants. Some mutants with normal expression but no ligand binding (therefore no signaling) are not included in this classification. Mutants that are normal in cell‐surface expression, ligand binding, and signaling are also not included. Our classification scheme is based on the life cycle of the receptors, from its biosynthesis to signaling, although we did not include mutants that affect processes downstream of receptor activation, such as desensitization, internalization, and resensitization. Defects in these processes can also cause dysfunctional receptor. A prominent example is a V2 vasopressin receptor mutant that is constitutively desensitized therefore demonstrating an apparent defect in signaling.149 Therefore, further refinement can be introduced. I suggested that the current system can also be used to classify mutations in other GPCRs that cause human diseases.133 Caution need to be exercised when extrapolating the data from in vitro experiments in heterologous cells such as HEK293 to the in vivo condition. In addition to the spare receptor phenomenon that was just mentioned, we need to remember that MC4R is expressed endogenously in neurons. The data obtained with heterologous non‐neuronal cells might not reveal any neuron‐ specific aspects of MC4R function.150 In summary, it is important to analyze the functional properties when new variant MC4Rs are identified in obese subjects in order to more accurately determine if the phenotype of the variant is indeed consistent with the clinical phenotype. A classification system such as one summarized here will be beneficial in cataloging the increasingly large array of MC4R mutations associated with severe childhood obesity. It is unfortunate that some of the recent reports did not study the cell‐surface expression and/or binding of the mutant receptors. Therefore from the data provided it is not possible to classify these mutants. If ever a personalized treatment is to be achieved, it is imperative to identify the exact defects.
F. Insights into the Structure and Function of the MC4R Naturally occurring mutations in GPCRs not only help us understand the etiologies of the associated diseases, these mutagenesis experiments of Mother Nature frequently provide important insights into the structure–function relationship of the GPCRs. Since most of the MC4R mutations associated with obesity are intracellularly retained, they cannot provide insights into the roles of the mutated residues in ligand binding and signaling. Misfolding mutations cause the receptors recognized by the cell’s quality control system therefore retained inside the cell, most likely in the endoplasmic reticulum. The majority of the mutations do not affect directly trafficking motifs.151 Indeed mutations that are retained intracellularly may occur in any part of the molecule, without any apparent pattern. One exception is the dileucine motif at the C terminus of
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MC4R.144 This motif, consisting of two hydrophobic residues such as leucine, isoleucine, valine, phenylalanine, or methionine preceded by an acidic residue, is highly conserved in GPCRs.152 Studies in several receptors, including V2 vasopressin receptor and lutropin receptor, showed that cell‐surface expression is impaired when the dileucine motif is mutated.152,153 MacKenzie and colleagues generated the naturally occurring mutation in this motif (I317T) as well as the corresponding mutation in I316. They showed that both mutants had decreased cell‐surface expression and there is additive effect in the double mutant.144 They suggested that this motif might be a necessary binding site for protein folding or a binding site for an adaptor protein involved in membrane trafficking. A subsequent study showed that in V2 vasopressin receptor, pharmacological chaperones could rescue these mutants suggesting that the mutants were misfolded, arguing against the suggestion that the motif is a transport signal.154 Similar experiments have not been done with the MC4R mutants. The few mutations that do result in a clear‐cut defect in either ligand binding or signaling are very interesting starting points for further mutagenesis experiments. The binding defective mutants are located in TM2, extracellular loop 1 and TM3, implicating this domain as important for ligand binding, consistent with previous site‐directed mutagenesis experiments.155,156 D90N is clearly defective in signaling (Ref. 76 and our unpublished observations). This mutation in the most highly conserved Asp in TM2 is interesting because previous site‐directed mutagenesis experiments in other GPCRs have shown that mutations of this conserved Asp frequently result in a defect in receptor‐G protein coupling.157–159 MC4R can be added to this list. We and two other labs showed that S136F is defective in signaling.91,97,142 This serine is 100% conserved in the MCR family members cloned so far (http://www.gpcr.org/7tm/classes/melanocortin/msa/) suggesting that it is very important functionally. To gain a better understanding of the role of this codon in receptor function, we generated seven additional mutants, including mutating S136 into hydrophobic amino acids such as alanine and leucine, polar amino acids such as threonine, tyrosine and cysteine, and charged amino acids such as aspartate and arginine. The results of these experiments showed that all seven mutants could signal in response to NDP‐MSH stimulation. Three mutants, including S136A, S136C, and S136R, had decreased maximal responses, and four mutants, including S136A, S136L, S136R, and S136Y, had significantly increased EC50s.142 Farooqi and colleagues showed that S136P is retained intracellularly with decreased maximal binding.97 The mutants we generated had normal maximal binding.142 Of interest is that in certain strains of pigs, the highly conserved Asp in MCRs in the N/DPxxY motif of TM7 is mutated to Asn.160 Earlier functional analyses suggested that pig D298N MC4R binds to NDP‐MSH normally, but is
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devoid of NDP‐MSH‐stimulated cAMP production.161 We were surprised by these data, because the majority of Family A GPCRs has Asn at this position. MCRs are some of the few Family A (rhodopsin‐like) GPCRs that have DPxxY sequence rather than NPxxY sequence. We reasoned that Asn in the MC4R should be able to confer normal signaling. Therefore we sought to replicate the studies by Kim and colleagues.161 Our data showed that indeed in both human and pig MC4Rs, D298N mutants have normal binding and signaling to NDP‐ MSH; the porcine MC4R D298N also has normal binding to AgRP and ‐ MSH and signals normally to ‐MSH stimulation with the same EC50 and maximal response as the WT receptor.150 The discrepancy between our data and those of Kim et al. might be due to the extremely low maximal signaling in their study. Another group independently reported that human MC4R D298N has normal cAMP response to NDP‐MSH stimulation.162 Several genotype– phenotype association studies on this polymorphism have been published since the original report.160 Divergent conclusions, with some studies supporting the original association163–166 while some studies failed to find an association,167–169 were reached, raising doubts about the original claim that this variant is associated with fatness, growth, and feed intake traits.
VI. Potential Therapeutic Strategies Previously, I suggested that depending on the defect, GPCR including MC4R mutants might be corrected via several approaches.133 Class I mutants due to nonsense mutations can potentially be corrected by aminoglycoside antibiotics. These compounds decrease the codon–anticodon fidelity, resulting in read‐through of the premature stop codon. Class III and Class IV mutants, those mutants that are expressed on the cell surface but could not bind the endogenous ligands or respond to these ligands, could potentially be corrected by synthetic ligands. These synthetic ligands might bind to residues different from endogenous ligands therefore activate some of the mutant receptors.148,170 Haskell‐Luevano and colleagues provided data demonstrating that some of the synthetic ligands, including peptides and small molecules, can indeed stimulate MC4R mutants that are impaired in responding to endogenous ligands.170 Since some of the MC4R mutants have residual activity in terms of hormone‐stimulated cAMP generation, approaches that result in their increased expression on the cell surface could potentially be of therapeutic value. Some of the mutations in V2 vasopressin receptor that cause nephrogenic diabetes insipidus also result in the mutant receptor trapped intracellularly.128 Recent studies from Bouvier and colleagues have identified small molecules of vasopressin analogs that can cross the cell membrane and act as
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pharmacological chaperones, increasing the cell‐surface expression of the mutant V2 vasopressin receptors.171 Similar results were achieved recently in naturally occurring mutations in gonadotropin‐releasing hormone receptor172,173 and the prototypical GPCR, rhodopsin.174,175 In addition, WT or laboratory‐generated mutants in several other GPCRs can also be corrected by pharmacological chaperones.176–182 The clinical utility of pharmacological chaperones was shown by a recent clinical trial. In this study, it was shown that treatment of patients with nephrogenic diabetes insipidus harboring transport‐ defective V2 vasopressin receptor mutations with the nonpeptide antagonist SR49059 decreased urine volume and water intake.183 We recently showed that a small molecule MC4R antagonist ML00253764 was able to rescue two MC4R mutants that are retained intracellularly, C84R and W174C.142 ML00253764, [2‐{2‐[2‐(5‐bromo‐2‐methoxyphenyl)‐ethyl]‐3‐ fluorophenyl}‐4,5‐dihydro‐1H‐imidazolium hydrochloride], with a molecular weight of 377.3, was originally described by Vos and colleagues.184 It was shown to be a MC4R inverse agonist in a subsequent study.185 The compound can cross the blood–brain barrier. We hypothesized that it might act as a pharmacological chaperone correcting the misfolding of intracellularly retained mutants. To test this hypothesis, we established stable cell lines expressing WT, C84R or W174C MC4Rs. Cells transfected with the empty vector pcDNA3.1 were used as a negative control. Cells were treated with 105 M ML00253764 for 24 h. Cell‐surface expression of the receptors was investigated with confocal microscopy and flow cytometry. The cell‐surface expressions of both mutants were increased to about 35% of that of the untreated WT MC4R. Interestingly, the cell‐surface expression of the WT MC4R was also increased to 152% after 24 h treatment with ML00253764, suggesting that WT MC4R maturation is not very efficient. Importantly, when the cells were stimulated with the superpotent ‐MSH analog, NDP‐MSH, the mutant receptors had increased cAMP production, up to 80% of the signaling generated by the untreated WT MC4R (Fig. 4).142 These data suggest that small molecule MC4R ligands could act as pharmacological chaperones assisting the trafficking of intracellularly retained mutant receptors. They could potentially be used to treat patients harboring these MC4R mutations. Since they could also increase the cell‐surface expression of the WT MC4R, they might also be used to treat common obesity when the patients do not have MC4R mutations.
VII. Conclusions Obesity is a multifactorial disease with genetic, behavioral, and environmental components. Although numerous quantitative trait loci and genetic variants have been found to predispose to obesity,186 the MC4R is one of the
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FIG. 4. Pharmacological rescue of two intracellularly retained MC4R mutants, C84R and W174C, by a small molecule MC4R antagonist, ML00253764. Human embryonic kidney 293 cells stably transfected with WT, C84R, or W174C MC4R or empty vector pcDNA3.1 (as a negative control) were incubated with 10 mM ML00253764 for 24 h at 37 C. Cells were then stained with anti‐myc monoclonal antibody to visualize the c‐myc epitope at the N terminus of the MC4R (A) or sorted with flow cytometry (B). To investigate whether the rescued mutants are functional, the cells were stimulated with 106 NDP‐MSH for 1 h, and intracellular cAMP levels measured (C). The cAMP levels in cells expressing WT MC4R and treated with NDP‐MSH were set as 100%. Star ($) indicates significant difference from corresponding vehicle treated controls ( p < 0.05). Reprinted with permission from Ref. 142. Copyright Blackwell Publishing Limited, 2009.
loci that have been undisputedly proved to be important for human obesity pathogenesis. Detailed studies on the defects of the mutants and identifying ways to correct these defects represent important steps toward personalized medicine. Pharmacological chaperones and novel ligands are promising approaches.
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194 Acknowledgments
My own studies on the MC4R were supported by American Heart Association (0265236Z), National Institutes of Health (R15DK077213), Diabetes Action Research and Education Foundation, Alabama Agricultural Experiment Station, Animal Health and Disease Research Program of Auburn University College of Veterinary Medicine, and startup funds from Auburn University. I thank Dr. Zhen‐Chuan Fan for contributing to the studies on the pharmacological chaperones.
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Index
A AAAS gene, 164–165 Achalasia, 163 ACTH. See Adrenocorticotropin hormone ACTH stimulation test, 159 Activated microglia, 20 Adaptor protein (AP-3) complex, 91 Adrenal cortex, 112, 156, 158, 164, 175 Adrenal insufficiency, 98 Adrenocorticotrophin resistance syndromes, 156, 161 Adrenocorticotropin hormone, 97, 156, 175 Adrenoleukodystrophy, 159 Agouti (Ay) mice, 179 Agouti-related peptide (AgRP), 175 Agouti signaling protein (ASP), 101–102 AgRP expression, 179 AgRP gene, 176 Alacrima, 163 Alacrima/achalasia/adrenal insufficiency/neurologic disorder, 164 function of, 164 mutations, 165 relation between AAAS gene mutations, 165 ALADIN. See Alacrima/achalasia/adrenal insufficiency/neurologic disorder Allgrove syndrome. See Triple A syndrome Angular cheilitis, 164 Anterior pituitary hypoplasia (APH), 58, 72–73 Arrestin knockout mice, 18 AT-1 angiotensin receptor, 159 Ataxia, 163 Autonomic nervous system, 163 Autosomal dominant retinitis pigmentosas (ADRPs), 3
B Bacterial artificial chromosome (BAC), 101 Basic fibroblast growth factor (bFGF), 120
Blonde hair color, 109 Bone tumors, 65 BRAF and NRAS oncogenes, 126 BRAF mutant melanoma, 125–126 BRAF mutations, 124 Breast cancer lines, 65
C Calcium-sensing receptor mutations, 185 Calcium signaling, 100–101 cAMP. See Cyclic adenosine monophosphate cAMP-response element binding protein (CREB), 64 cAMP-responsive element (CRE), 157–158 Cardiovascular disease, 174 CDKN2A gene, 125 CDKN2A mutations, 125 Central nervous system, 20, 49, 156, 179 Central precocious puberty (CPP), 49–50 Chemical chaperones, 129 CHO cells, 162. See also Familial glucocorticoid deficiency Circular dichroism (CD), 10 CNS. See Central nervous system Cone cell degeneration, 13 metabolic support, 21–22 toxic factors, 20–21 trophic factors, 18–20 Cone photoreceptors, 2, 18, 22 Convulsion, 158 C-reactive protein, 72 Cre-lox technology, 179 Cyclic adenosine monophosphate, 58, 70 and Ca2þ ions concentration, 61 heterologous cells transfected with MC1R exhibiting, 119 hormone-stimulated, 191 increased intracellular cAMP levels, 184 kisspeptin modifying basal/forskolininduced, 39
205
206
index
Cyclic adenosine monophosphate (cont.) lit mutation and, 66 MC1R-mediated signaling, 98–100 MC2R signals and, 157 measurement, 184 pathways, for MC2R signals, 157 PRL gene transcription and, 71 in P347S retinas, 15 receptor activation, 175 reduced function, 105 responses, of RHC variants, 106 responsive luciferase reporter assay, 161 signaling cascade, 98 Cyclic nucleotide gated (CNG) channels, 6–7 Cyclobutane pyrimidine dimers (CPD), 96, 125
D DCT. See DOPAchrome tautomerase DCT gene, 91 -Defensin 3, 102 D294H variant receptor, 111 5,6-Dihydroxyindole-2-carboxylic acid (DHICA), 94 DNA damage, 89, 96–98, 124–125, 130. See also Skin cancer; UV-induced DNA damage D90N mutation, 185 DOPAchrome, 91 DOPAchrome tautomerase, 90–92, 94, 98–99, 111 Drosophila model, expressing Rh1P37H, 11–12 Dwarfism, 72 Dysarthria, 163 Dyslipidemia, 174
E Eating disorder, 184 Ectopia of posterior pituitary (PPE), 58 Electroretinogram (ERG), 2 Endoplasmic reticulum (ER), 4 Endothelin-1 (EDN1), 120 Energy homeostasis, 113, 156, 175, 179, 183 Ephelides. See Freckles Estrogen receptor element (ERE), 64 Eumelanin, 89–91, 94, 98–99, 101–103, 119, 124, 175
Exocytosis, 88 Extracellular signal-regulated kinase (ERK), 101
F Familial glucocorticoid deficiency, 156 clinical features, 158–159 diagnosis, 159 etiology, 160–162 MC2R missense and nonsense mutations, 160 MRAP gene and mutations, 163 Fat/fat mice, 177 Faulty GHRHR alleles, 70 FGD. See Familial glucocorticoid deficiency Flow cytometry, 184 Follicle stimulating hormone (FSH), 35 Fovea, 2 Freckles, 126
G Ganglion cell layer (GCL), 4 Gastroenteropancreatic carcinomas, 65 Gene expression arrays, 21 Gene therapy, 22–25 GH-releasing peptide (GHRP), 62 GH replacement therapy, 71–72 GHRH antagonists, 65 GHRH desensitization, 62 GHRHR biology, 60 and ligand, 60 cascade of events, 61 mutations, 66–70 (see also GHRHR mutations) organization and regulation, 64–65 polymorphisms, 70 signal transduction, 61–62 splice variants detected in, 65 responsive to GHRH antagonists, 65 structure, 62–63 alignment of amino acid sequences, 63 and localization of mutations, 69 GHRHR mutations, 60, 68 diseases associated with heterozygous carriers, 73–74 homozygous individuals, 70–73
207
index GHRH-secreting tumors, 62 GHRH stimulation, 62 GH therapy, 72 Glioblastoma, 65 Glucocorticoid deficiency, 163 Glucocorticoid response element (GRE), 64 Glycosylation, 26 GnRH/GnRH-receptor system, 42 GnRH neurons, 35 Gonadotropin-releasing hormone (GnRH), 35 analog therapy, 71 Gonadotropins, 178 GPCR mutants, 128 GPCRs. See G protein-coupled receptors GPR54/kisspeptin signaling, 40 gpr54 knockout mice, 44 G protein, 6, 45, 62 coupling defective, 110 coupling/recognition, 116–117 uncoupling, 116 G protein-coupled receptor 54 (GPR54). See also Kisspeptin and cancer, 40–41 endogenous ligand, 37–38 and KISS1 genes in evolution, 38–39 loss-of-function mutations, 44–49 role, 49–51 signaling pathway, 39–40 structure and expression, 35–37 G protein-coupled receptor kinase (GRK), 62 G protein-coupled receptors, 3, 36, 54, 97, 156, 175 activation and inactivation, 45 binding of endogenous ligand, 34 carboxyl-terminal tail, 51 loss-of-function mutations, 34 MRAP, accessory protein, 157 of rod photoreceptor cells, 3 role in body homeostasis and, 34 signaling, 34 structural abnormalities, of genes encoding, 34 transmembrane regions, 35 Grafted E48 pig retinas, 27 Griscelli syndrome, 88 Growth hormone (GH), 58
H Hermansky–Pudlak syndrome type 2 (HPS-2), 91 Homeostasis model assessment of insulin resistance (HOMAIR), 73–74 Human agouti hair pattern, 101 analog of little mouse, 66 associated with SILV mutations, 94 chromosome 21q22.1, 158 disease in Aaas-/-mice, 164 diseases associated with SILV mutations, 94 GHRH receptor (GHRHR) gene, 60 GPR54 gene, 36–37 GPR54 inactivating mutations, 47 GPR54 receptor, 36 injection of MSH, 98 kisspeptin-10, 10 MC1R polymorphism, 104, 114 monogenic obesity, 156 MRAP gene, 158 -MSH for energy homeostasis in, 178 mutations in GH gene (GH1), 60 POMC gene, 177 RAB27A, MYO5A, or MLPH genes, 88 TYR gene, 91 pigmentation genes, 92–93 variation, 103 rhodopsin P23H, 11–12 role ASIP, 102 17-Hydroxiprogesterone, 159 21-Hydroxylase deficiency, 159 Hyperparathyroidism, 185 Hyperphagia, 183 Hyperpigmentation, 158, 163 Hypertension, 174 Hypoglycemia, 158
I IGF-1 binding protein-3 (IGFBP-3), 71 Immunocytochemistry, 184 Inflammation, and MC1R, 127 Inner nuclear layer (INL), 4 Insulin-like growth factor-1 (IGF-1), 58, 74 IRF4 gene, 95
208
index
IRF4 protein, 95 Isolated growth hormone deficiency (IGHD), 58 categories, 59 GH1 mutations prevalence, 60
J Jaundice, 158
L LDL-cholesterol, 72 LEP gene, 177 LEPR gene, 177 Leptin (LEP) gene, mutations, 177 Leptin-regulated melanocortin circuit, 175–178 L144H mutation, 67 Linkage analysis, of FGD, 161 little (lit/lit) mouse, 66 Luteinizing hormone (LH), 35
K K296E-mediated degeneration, 18 K296E mutant rhodopsin, 16–17 persistent photosignaling, 17 rhodopsin–arrestin complex formation, 18 KISS1 gene, 37, 39 expression after gonadectomy and, 43 in nonmetastatic melanoma cells, 40 in placenta, 43 genes in evolution, 38–39 mapping, 38 overexpression to demonstrate kisspeptin secretion, 41 in fibrosarcoma, 40 into human metastatic breast cancer cell line, 41 products, 37–38 KISS2 gene, 39 KiSS1/GPR54 complex, 49. See also Kisspeptin kiss1 knockout mice, 44 Kisspeptin, 35, 37–46, 48, 50–51 activation of GPR54 receptor, 39 affinity for, 48 binding to GPR54, 41 effects on LH, 42, 51 excitatory neuroregulator system, 35 and GPR54 coexpression, 44 immunoreactivity, 43 induced GnRH secretion, 39 mammalian, 39 MAPK pathway activation for, 39 secretion required, 41 stimulation, 39, 51 Kisspeptin/GPR54 system, 41 KITLG (KIT ligand) gene, 96 KIT receptor signaling, 96
M Mammalian target of rapamycin (mTOR) pathway, 21 MAPK/ERK pathway, 101 MATP protein, 95 MC1R. See also MC1R gene; MC1R motifs; MC1R signaling and BRAF mutant melanoma, 125–126 and CDKN2A familial melanoma, 125 cell-surface fluorescence, 105 and DNA repair, 125 and melanocytic morphology, 127 and nonpigmentary functions, 127–128 inflammation, 127 multiple sclerosis, 127 pain sensitivity, 128 vitiligo, 128 therapeutic applications, 128–130 chaperone-based drugs, 130 MC2R. See Melanocortin-2 receptor MC4R. See Melanocortin-4 receptor MC3/4R agonist melanotan II, 178 MC4R agonist melanotan II, 179 MC3/4R antagonist SHU 9119, 178 Mc1r deficient mice, 101 Mc1re recessive yellow mice (Mc1r null), 109 MC1R gene, 103 discovery, 97 isolation of full-length, 97 polymorphism, in Asian population, 104 and red hair phenotype, 103 and population allele frequencies, 103–104 role in regulation of pigmentation, 97–98 agouti signaling protein, 101–102
index -defensin 3, 102 calcium signaling, 100–101 MAPK/ERK pathway, 101 MC1R-mediated cAMP signaling, 98–99 MC1R signaling pathways, alternative, 99–100 pheomelanin to eumelanin switch, 99 tanning, 98 and skin cancer risk (see also Skin cancer) MC1R variant alleles and phenotypes, 123 structure, activity, and regulation (see MC1R motifs) variant alleles (see MC1R variant allele) MC4R genotype–sex interaction, 182 Mc4r knockout mice, 185 MC1R-mediated cAMP signaling, 98–99 MC2R missense, and nonsense mutations, 160. See also Missense mutation MC1R motifs constitutive activity, 119 desensitization and internalization, 118 dimerization and dominant negative effects, 119–120 E/DRY motif, 115–116 G-protein coupling, 117 ligand binding, 117–118 N/DPXXY motif, 116–117 phosphorylation sites, 118–119 recycling and endosomal signaling, 118 regulation of gene expression, 120 RXR motif, 117 MC4R mutants. See also MC4R mutations functional characterizations, 184–186 molecular classification, 186–189 binding defective mutants, 187 intracellularly retained mutants, 186–187 null mutations, 186 signaling defective mutants, 187–188 variants with unknown defect, 188 receptors, 114 MC2R mutations, 114 MC4R mutations clinical phenotypes of patients harboring, 182–184 identification, 179–181 and obesity, 156 prevalence, 181–182 in Asians, 182 obese German children and adolescents, 181
209 obese North Americans, 182 structure and function, associated with, 189–191 binding defective mutants, 190 dileucine motif, mutation, 190 D90N and S136F, defective in signaling, 190 human MC4R D298N, 191 misfolding mutations causing, 189 therapeutic strategies, 191–192 MC1R RHC alleles, 126 MCRs. See Melanocortin receptors MC1R signaling, 96, 99–100, 103 pathways, alternative, 99–100 MC2R signaling, 157, 177 MC1R variant allele categories, 110 altered internalization, 111 altered ligand binding, 110 function in coculture, 111 G-protein coupling defective, 110 intracellularly retained, 110 null alleles, 111 pseudo-wild type, 110 SNPs in melanocortin receptor family, 111–114 function, 104–110 association of V92M with, 107 cAMP signaling responses, 106–107 cell-surface expression, 106 D294H cell-surface localization, 106 dominant negative activity on, 109 functional/signaling ability, 106 heterozygote effect on, 109 residual functional activity, 109 surface levels, quantification, 105 in vitro functional ability, 105 impact on MC1R expression, maturation, and, 112 and skin cancer phenotypes, 123 Mc1r with point mutations, 97 Melanin function, 89, 96 types, 89 Melanocortin hormones, 97 Melanocortin-2 receptor, 156–157 activation by ACTH, 157 signaling, 157–158 Melanocortin-4 receptor and energy homeostasis, 178–179
210 Melanocortin-4 receptor (cont.) structure and function, 189–191 Melanocortin-2 receptor accessory protein, 158 Melanocortin receptors, 99, 111–114, 175 Melanocortin receptors 1–5 (MC1R–MC5R), 175 Melanocortin system, 174–175 Melanocyte, 87–89 apoptosis, 125 and tanning response, 88 Melanocyte stimulating hormone, 157, 174 -Melanocyte stimulating hormone, 97–98, 126–127 Melanogenic enzymes, and pigmentation genes KIT and KITLG, 96 OCA2/P-protein, 94 Pmel17, 94 transcription factors, 95 transporter proteins, 95 TYR and TYR-related proteins, 91–94 Melanomas, of RHC homozygotes, 127 Melanosome, 88–91 maturation—stages I–IV, 90 Membrane-associated transporter protein (MATP), 95 Mendelian inheritance patterns, 86 Metastasis suppressor gene, 37 Microarray analysis, of mouse melanocytes, 102 Microcephaly, 164 Microglia, 20 Micropthalmia-associated transcription factor (MITF), 95–96 Mineralocorticoid replacement, 163 Mislocalization, of GPCR mutants, 128 Missense mutation, 57, 63, 66–68, 160–161 MITF gene, mutations, 95 Mitogen-activated protein kinases (MAPK), 39 Monogenic obesity, in humans. See Leptinregulated melanocortin circuit Morbid obesity, 174 Motor neuropathy, 163 Mouse agouti, 179 Mouse extension locus, 97 Mouse 3T3-L1 cells, 158 MRAP. See Melanocortin-2 receptor accessory protein MRAP gene and mutations, in FGD type 2 patients, 163 MRAP gene, human, 158, 163 MSH. See Melanocyte stimulating hormone
index -MSH. See -Melanocyte stimulating hormone MSH-mediated MC1R signaling, 125 -MSH, role in energy homeostasis, 178 Multiple sclerosis (MS), and MC1R, 128 Murine Mc1r, 97 Mutant rhodopsins, 7–8 Mutation (E72X), 66–68
N NCKX5 protein, 95 NDP-MSH stimulation, 185 Neuropeptide Y, 176 Nevi, 126 NF-kB activation, 127 Nitric oxide (NO), 73 non-Hodgkin’s lymphomas, 65 Nuclear factor 1 (NF-1), 64
O Obesity, 173–174. See also MC4R mutations causes, 174 importance, 174 multiple components in circuit cause, 176 mutations in POMC gene and, 177–178 in United States, 174 Ob/ob and db/db mice, 177 OCA3 in humans, 91 OCA4 in humans, 95 OCA2/P-protein, 94 Ocular melanocytes, 89, 97 Oculocutaneous albinism type 1 (OCA1), 91 Olfactory abnormalities, 35 Optic atrophy, 163 Osteoporosis, 164 Outer nuclear layer (ONL), 4 Outer plexiform layer (OPL), 4 Oxidative damage, 21 Oxidative stress, in retinal degeneration, 21 Oxygen levels, in outer retina, 20
P Pain sensitivity, MC1R role, 128 Palmo-plantar hyperkeratosis, 164 Pancreatic cancer, 65 PCSKI gene, 178
211
index Pearl and Mocha mouse strains, 91 PEST sequence, 38 Phagocytosis, 88 Pharmacological chaperones, 129 Pharmacoperones, 129 P23H difference spectra, 10 Pheomelanin, 89, 91, 99 Pheomelanin:eumelanin ratio, 99 P23H mutant. See also P23H rhodopsin mutant rhodopsin in OPL, 12 rhodopsin, in vitro expression, 9 rhodopsin localization in vivo, 12 Photopsia, 2 Photoreceptors, 3, 18, 21, 23–24. See also Cone photoreceptors; Rod photoreceptors Photosensitizer, 89 Phototransduction cascade, 6 P23H rhodopsin mutant misfolding, 9–11 mislocalization, 11–12 retinas, 19 P23H/VPP mutant rhodopsin mouse, 21 Piebaldism, 96 Pigmentation genes, 91–94 Pit-1 transcription factor, 64, 69 Pituitary hormone deficiency, 58 Pituitary stalk agenesis (PSA), 58 P347L mutant rhodopsin, transgenic pig retinas, 21 p38 MAPK signaling, 101 Pmel17 protein, 94 Point mutations associated with ADRP, 8 (see also Rhodopsin protein) of proline at residue 347 in rhodopsin, 12 Polymorphism, 111, 181 in melanocortin-1 receptor (MC1R) gene, 87 POMC, human, 175 POMC mutations, 177 Progastrin, 178 Proglucagon, 178 Progondotropin-releasing hormone, 178 Prohormone convertases 1 and 2 (PC1 and PC2), 175 Proinsulin, 178 Proline at residue 23 (P23), 9 Proopiomelanocortin (POMC), 97, 175 Prostate cancers, 65 Protein kinase A (PKA), 98, 157 P347S mutant rhodopsin, 12–13
cell sorting, 14 nonautonomous degeneration, 13 rhodopsin stability and signaling, 15–16 P347S protein stability, 16 Pyrimidine (6–4) pyrimidone photoproducts (64PP), 96
R Ras/Raf/ MEK/ERK pathway, 101 Reactive oxygen species (ROS), 89, 126 Red hair phenotype, 103 Renal cell carcinomas, 65 Retinal pigment epithelial (RPE), 3–4, 21–22 Retinal transplantation, 26–27 Retina structure, 3–5 Retinitis pigmentosa (RP), 2, 185 R307G polymorphism, 111 R142H and D294H variant alleles, 110 RHC phenotype, 87, 103 Rhodopsin–arrestin complex formation, 18 Rhodopsin function, 3 Rhodopsin-mediated ADRP, treatment, 22 gene therapy, 22 adenovirus expressing, apoptosis inhibitor, 24 use of ribozymes to treat, 23–24 using recombinant adeno-associated virus, 23 pharmacological chaperones application, 26 retinal transplantation, 26–27 supplementation with vitamin A, 25–26 Rhodopsin mutants. See P23H rhodopsin mutant Rhodopsin mutations, 3, 185 classification, 7–9 Rhodopsin protein, 3 secondary structure, 8 stability, and signaling, 15–16 Rod cell-specific expression, 5–7 CNG channels, role, 7 conformational change of rhodopsin (R*), 5–6 inactivation of rhodopsin, 7 lowering intracellular levels, of cGMP, 7 transducin activation, 6 visual arrestin, role, 7 Rod degeneration, mechanisms, 9 Rod-derived cone viability factor (RdCVF), 19
212
index
Rod-derived cone viability factor-2 (RdCVF2), 19 Rod photoreceptors, 2, 5, 20–21 R163Q allele, 104 R163Q r variants, 113
S 5-S-CysteinylDOPA, 91 Seizures, 163 Sensory impairment, 163 Serum prolactin (PRL) level, 71 Severe combined immunodeficiency (SCID) syndrome, 25 Short stature, 164 SILV gene, 94 Single nucleotide polymorphisms (SNPs), 87, 96, 104, 181 Skin cancer, 96 DNA damage and, 96 environmental cause, 96 haplotypes near ASIP and, 102 MC1R RHC variant alleles, 124 MC1R targeting drugs and, 129 MC1R variant alleles with, 104, 121–122 photoaging and, 97 related mortality, 87 risk, in Caucasian population, 86 V92M with, 107 Skin color. See Melanin; Melanosome Skin pigmentation, and photosensitivity, 89 SL24A4 gene, 95 Small cell lung carcinomas, 65 Somatostatin, 58 Steroidogenic acute regulatory protein (StAR), 156
Triple A syndrome, 156 clinical features, 161, 163–164 etiology of, 164–165 Type II diabetes mellitus, 174 TYR enzyme, 91 Tyrosinase related protein-1 (TYRP1), 90 Tyrosinase (TYR), 91 TYR protein, role in melanin production, 91, 94
U Ubiquitin pathway, 11 Ultraviolet radiation (UVR)-induced skin cancer. See also Skin cancer and human pigmentation phenotype, link between, 86 and MC1R, 97 UVR-induced DNA damage, 96–97 Unfolded protein response (UPR), 9 Upstream stimulatory factor (USF), 64 UV-induced DNA damage, 87, 89 UV-induced skin cancer, 87 UV-irradiated melanocytes, 101 UVR-induced melanogenesis. See Tanning
V V103I MC4R polymorphism, 183 Visual arrestin, 7 Visual dysfunction, 2 Vitamin D, 97 Vitiligo, and MC1R, 128 V60L variant receptor, 109 V92M allele, 107 V92M receptor, 110 V2 vasopressin receptor mutations, 185
W
T Tanning, 98 Teleost kisspeptin-1, 39 Thyroxine (T4) replacement therapy, 64 Tietz syndrome, 95 Toxic factors, from degenerating rods cells, 20 TPCN2 gene, 95 Transcription factor Pit-1 (Pou1f1), 64 Transducin, 6 Trans-Golgi network (TGN), 90 Transporter proteins, 95 Triiodothyronine (T3), 64
Waardenburg syndrome type 2, 95 WD-repeat domains, 164
X Xerostomia, 164 X-linked inhibitor of apoptosis (XIAP), 24
Y Y302F mutant, 186
