Leptin and Leptin Antagonists

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MEDICAL INTELLIGENCE UNIT

MEDICA L I N T E L L I G E N C E UNIT

The chapters in this book, as well as the chapters of all of the five Intelligence Unit series, are available at our website.

Biotechnology Intelligence Unit Medical Intelligence Unit Molecular Biology Intelligence Unit Neuroscience Intelligence Unit Tissue Engineering Intelligence Unit

GERTLER

INTELLIGENCE UNITS

Arieh Gertler

MIU

Leptin and Leptin Antagonists


Medical Intelligence Unit

Leptin and Leptin Antagonists Arieh Gertler, PhD

Institute of Biochemistry, Food Science and Nutrition The Hebrew University of Jerusalem Rehovot, Israel

Landes Bioscience Austin, Texas USA

Leptin and Leptin Antagonists Medical Intelligence Unit Landes Bioscience Copyright ©2009 Landes Bioscience All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the USA. Please address all inquiries to the publisher: Landes Bioscience, 1002 West Avenue, Austin, Texas 78701, USA Phone: 512/ 637 6050; Fax: 512/ 637 6079 www.landesbioscience.com ISBN: 978-1-58706-320-6 While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Leptin and leptin antagonists / [edited by] Arieh Gertler. p. ; cm. -- (Medical intelligence unit) Includes bibliographical references and index. ISBN 978-1-58706-320-6 I. Gertler, Arieh. II. Series: Medical intelligence unit (Unnumbered : 2003) [DNLM: 1. Leptin. 2. Leptin--Antagonists. 3. Leptin--antagonists & inhibitors. 4. Receptors, Leptin. WK 185 L6113 2009] QP572.L48L467 2009 572'.633--dc22 2008051305

Dedication I would like to dedicate this book to my beloved wife Anna who always encouraged me along the way.

About the Editor...

ARIEH GERTLER is a Professor-Emeritus in Agricultural Biochemistry, holder of the Karl Bach Chair in Agricultural Biochemistry. His main interests include the physiological and molecular action of several cytokines such as growth hormone, prolactin and placental lactogen. In the last decade his main research effort was in the field of leptin and culminated in development of leptin mutants devoid of agonistic activity but retaining their full capacity of binding to leptin receptors and just acting as potent competitive leptin antagonists. Upon his retirement in 2003 Arieh Gertler founded a small biotech company (Protein Laboratories Rehovot) which produces recombinant proteins for research purposes. Arieh Gertler received all his academic degrees (BSc, MSc and PhD) in the Hebrew University of Jerusalem, Israel.

CONTENTS Preface......................................................................................................... xv

Part I: Molecular Aspects of Leptin Action 1. Leptin Signal Transduction—A 2009 Update .............................................1 Walter Becker Leptin and the Leptin Receptor.............................................................................1 JAK Kinase .................................................................................................................2 Activation of STAT3 ................................................................................................3 Leptin-Regulated Genes ..........................................................................................4 Activation of Other STAT Factors .......................................................................4 Activation of the ERK Pathway .............................................................................7 Activation of the IRS/PI3K/PDE3B Pathway...................................................8 Activation of ATP-Sensitive K+ Channels ..........................................................8 Regulation of AMPK (AMP-Activated Protein Kinase) and mTOR (Mammalian Target of Rapamycin) ..........................................9 Perspective...................................................................................................................9 2. Insights in the Activated LR Complex and the Rational Design of Antagonists ................................................................................15 Frank Peelman, Lennart Zabeau and Jan Tavernier Leptin as a Disease-Promoting Factor: Rationale for Leptin Antagonists ......................................................................................16 Structure of Leptin and Its Receptor Homology with the IL-6 and G-CSF Receptor Systems..........................................................................16 Evidence for Receptor Oligomerisation and Higher Order Clustering ................................................................................................17 Three Binding Sites in Leptin...............................................................................18 Models of the CRH2-Leptin Complex............................................................. 20 Models for the Ig-Like and CRH1 Domains....................................................21 Homology Model for a Hexameric 2:4 Leptin:LR Complex....................... 22 Homology Models for the Fibronectin Type III Domains........................... 22 Mechanism of LR Activation .............................................................................. 24 Development of Leptin-Based Antagonists ......................................................25 Optimization of Leptin-Based Antagonists..................................................... 26 Concluding Remarks ............................................................................................. 26 3. Study of Leptin: Leptin Receptor Interaction by FRET and BRET .........30 Julie Dam, Cyril Couturier, Patty Chen and Ralf Jockers Activation Mechanism of OB-R Studied with Biochemical Methods .......31 Methodological Introduction to FRET/BRET ...............................................33 Activation Mechanism of OB-R Monitored by FRET and BRET ............ 34 The OB-R BRET Assay, a Screening Tool for the Identification of New OB-R Ligands ......................................................................................39 Conclusion ................................................................................................................39

Part II: Leptin Involvement in Physiological and Pathological Processes 4. Is Leptin a Pro- or Anti-Apoptotic Agent? ................................................43 Srujana Rayalam, Mary Anne Della-Fera, Suresh Ambati and Clifton A. Baile Apoptosis: A Basic Biologic Phenomenon ........................................................ 44 Anti-Apoptotic Effects of Leptin ........................................................................45 Pro-Apoptotic Effects of Leptin ..........................................................................47 Conclusions ............................................................................................................. 49 5. Leptin Actions in the Gastrointestinal Tract .............................................54 Sandra Guilmeau, Thomas Aparicio, Robert Ducroc and André Bado Gastric Leptin Directly Activates Vagal Afferent Neurons...........................55 Leptin and Intestinal Physiology .........................................................................55 Leptin in Gastrointestinal Pathologies ............................................................. 58 Conclusions and Perspectives ...............................................................................59 6. Leptin as a Novel Marker in Breast and Colorectal Cancer .......................63 Eva Surmacz and Mariusz Koda Leptin and Breast Cancer ......................................................................................63 Leptin and Colorectal Cancer............................................................................. 68 Summary and Perspectives ....................................................................................69 7. The Role of Leptin in Cardiac Physiology and Pathophysiology ..............73 Morris Karmazyn, Daniel M. Purdham, Venkatesh Rajapurohitam and Asad Zeidan Leptin Synthesis and Structure ............................................................................74 Leptin Resistance ....................................................................................................74 Is Leptin a Possible Link between Obesity and Increased Cardiovascular Risk? .........................................................................................74 Expression of Leptin Receptors in Cardiovascular Tissues ...........................75 Effect of Leptin on Cardiomyocyte Function ..................................................76 Cardiomyocyte Hypertrophic Effects of Leptin ..............................................76 Leptin as a Cardioprotective Agent ................................................................... 77 Post Receptor Leptin Signaling .......................................................................... 77 Conclusions: Potential of Leptin Modulators as Therapeutic Agents ........78 8. The Role of Leptin in Bone Development and Growth .............................83 Efrat Monsonego Ornan and Michal Ben-Ami The Effect of Leptin on the Skeleton ..................................................................83 Leptin and Growth .................................................................................................85 Central Effect of Leptin.........................................................................................85 Peripheral/Direct Effect of Leptin ..................................................................... 86 Synopsis .................................................................................................................... 88

9. Involvement of Leptin in Arterial Hypertension .......................................91 Jerzy Beltowski Physiological Effects of Leptin Relevant for Blood Pressure Regulation...... 92 Selective and Peripheral Leptin Resistance ...................................................... 95 Prohypertensive Effects of Chronic Hyperleptinemia................................... 98 Conclusions and Future Perspectives .............................................................. 102 10. Involvement of Leptin in the Endometrial Function ...............................108 Ana Cervero and Carlos Simon Overview of the Leptin System ......................................................................... 108 Leptin System in the Endometrium................................................................. 109 Leptin System in the Implantation Process.................................................... 109 Leptin System in the Endometriosis .................................................................111 Summary and Conclusions ................................................................................ 113 11. The Use of Leptin for the Treatment of Lipodystrophy ...........................116 Angeline Y. Chong, Elaine K. Cochran and Phillip Gorden Metabolic Effects of Leptin Therapy.................................................................117 Endocrine Effects of Leptin Therapy ............................................................... 120 Hepatic and Muscular Effects of Leptin Therapy ......................................... 123 Conclusion ............................................................................................................. 124 12. Use of Anti-Leptin or Anti-Leptin Receptor Antibodies as Blockers of Immune Response ................................................................................126 Giuseppe Matarese and Veronica De Rosa Leptin Has Multiple Functions in Immunity ............................................... 127 Leptin Is Involved in the Development of Various Diseases ...................... 127 Immunotherapeutic Applications Targeting Leptin: Current Evidence and Hypotheses .............................................................. 127 Leptin Neutralization: Novel Strategies to Block Autoimmunity and to Improve Leptin Resistance Observed in Obesity........................ 129 Leptin-Receptor Neutralization ....................................................................... 131 Conclusions and Future Perspectives .............................................................. 131 13. Use of Leptin Antagonists as Anti-Inflammatory and Anti-Fibrotic Reagents ......................................................................133 Eran Elinav and Arieh Gertler Results .....................................................................................................................134 Chronic Hepatitis and Fibrosis ......................................................................... 136 Conclusions ........................................................................................................... 138 14. The Role of Leptin during Early Life in Imprinting Later Metabolic Responses ................................................................................141 Mark H. Vickers, Stefan O. Krechowec, Peter D. Gluckman and Bernhard H. Breier Background............................................................................................................ 141 Leptin and Developmental Programming...................................................... 142 Evidence from Animal Models ......................................................................... 143

Epidemiological and Clinical Evidence........................................................... 146 Leptin in Early Life and Catch-Up Growth .................................................. 147 Potential Mechanisms ......................................................................................... 148 Developmental Programming and Gender Differences in Leptin Sensitivity........................................................................................ 150 Leptin in the Perinatal Period—A Therapeutic Window of Intervention?................................................................................................ 150 Extrapolation from Animal Models to the Clinical Setting .......................152 Discussion ...............................................................................................................153 Index .........................................................................................................163

EDITOR Arieh Gertler

Institute of Biochemistry, Food Science and Nutrition The Hebrew University of Jerusalem Rehovot, Israel Email: [email protected] Chapter 13

CONTRIBUTORS Note: Email addresses are provided for the corresponding authors of each chapter. Suresh Ambati Department of Animal and Dairy Science University of Georgia Athens, Georgia, USA Chapter 4

Thomas Aparicio INSERM, U773 Centre de Recherche Biomédicale Bichat Beaujon Paris, France Chapter 5

André Bado INSERM, U773 Centre de Recherche Biomédicale Bichat Beaujon Paris, France Email: [email protected] Chapter 5

Clifton A. Baile Department of Animal and Dairy Science University of Georgia Athens, Georgia, USA Email: [email protected] Chapter 4

Walter Becker Institute of Pharmacology and Toxicology Medical Faculty of the RWTH Aachen University Aachen, Germany Email: [email protected] Chapter 1

Jerzy Beltowski Department of Pathophysiology Medical University Lublin, Poland Email: [email protected] Chapter 9

Michel Ben-Ami Department of Biochemistry and Nutrition The Hebrew University Jerusalem, Israel Chapter 8

Bernhard H. Breier Liggins Institute and The National Research Centre for Growth and Development The University of Auckland Auckland, New Zealand Chapter 14

Ana Cervero Fundación IVI Valencia, Spain Chapter 10

Patty Chen Institut Cochin Department of Cell Biology Université Paris Descartes Paris, France Chapter 3

Angeline Y. Chong Clinical Endocrinology Branch National Institute of Diabetes, Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland, USA Chapter 11

Elaine K. Cochran Clinical Endocrinology Branch National Institute of Diabetes, Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland, USA Chapter 11

Cyril Couturier Centre National de la Recherche Scientifique Université Lille Lille, France Chapter 3

Julie Dam Institut Cochin Department of Cell Biology Université Paris Descartes Paris, France Chapter 3

Mary Anne Della-Fera Department of Animal and Dairy Science University of Georgia Athens, Georgia, USA Chapter 4

Veronica De Rosa Laboratorio di Immunologia Istituto di Endocrinologia e Oncologia Sperimentale Consiglio Nazionale delle Ricerche Napoli, Italy Chapter 12

Robert Ducroc INSERM, U773 Centre de Recherche Biomédicale Bichat Beaujon Paris, France Chapter 5

Eran Elinav Gastroenterology and Liver Institute Tel Aviv Sourasky Medical Center (TASMC) Tel Aviv, Israel Email: [email protected] Chapter 13

Peter D. Gluckman Liggins Institute and The National Research Centre for Growth and Development The University of Auckland Auckland, New Zealand Chapter 14

Phillip Gorden Clinical Endocrinology Branch National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health Bethesda, Maryland, USA Email: [email protected] Chapter 11

Sandra Guilmeau INSERM, U773 Centre de Recherche Biomédicale Bichat Beaujon Paris, France Chapter 5

Ralf Jockers Institut Cochin Department of Cell Biology Université Paris Descartes Paris, France Email: [email protected] Chapter 3

Morris Karmazyn Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London, Ontario, Canada Email: [email protected] Chapter 7

Mariusz Koda Department of Pathomorphology Medical University of Bialystok Bialystok, Poland Chapter 6

Stefan O. Krechowec Liggins Institute and The National Research Centre for Growth and Development The University of Auckland Auckland, New Zealand Chapter 14

Giuseppe Matarese Laboratorio di Immunologia Istituto di Endocrinologia e Oncologia Sperimentale Consiglio Nazionale delle Ricerche Napoli, Italy Email: [email protected] Chapter 12

Efrat Monsonego Ornan Department of Biochemistry and Nutrition The Hebrew University Jerusalem, Israel Email: [email protected] Chapter 8

Frank Peelman Department of Medical Protein Research, VIB Department of Biochemistry Ghent University Ghent, Belgium Chapter 2

Daniel M. Purdham Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London, Ontario, Canada Chapter 7

Venkatesh Rajapurohitam Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London, Ontario, Canada Chapter 7

Srujana Rayalam Department of Animal and Dairy Science University of Georgia Athens, Georgia, USA Chapter 4

Carlos Simon Fundacion IVI Valencia, Spain Email: [email protected] Chapter 10

Eva Surmacz Sbarro Institute for Cancer Research and Molecular Medicine Temple University Philadelphia, Pennsylvania, USA Email: [email protected] Chapter 6

Jan Tavernier Department of Medical Protein Research, VIB Department of Biochemistry Ghent University Ghent, Belgium Email: [email protected] Chapter 2

Mark H. Vickers Liggins Institute and The National Research Centre for Growth and Development The University of Auckland Auckland, New Zealand Email: [email protected] Chapter 14

Lennart Zabeau Department of Medical Protein Research, VIB Department of Biochemistry Ghent University Ghent, Belgium Chapter 2

Asad Zeidan Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London, Ontario, Canada Chapter 7

PREFACE The discovery of leptin, the obese (ob) gene product which is not expressed as a functional protein in ob/ob mice, focused the scientific community’s attention on its role as an anorexic hormone involved in the negative regulation of food intake. Almost 14 years after this breakthrough discovery and over 14,000 leptin-related publications later, leptin is now known to participate in a wide range of biological functions that include, in addition to its early envisaged function as an adipostat, glucose metabolism, glucocorticoid synthesis, CD4+ T-lymphocyte proliferation, cytokine secretion, phagocytosis, hypothalamic-pituitary-adrenal axis regulation, reproduction, cardiovascular pathology, bone formation, apoptosis and angiogenesis. In short, it is now well-documented that leptin acts like a cytokine hormone with many pleiotropic effects. Furthermore, in recent years, it has become more and more apparent that many of leptin’s effects are acquired not only through its central action, but also through its systemic action on a peripheral level. This book focuses mainly on the relatively novel aspects of leptin’s actions. In parallel to the discovery and exploration of leptin’s physiological action, extensive research has been aimed at clarifying leptin signal transduction. Although many transduction pathways have been discovered, the structural aspects of the leptin:leptin receptor interaction have remained mostly speculative and based mainly on models, due to the lack of any valid structural information on the leptin receptor. Nevertheless, modeling of this interaction has enabled a better understanding of the leptin:leptin receptor interaction and has led to the rational development of leptin antagonists. This book is divided into two parts: Part I deals with the molecular aspects of leptin’s action, whereas Part II is devoted to various central and peripheral physiological activities, with an emphasis on its potential involvement in different pathologies. In the first chapter of this book Becker sums up the recently acquired knowledge on leptin’s action. He provides updated information on various leptin-activated transduction signaling pathways, including novel and relatively little investigated phenomena such as activation of ATP-sensitive K+ channels and AMPK-mediated effects leading to activation of mTOR (mammalian target of rapamycin). This chapter also gives an updated compilation of leptin-induced genes (or: “target genes”). In Chapter 2, Tavernier and his colleagues contribute deep insight into the possible models of the leptin:leptin receptor interaction. They review the experiments which led to the hypothesis that leptin interaction with its receptor resembles that of interleukin 6, namely that the leptin:leptin receptor complex is a hexamer composed of two leptin and four leptin receptor molecules. This model led to the identification of leptin’s site III interaction site, which interacts with the Ig domain of the receptor, a breakthrough discovery that led to the development of leptin antagonists. In the third chapter of the Part I, Jockers and his colleagues review the resonance energy transfer (RET)

methodologies used to study the leptin:leptin receptor interaction, which led to the conclusion that leptin receptors exist as preformed homodimers. Upon leptin binding, the receptors undergo a conformational change and possibly, further aggregation, leading to their activation. This activation can, however, be prevented by leptin antagonists. These authors also review the use of RET technology for the screening of small molecules affecting the leptin:leptin receptor interaction. The second part of this book is devoted to various aspects of leptin’s action beyond its direct regulatory effect on food intake. In the last 40 years, apoptosis has become a major field of study. Leptin’s involvement as an anti-apoptotic agent was described in as early as 1999 and in Chapter 4, Baile and his colleagues summarize leptin’s dual involvement: via direct anti-apoptotic action, mostly in the periphery, and indirect pro-apoptotic action, which affects mainly the adipose tissue and most likely mediated via increased sympathetic activity. Although adipose tissue is the main source of leptin, in 1998, Bado and his colleagues had identified expression of leptin gene in the stomach, the distribution of its receptors throughout the gastrointestinal tract, and the production of leptin by gastric epithelial cells within the gastric mucosa in rodents and humans. In Chapter 5, Bado and his colleagues review these aspects of leptin action, suggesting that gut leptin may act locally to influence gastrointestinal functions. The association between obesity and cancer is now well established, based mostly on epidemiological data; however, the precise underlying mechanism remains elusive. In Chapter 6, Surmacz and Koda review the putative involvement of leptin in breast and colorectal cancer. Compiling a wide array of studies using several in-vivo and in-vitro models, they conclude that leptin acts as a mitogen and survival factor, and may promote anchorage-independent growth, migration and invasion of breast and colorectal cancer cells. Although the association between circulating leptin levels and cancer is yet unclear, the authors suggest that leptin effects can be attributed to overexpression of leptin receptors in breast and colorectal cancer tissues as well as enhanced intratumoral leptin synthesis. Leptin’s involvement in cardiac physiology and pathology is the subject of a relatively new field of investigation originating from the well-documented relationship between obesity and increased risk of cardiovascular disease. This field is reviewed in Chapter 7 by Karmazyn and his colleagues, who highlight leptin’s hypertropic effects in cardiomyocytes and discuss the complex and sometime controversial role of leptin in cardiac pathology. These authors also outline the novel RhoA/ROCK transduction pathway activated by leptin. The next chapter (Chapter 8) reviews the controversial issue of leptin’s putative role in bone elongation. The authors, Monsonego-Ornan and Ben-Ami discuss its specific involvement in the process of endochondral ossification, and review the still unresolved question of whether these effects are limited to central leptin action or are also mediated by systemic leptin, acting in the periphery and

affecting chondrocyte proliferation, differentiation, mineralization and apoptosis. The issue of leptin’s putative involvement in arterial hypertension is reviewed in Chapter 9 by Beltowski. The author discusses the dual contradictory acute activity of exogenously administered leptin versus chronic hyperleptinemia, sums up the various related molecular pathways affected by leptin and suggests possible therapeutic strategies and a relationship to obesity. Involvement of leptin in endometrial function is then reviewed by Cervero and Simon in Chapter 10. These authors begin by outlining the leptin system in the endometrium, then discuss leptin’s controversial role in implantation by comparing humans and mice, and conclude with an evaluation of leptin’s function in endometrial pathologies. Chapter 11 is unique in addressing the use of leptin as a therapeutic agent in lipodystrophy. The authors, Chong, Cochran and Gorden review leptin’s various effects on glycemic control, lipid metabolism and body composition. They then address the various effects of leptin therapy on gonadal function, growth hormone, thyroid and adrenal axis, and the muscle and liver, and conclude with a discussion on the possible utilization of leptin in various therapies. Chapters 12 and 13 are devoted to reviewing leptin’s effects on the immune system. First, Matarese and Rosa in Chapter 12, provide an extensive review of leptin’s involvement in modulating the immune response, with a special emphasis on its Th1-promoting effects, which have been linked to enhanced susceptibility to experimentally induced autoimmune diseases. They suggest that leptin also exerts a negative signal for the proliferation and expansion of regulatory T cells (Tregs), a specific subset of cells involved in the control of immune and autoimmune responses. They also review the possibility of using either anti-leptin or anti-leptin receptor antibodies as blockers, i.e., the action that is being blocked is the breaking of self-tolerance. Then, in Chapter 13, Elinav and Gertler report on the first use of a recently developed competitive leptin antagonist acting as an anti-inflammatory agent in mice models of acute and chronic T-cell-mediated liver inflammation and chronic liver fibrosis. Their recent results suggest that this beneficial effect may be mediated by both the direct modulation of T-cells and the inhibition of hepatic stellate cells activation and function. Leptin’s involvement in early postnatal imprinting has led to new insight into developmental programming. This highly novel aspect of leptin’s action is reviewed extensively in the final chapter of this book by the Auckland group, Vickers, Krechowec, Gluckman and Breier. In the last five years, it has been shown that at least in rodents, leptin acts as an important neurotrophic factor promoting the early postnatal maturation of neural pathways within the hypothalamus. The authors review experimental evidence, originating largely from their own work, which shows that therapeutic intervention with leptin in the rodents’ early postnatal life can potentially reverse or substantially ameliorate the consequences of developmental malprogramming, and that this effect is highly influenced by both gender and postnatal diet.

In conclusion, my colleagues who contributed to this book and I hope that this extensive review of the recent advances in leptin research will be of help and interest to the scientific community at large, particularly those whose field of study involves this multifaceted hormone. Arieh Gertler, PhD Institute of Biochemistry, Food Science and Nutrition The Hebrew University of Jerusalem Rehovot, Israel

Chapter 1

Leptin Signal Transduction— A 2009 Update Walter Becker*

Abstract

L

eptin is an adipocyte-secreted hormone that informs the brain about the status of the body’s energy stores. Leptin controls energy homeostasis through effects on satiety and energy expenditure but also regulates other processes, including reproduction, glycemic control, immune function and wound healing. The leptin receptor exists in multiple alternatively spliced isoforms, of which only the long form (LEPRb) associates with Janus kinase 2 ( JAK2) to mediate intracellular signaling. Upon leptin binding, LEPRb initiates multiple intracellular signal transduction pathways that result in the activation of STAT family transcription factors, extracellular signal-regulated kinases (ERK), phosphoinositol-3 kinase, AMP-activated kinase and ATP-sensitive potassium channels. This chapter gives a delineation of our current knowledge about leptin signal transduction, with a particular focus on the role of individual signaling pathways in vivo and the changes in gene expression induced by leptin.

Leptin and the Leptin Receptor

Leptin is a 16 kDa polypeptide secreted from adipocytes that is often referred to as an adipokine because of it is structurally related to the long-chain four helix bundle family of cytokines, which includes interleukin 6 (IL-6), oncostatin M and others. The cytokine character of leptin is reflected by the pleiotropic actions of leptin and the widespread expression of the leptin receptor. In addition to its central function as a regulator of food intake and energy expenditure in hypothalamic nuclei, leptin is involved in many additional physiological processes. Adequate leptin levels are required to permit energy consuming processes such as reproduction, angiogenesis, wound healing, hematopoiesis, bone development and activation of the immune systems (see also part 2 of this book).1-3 Leptin also regulates glucose homeostasis and lipid metabolism independently of its central weight regulatory function, partly via direct action on pancreatic -cells and hepatocytes.4-6 Cloning of the leptin receptor identified it as a single membrane-spanning receptor of the class I cytokine family.7 The murine leptin receptor exists in at least six isoforms that are alternative splicing products derived from a single Lepr gene.7 Each of LEPRa—LEPRf are identical in their extracellular domain that binds leptin with an affinity in the nanomolar range.8 The molecular interaction of leptin with the extracellular domain of the leptin receptor is dealt with in detail in the ensuing chapters.9,10 The shortest isoform (LEPRe) lacks the transmembrane region and forms a soluble, secreted form of the receptor. The extracellular domains of the membrane-bound receptors can also shed into the circulation by the action of surface proteases.11 These soluble receptors determine the proportion of free and protein-bound leptin in the circulation and appear to alter leptin clearance without directly affecting leptin action.12,13 *Walter Becker—Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany. Email: [email protected]

Leptin and Leptin Antagonists, edited by Arieh Gertler. ©2009 Landes Bioscience.

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Four splicing variants of the leptin receptor, i.e., LEPRa, LEPRc, LEPRd and LEPRf, share the membrane-spanning region and 29 intracellular amino acids and diverge thereafter, comprising only 3-11 additional residues at their cytosolic domain. Only LEPRb has an extended intracellular domain of approximately 300 amino acids, which comprise the typical structural elements of cytokine receptors.7 LEPRa is the most abundant isoform of the leptin receptor and exhibits some signalling capacity in overexpression systems.14 However, the specific lack of LEPRb in Lepr db/db mice results in an obesity phenotype very similar to that in the leptin-deficient Lepob/ob mice, indicating that LEPRb is crucial for the function of leptin in vivo. Furthermore, LEPRa does not form functional heterodimers or -oligomers with LEPRb.15,16 Taken together, there is no evidence that any of the short isoforms of the leptin receptor can elicit intracellular effects and this review focuses on leptin signaling via LEPRb. It should however be noted that LEPRa and LEPRc have been proposed to play a role in leptin uptake or efflux from cerebrospinal fluid and in receptor-mediated transport of leptin through the blood brain barrier.17,18 This review summarizes the current knowledge about the intracellular signal transduction pathways initiated by LEPRb (see Fig. 1). Mechanism of negative regulation of leptin signaling are not within the scope of this chapter but are covered by excellent recent reviews.19-21

JAK Kinase

Like other cytokine receptors, LEPRb does not have intrinsic tyrosine kinase activity but signals by activating a noncovalently associated tyrosine kinase of the Janus kinase family, JAK2.22 Association of LEPRb with JAK2 does not depend on ligand binding, but rather the receptor polypeptide and the kinase form a constitutive complex.22,23 In contrast to the receptors of the IL-6 receptor family, LEPRb do not form heteromeric complexes with other receptor chains and does not require nonsignaling receptor subunits such as CNTFR, IL11R or IL-6R for JAK activation.24 JAKs bind to the membrane-proximal region of cytokine receptors, which contains an essential

Figure 1. Intracellular signal transduction pathways regulated by LEPRb. The cartoon illustrates the activation of different pathways by recruitment of SH2-domain containing proteins to the intracellular tyrosine residues of LEPRb (pTyr985, pTyr1077, pTyr1138) or JAK2. See the text for further details.

Leptin Signal Transduction—A 2008 Update

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and conserved so-called box1 motif and a less well defined box2 motif that also contributes to JAK binding.24 The box1 sequence is located in the short piece of sequence common to the long and the short isoforms of the leptin receptor, but additional residues specific to LEPRb have been identified that are required for JAK activation.23,25 Recently, the activation of JAK2-independent pathways by LEPRb has been described and attributed to kinases of the Src family.129 However, it remains to be determined whether other JAK family kinases may compensate for the lack of JAK2 in this experimental system. Binding of leptin to the extracellular domain of LEPRb leads to trans-autophosphorylation of the associated JAK on at least 13 tyrosines, including Tyr1007 and Tyr1008 in the activation loop that cause the activation of these kinases.26,27 The requirement of at least two JAK molecules within a receptor complex has been experimentally shown using chimeric LEPRb constructs.23,28 Although it is not fully understood how cytokine receptors transmit the signal through the membrane, conformational changes of the receptor somehow propagate through the membrane and orientate the JAK molecules correctly to allow their reciprocal phosphorylation.29 In addition to autophosphorylation, activated JAK2 also phosphorylates three conserved intracellular tyrosine residues on LEPRb (Tyr985, Tyr1077 and Tyr1138 in murine LEPRb).30-34 In some species including human, LEPRb contains additional tyrosines in the cytoplasmic domain that are not evolutionary conserved and thus are unlikely to play a role in signal transduction. The phosphorylated tyrosine residues in LEPRb and in JAK2 then provide docking sites for signal transduction proteins with specialized phosphotyrosines-binding domains called Src homology 2 (SH2) domains. In general, SH2 domains from different proteins specifically recognize phosphotyrosines in different sequence motifs. Thus, each tyrosine phosphorylation site recruits specific downstream signaling proteins depending on the surrounding amino acids.

Activation of STAT3

The best known downstream targets of the JAKs are the members of the STAT (Signal Transducers and Activators of Transcription) family of transcription factors. STATs are transiently recruited to specific phosphotyrosine motifs, are themselves phosphorylated on a single tyrosine residue by the receptor-associated JAK kinases, dimerize, translocate to the nucleus and modulate the transcription of target genes. LEPRb is most closely related with IL-6-type cytokine receptors (gp130, OSMR, LIFR; ref. 19), which signal via activation of STAT3, and the phosphorylation of STAT3 on Tyr705 is indeed the most robust downstream effect of leptin receptor activation. Tyrosine phosphorylation and/or nuclear translocation of STAT3 upon leptin treatment was observed in most if not all leptin-responsive cells, including hypothalamic neurons, hepatocytes, hepatic stellate cells, T-cells, insulin-secreting-cells, macrophages, endothelial cells and many others.35-42 In addition to phosphorylation of Tyr705, phosphorylation of Ser727 in the activation domain appears to be necessary for the full transcriptional activity of STAT3 at least in certain systems and was shown to be biologically important in STAT3S727A knock-in mice.43,44 Leptin-induced phosphorylation of STAT3 on Ser727 has not yet been extensively studied but was observed in the J744.2 macrophage cell line (ref. 41) and in RINm5F insulinoma cells (unpublished results from our lab). Numerous protein kinases have been implicated in the phosphorylation of Ser727 in different systems (ref. 45), but the effect of leptin in the macrophages was dependent on the activation of the ERK (extracellular signal-regulated kinase) pathway.41 LEPRb contains a canonical STAT3 binding motif (box3, Tyr-x-x-Gln) at position 1138-1141 that is essential for the leptin-induced activation of STAT3 in vitro and in vivo.46,47 Homologous replacement of Tyr1138 with serine in transgenic mice (Lepr S1138) completely abolished leptin-induced activation of STAT3 and provided an excellent model for assessing the specific contribution of this pathway to the different biological effects of leptin.47-49 Lepr S1138 homozygous mice were obese and hyperphagic like Lepr db/db, indicating that activation of STAT3 is indispensable for the hypothalamic effects of leptin on appetite control and regulation of energy expenditure. However, Lepr S1138/S1138 mice are less hyperglycemic compared to Lepr db/db mice and display nearly normal reproductive function.47,49 These elegant studies provide clear evidence that important effects of

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leptin such as fertility and glycemic control are mediated via STAT3-independent effector systems. Using a different approach to elucidate the requirement of STAT3 for the effects of leptin, Buettner et al inhibited STAT3 activation by stereotaxic intracerebroventricular application of a peptide inhibitor of STAT3 activation.50 This acute inhibition of leptin-induced STAT3 activation in the hypothalamus prevented the effect of leptin on food intake and hepatic glucose metabolism. Surprisingly, the restoration of the luteinizing hormone (LH) surge in food-deprived female rats by leptin was also abolished by inhibition of STAT3 activation in the hypothalamus. This observation contrasts with the fertility of the Lepr S1138/S1138 mice but can possibly be explained by the fact that the peptide inhibited activation of STAT3 by cytokines other than leptin. Recently, mice with a specific deletion of STAT3 in Lepr expressing cells were shown to exhibit normal fertility.51 Concerning peripheral leptin effects, mice with an adipocyte-specific disruption of STAT3 have increased adiposity and an impaired lipolytic effect of leptin.52 In contrast, leptin stimulates vascular smooth muscle cell proliferation independent of STAT3 activation.53 Taken together, these results clearly establish STAT3 as a key effector of leptin’s physiological functions but show that other pathways are also involved. Furthermore, many effects of leptin have not yet been studied in the Lepr S1138 mice, e.g., the immune-modulatory function and the action on pancreatic -cells.

Leptin-Regulated Genes

The requirement of STAT3 signalling for the weight lowering effect of leptin raises the question which target genes of STAT3 are involved (Table 1). Leptin is well known to increase transcription of the proopiomelanocortin gene (Pomc) in a specialized population of hypothalamic neurons.54 Proopiomelanocortin is the precursor for –melanocyte-stimulating hormone (-MSH), which has anorectic effects by activating melanocortin receptors (MC3R, MC4R). Pomc gene expression is directly induced by leptin via a STAT3 response element in the promoter in vitro and in vivo.55 Interestingly, genetic inactivation of STAT3 in POMC neurons caused only mild obesity and did not completely abolish the appetite-suppressing effect of leptin, indicating that STAT3-dependent effects in other cells are also involved.56 A second well-characterized effect of leptin is the negative regulation of the orexigenic peptides neuroeptide Y (NPY) and agouti-related protein (AgRP). Interestingly, genetic disruption of STAT3 in hypothalamic AgRP/NPY neurons revealed that STAT3 in these neurons contributes also to the regulation of energy homeostasis.128 Different mouse models have yielded conflicting results concerning the role of STAT3 in the leptin-induced downregulation of NPY and AgRP.47,128 Another effect of leptin directly transmitted through STAT3 is the upregulation of thyreotropin-releasing hormone (TRH) that enhances thyroid function and results in an increased energy expenditure.57-59 Activation of the JAK/STAT pathway by leptin is expected to result in extensive changes in gene expression. Apart from the above-mentioned neuropeptides, surprisingly few leptin-induced genes have been linked to the multitude of leptin effects in the different target organs. Although numerous leptin-regulated transcripts have been identified in various tissues, many of these changes are a consequence of metabolic reprogramming (e.g., ref. 60). Among the direct targets of leptin, suppressor of cytokine signaling 3 (SOCS3) is a feedback inhibitor that is rapidly induced after activation of STAT3 and downregulates receptor activity by inhibiting the receptor-associated JAK kinase.61 Most of the known leptin-induced genes are regulated by STAT3, including several genes encoding inflammation-related proteins (-fibrinogen, plasminogen activator, tissue-type (tPA), pancreatitis-associated protein, lipocalin-2, preprotachykinin, superoxide dismutase 2, see Table 1).62 However, in most cases the functional consequences of their upregulation by leptin have not been defined in vivo.

Activation of Other STAT Factors

In addition to STAT3, leptin can induce tyrosine phosphorylation and activation of STAT1, STAT5 and STAT6.33,46,62 The biological role of these STAT factors in leptin signalling is less clear than that of STAT3. STAT1 is recruited to the same docking site as STAT3 (Tyr1138 in mouse LEPRb) and can form heterodimers with STAT3 once activated.46 This promiscuity of the box3

Experimental Evidence Gene Symbol

Gene Product

Tissue or Cell Line

Method

Upregulation of...

Pathway

Ref

Pomc

proopiomelanocortin

Hypothalamus AtT20*, HEK*

LEPRb-mut, dnSTAT3

mRNA, promoter activity

STAT3

47,55

Trh

thyreotropin-releasing hormone

Hypothalamus 293T*

ChIP

STAT3 binding, Promoter activity STAT3

58,59

Socs3

suppressor of cytokine signalling 3 (SOCS3)

Hypothalamus 32D cells* INS1

ChIP, LEPRb-mut, EMSA

mRNA STAT3/5 binding, Promoter activity

STAT3 (STAT5)

58,31,122

Fos

c-fos proto-oncogene#

Hypothalamus 293T*

LEPRb-mut, PD98059

Protein mRNA

ERK

78,31

Egr1

early growth response 1

CHO*

dnSHP2

Promoter activity

ERK

75

Il1b

Interleukin 1β (IL-1β)#

Hypothalamus microglia cells

Lepr db/db Peptide inh.

mRNA, Secreted IL-1β

Non-STAT3§ STAT3

123, 124

Il1rn

IL-1 receptor antagonist#

HepG2*

PD98059, U0126

Promoter activity

ERK

125

ERK, STAT3

38,126

Timp1

Tissue inhibitor of metalloproteinase-1#

LX-2

PD098059, EMSA

mRNA, STAT3 binding

$


Table 1. Leptin target genes and signaling pathways involved in their regulation

continued on next page

5

6

Table 1. Continued Experimental Evidence Gene Symbol

Gene Product

Tissue or Cell Line

Method

Upregulation of...

Pathway

Ref

Pap

Pancreatitis-associated protein#

RINm5F*, PC12*

dnSTAT3 LEPRb-mut


STAT3

127

Lcn2

Lipocalin-2#

RINm5F*, HIT-T15*

LEPRb-mut


STAT3

61

#

Tac1

preprotachykinin

LEPRb-mut


STAT3

61

Sod2

superoxide dismutase 2# RINm5F*

RINm5F*, HIT-T15*

LEPRb-mut

mRNA

STAT3

61

Fbgn

fibrinogen β#

RINm5F*

LEPRb-mut

mRNA

STAT3

61

Plat

plasminogen activator, tissue-type (tPA)#

RINm5F*

LEPRb-mut

mRNA

STAT3

61


*cell line ectopically expressing recombinant LEPRb; §the reported upregulation of IL-1β in Lepr db/db mice implies a tyrosine-independent mechanism and is in contrast with the study by Pinteaux (ref. 124); $indirect binding via association with Sp1; #gene products involved in inflammatory processes. Evidence for the contribution of either the JAK/STAT3 or the ERK pathway was obtained using LEPRb mutants lacking specific tyrosine residues (LEPRb-mut), overexpression of dominant negative mutants of STAT3 (dnSTAT3) or SHP2 (dnSHP2), specific inhibitors of ERK activation (PD98059, U0126) or by detecting promoter binding of STAT3 to the promoter of the target gene by chromatin immunoprecipitation (ChIP) or electrophoretic mobility shift assay (EMSA). In one study, STAT3 activation was blocked with help of a specific STAT3 peptide inhibitor (peptide inh). The stimulatory effect of leptin was determined as upregulation of mRNA levels (Northern blot or RT-PCR), promoter activity (reporter gene assay), protein levels (ELISA or immunocytochemistry) or enhanced binding of transcription factors to the promoter.


7

motif is also known for the gp130 receptor subunit of the IL-6 type cytokine receptors that also activates both STAT3 and STAT1 via the same phosphotyrosines.24 In a direct side-by-side comparison in the insulinoma cell line RINm5F, leptin induced a much stronger tyrosine phosphorylation of STAT1 than IL-6 under conditions of similar STAT3 phosphorylation.33 Although the activation of STAT1 might suggest that leptin can induce an IFN-like response, leptin did not upregulate the paradigmatic STAT1 target gene, IRF1 (interferon regulatory factor 1) under these conditions.62 Like in IL-6 signaling, the STAT1 response is probably suppressed via a STAT3-dependent mechanism and would only take effect in the absence of STAT3.63 Consistently, leptin strongly activates STAT1 in STAT3-deficient but not in wild-type mouse adipocytes.52 However, leptin induced tyrosine phosphorylation of STAT1 in normal rat adipose tissue.64 STAT5 is activated by leptin in many cell lines, including HIT-T15 and RINm5F insulinoma cells, neuronal GT1-7 cells, enterocyte-like CACO2 cells, H35 hepatoma cells and LX-2 hepatic stellate cells.33,65-68 However, early in vivo-studies failed to detect leptin-induced STAT5 phosphorylation in the hypothalamus of mice and rats.35,69 Recent studies have now succeeded to demonstrate STAT5 phosphorylation in mouse hypothalamus and nuclear translocation of STAT5 in rat hypothalamic nuclei.70,71 Either Tyr1077 or Tyr1138 in LEPRb can mediate the activation of STAT5, with no preferential activation of either STAT5A and STAT5B.33,68 Phosphorylation of Tyr1077 in LEPRb proved difficult to detect with most phosphotyrosine-specific antibodies (refs. 31,72) and was controversial until recently. Gong et al68 have now unambiguously shown that this residue is phosphorylated after receptor activation. Notably, the amino acids around Tyr1077 are phylogenetically conserved in vertebrates, indicating that the role of this tyrosine in signaling is not redundant.33 So far no target gene nor any effect of leptin is known to be regulated via STAT5. In vitro, leptin can activate STAT5-dependent promoters and thus might have gene regulatory effects overlapping with those other STAT5 recruiting hormones such as growth hormone (GH) or prolactin, given that the receptors are expressed on the same cells (e.g., hepatocytes or pancreatic -cells).33 It will be difficult to reveal the physiological effects of leptin-induced STAT5 activation because the targeted mutation of Tyr1077 in transgenic mice will still allow STAT5 activation via Tyr1138. Taken together, even if the poorly characterized activation of STAT6 is neglected, LEPRb activates a broader spectrum of STAT factors than most other cytokine receptors.

Activation of the ERK Pathway

Like many other cytokines, leptin activates the RASRAFMEKERK pathway.14,31,73 Other members of the MAPK family (p38, JNK) have also been reported to be activated by leptin (e.g., refs. 74,75), but the relevant pathways have not been well characterized. Although the complete chain of reactions leading to the activation of ERK1 and ERK2 has not been specifically dissected in leptin signaling, LEPRb most likely exploits the same pathway as the signal transducing subunit of the IL-6-type cytokine receptors, gp130.24 Phosphorylation of the most proximal intracellular tyrosine residue, Tyr985 in LEPRb or Tyr759 in gp130, creates a binding site for the carboxyterminal SH2 domain of the tyrosine phosphatase SHP2.72,76 SHP2 becomes itself phosphorylated on C-terminal tyrosines, which then recruit the adapter protein GRB2 (growth factor receptor-bound protein-2) to the receptor complex.31 In the canonical ERK pathway, GRB2 forms a complex with SOS, the GTP exchange factor for RAS and initiates the RAFMEKERK pathway, wherein each kinase activates the downstream kinase by phosphorylation. Leptin can also induce a lower level of ERK activation independent of Tyr985 and SHP-2, possibly mediated by direct binding of GRB2 to JAK2.31 Interestingly, catalytically inactive SHP2 did not support LEPRb mediated ERK activation.77 Of the many possible downstream effects of ERK, upregulation of the immediate early genes egr-1 and c-fos has been demonstrated in cell culture and in vivo in the hypothalamus (Table 1).77,78 The same genes are also upregulated by IL-6 via Tyr759 in gp130, SHP2 and ERK.79 Analysis of the Lepr S1138/S1138 knock-in mice confirmed that upregulation of c-fos does not depend on STAT3 activation.80 It is not clear how activation of ERKs translates into physiological effects of leptin,

8


but upregulation of c-fos is a marker for neuronal activity.78 Knock-in mice homozygous for a Tyr985Leu point mutation have not yet revealed a biological function of leptin-induced ERK activation.81 These mice exhibit increased leptin sensitivity, consistent with the known role of Tyr985 as a binding site for the site for autoinhibitory SOCS3, and their phenotype demonstrates that Tyr985 is not essential for regulation of growth or reproduction. Another function of ERK1/2 is the phosphorylation and activation of RSK (ribosomal protein S6 kinase). Phosphorylation of S6 by RSK enhances cap-dependent translational initiation and protein synthesis.70 Leptin-dependent phosphorylation of S6 has been demonstrated in vivo in the hypothalamus (ref. 82) and in vitro-studies have shown that Tyr985 and ERK activation are required for this effect of LEPRb.70

Activation of the IRS/PI3K/PDE3B Pathway

PI3K (phosphoinositide 3-kinase) is a key signaling molecule that transmits downstream effects of insulin. Activation of PI3K by receptor tyrosine kinases is mediated via phosphorylation of IRS (insulin receptor substrate) proteins, which then associate with the SH2 domain of the regulatory subunit of PI3K, p85. Leptin reportedly stimulates tyrosine phosphorylation of IRS1 and IRS2 and activation PI3K in different cell types.14,64,88 This pathway does not depend on STAT3 activation but is initiated by direct binding of IRS proteins to phosphorylated JAK2.73 Recently, leptin was found to recruit IRS4, which binds to phosphorylated Tyr1077 and can also associate with p85 to recruit PI3K.83 Although leptin signaling via the IRSPI3K pathway suggests that leptin may have insulin-like effects in cells that express both receptors, the crosstalk between these hormones is complicated by many indirect effects. In liver, the acute lipid-lowering effect of leptin and inhibition of gluconeogenesis depend on PI3K activity.84,85 However, results obtained in myoblasts and even in different hypothalamic neurons are inconsistent and indicate that cell type-specific mechanisms determine the actual interaction between these pathways.86-90 It is also important to note that the magnitude of PI3K stimulation in response to leptin in vivo is much lower than that seen with insulin.64 At least in some cell types, leptin increases the levels of PIP3 (phosphoinositide3,4,5 trisphosphate), the reaction product of PI3K, mainly by inhibition of the lipid phosphatase, PTEN.91 Downstream of PI3K, PIP3 stimulates protein kinases such as PDK1 and PKB/Akt. PKB/Akt has been implicated in insulin-induced phosphorylation and activation of membrane-associated phosphodiesterase 3B (PDE3B).92 PDE3B activation reduces intracellular cAMP levels and thus leptin-induced activation of PDE3B antagonizes the cAMP-mediated effects of glucagon-like peptide-1 (GLP-1) in pancreatic -cells and glucagon in hepatocytes.92,93 Leptin-induced activation of PDE3B has also been shown in the hypothalamus and intracerebroventricular injection of the PDE3 inhibitor, cilostamide, blocked the inhibitory effect of leptin on food intake.94 In vivo studies implicate the LEPRPI3KPDE3B pathway in the suppression of NPY neurons in the arcuate nucleus.95-97 Thus, this pathway may be responsible for STAT3-independent the gene regulatory effects in the hypothalamus. In contrast, activation of PI3K appears less important in POMC neurons, because genetic ablation of IRS2 in POMC neurons did not cause obesity.98 SH2B1 (a.k.a. SH2-B) is an SH2-domain containing adapter protein that increases the leptin-induced tyrosine kinase activity of JAK2 by binding to the autophosphorylated pTyr830.99,100 In addition, SH2B1 enhances the activation of IRS-dependent pathways by recruiting IRS proteins to the receptor complex.100,101 Transgenic mouse models have revealed an important role of neuronal SH2B1 in the control of leptin sensitivity and energy homeostasis.102

Activation of ATP-Sensitive K Channels

Activation of ATP-sensitive K channels by leptin was first observed in certain hypothalamic neurons and pancreatic -cells.103,104 Hyperpolarization due to the enhanced K conductance results in reduced neuronal firing and inhibition of insulin secretion from -cells, respectively. Activation of the ATP-sensitive K channels depends on the IRSPI3KPIP3 pathway and is mediated by direct binding of PIP3 to the ATP binding site of the channel.91,105,107 In the hypothalamus, this pathway


9

can explain the leptin-induced hyperpolarization of NPY/AgRP neurons, whereas depolarization of POMC neurons must obviously be accomplished by a different pathway. As far as known, the activation of ATP-sensitive K channels is a unique feature of LEPRb as compared with other cytokine receptors and may reflect the predominantly neuronal action of leptin.

Regulation of AMPK (AMP-Activated Protein Kinase) and mTOR (Mammalian Target of Rapamycin)

AMPK acts as a sensor of cellular energy status and also regulates whole body energy homeostasis by integrating nutrient and hormonal signals in the hypothalamus.108 Leptin regulates AMPK activity in a tissue-specific manner: leptin activates AMPK in muscle and liver, causing suppression of ATP-consuming metabolic pathways (e.g., hepatic glucose production, fatty acid synthesis) and stimulation of ATP-regenerating pathways (e.g., oxidation of intracellular fatty acids).109,110 Thereby leptin improves glucose tolerance and has an antisteatotic effect that protects tissues from the lipotoxicity that is a consequence of leptin deficiency.111 Interestingly, the oral antidiabetic drug, metformin, acts via stimulation of hepatic AMPK activity and thus has leptin-like effects.112 The mechanism of AMPK activation by leptin is unknown but requires JAK kinase activity and does not appear to depend on intracellular tyrosine motifs.110 Surprisingly, no effect of leptin was observed in insulinoma cell lines although AMPK was readily activated by glucose deprivation.33,113 In contrast to its action in muscle and liver cells, leptin reduces AMPK activity in hypothalamic neurons and thus suppresses the stimulatory effect of AMPK on food intake.114,115 However, recent results indicate that the targeted deletion of AMPK activity in POMC and NPY/AgRP neurons did not affect the appetite suppressing effect of leptin but specifically prevented glucose sensing.116 One downstream effect of AMPK is the inhibition of the protein kinase mTOR, which also integrates responses to changes in cellular energy status. Intracerebroventricular administration of leptin was reported to activate hypothalamic mTOR, possibly by preventing its inhibition by AMPK. Inhibition of mTOR by rapamycin caused an increase in food intake, demonstrating the role of this pathway in appetite regulation.80 Further studies will be necessary to fully elucidate the role of mTOR in leptin signaling.

Perspective

In the recent years, the analysis of transgenic mouse models has advanced our understanding of the signaling pathways that are important for the weight regulatory effect of leptin. In contrast, very little is known about the molecular mechanisms by which potentially negative effects are controlled, in particular enhanced immune responses in autoimmune diseases.117 The development of leptin antagonists to block the unwanted effects of leptin emphasizes the need to understand the mechanisms by which LEPRb produces these effect (refs.118,119, see also the other chapters of this book120,121). Although several inflammation-related genes have been found to be upregulated by leptin (Table 1), their role in vivo has yet to be determined. The analysis of transgenic mice with specific mutations of individual tyrosine residues in the intracellular part of LEPRb should offer valuable insight in the molecular mechanisms of leptin’s immune-modulatory and other peripheral effects and provide potential new targets for drug development.

Acknowledgements

I am grateful to Hans-Georg Joost for having introduced me to the study of leptin and its receptor. I wish to thank all past and present members of my lab and collaborating groups for contributing to our effort to understand leptin signaling. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 542 TP-B3). This article is dedicated to Professor Hans-Georg Joost on occasion of his 60th birthday.

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57. Harris M, Aschkenasi C, Elias CF et al. Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling. J Clin Invest 2001; 107:111-120. 58. Guo F, Bakal K, Minokoshi Y et al. Leptin signaling targets the thyrotropin-releasing hormone gene promoter in vivo. Endocrinology 2004; 145:2221-2227. 59. Huo L, Münzberg H, Nillni EA et al. Role of signal transducer and activator of transcription 3 in regulation of hypothalamic trh gene expression by leptin. Endocrinology 2004; 145:2516-2523. 60. Liang CP, Tall AR. Transcriptional profiling reveals global defects in energy metabolism, lipoprotein and bile acid synthesis and transport with reversal by leptin treatment in ob/ob mouse liver. J Biol Chem 2001; 276:49066-49076. 61. Bjørbæk C, Lavery HJ, Bates SH et al. SOCS3 mediates feedback inhibition of the leptin receptor via Tyr985. J Biol Chem 2000; 275:40649-40657. 62. Hekerman P, Zeidler J, Korfmacher S et al. Leptin induces inflammation-related genes in RINm5F insulinoma cells. BMC Mol Biol 2007; 8:41. 63. Costa-Pereira AP, Tininini S, Strobl B et al. Mutational switch of an IL-6 response to an interferon-gamma-like response. Proc Natl Acad Sci USA 2002; 99:8043-8047. 64. Kim YB, Uotani S, Pierroz DD et al. In vivo administration of leptin activates signal transduction directly in insulin-sensitive tissues: overlapping but distinct pathways from insulin. Endocrinology 2000; 141: 2328-2339. 65. Kaszubska W, Falls HD, Schaefer VG et al. Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line. Mol Cell Endocrinol 2002; 195:109-118. 66. Morton NM, Emilsson V, Liu Y-L et al. Leptin action in intestinal cells. J Biol Chem 1998; 273:26194-26201. 67. Wang Y, Kuropatwinski KK, White DW et al. Leptin receptor action in hepatic cells. J Biol Chem 1997; 272:16216-16223. 68. Cao Q, Mak KM, Ren C et al. Leptin stimulates tissue inhibitor of metalloproteinase-1 in human hepatic stellate cells: respective roles of the JAK/STAT and JAK-mediated H2O2-dependant MAPK pathways. J Biol Chem 2004; 279:4292-4304. 69. McCowen KC, Chow JC, Smith RJ Leptin signaling in the hypothalamus of normal rats in vivo. Endocrinology 1998; 139: 4442-4447. 70. Gong Y, Ishida-Takahashi R, Villanueva EC et al. The long form of the leptin receptor regulates STAT5 and ribosomal protein S6 via alternate mechanisms. J Biol Chem 2007; 282:31019-31027. 71. Mütze J, Roth J, Gerstberger R et al. Nuclear translocation of the transcription factor STAT5 in the rat brain after systemic leptin administration. Neurosci Lett 2007; 417:286-291. 72. Li C, Friedman JM. Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction. Proc Natl Acad Sci USA 1999; 96:9677-9682. 73. Myers MG. Leptin receptor signaling and the regulation of mammalian physiology. Recent Prog Horm Res 2004; 59:287-304. 74. Shin HJ, Oh J, Kang SM. Leptin induces hypertrophy via p38 mitogen-activated protein kinase in rat vascular smooth muscle cells. Biochem Biophys Res Commun 2005; 329:18-24. 75. Cui H, Cai F, Belsham DD. Leptin signaling in neurotensin neurons involves STAT, MAP kinases ERK1/2 and p38 through c-Fos and ATF1. FASEB J 2006; 20:2654-2656. 76. Carpenter LR, Farruggella TJ, Symes A et al. Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor. Proc Natl Acad Sci USA 1998; 95:6061-6066. 77. Bjørbaek C, Buchholz RM, Davis SM et al. Divergent roles of SHP-2 in ERK activation by leptin receptors. J Biol Chem 2001; 276:4747-4755. 78. Elias CF, Aschkenasi C, Lee C et al. Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron 1999; 23:775-786. 79. Kim H, Baumann H. Dual signaling role of the protein tyrosine phosphatase SHP-2 in regulating expression of acute-phase plasma proteins by interleukin-6 cytokine receptors in hepatic cells. Mol Cell Biol 1999; 19:5326-5338. 80. Münzberg H, Jobst EE, Bates SH et al. Appropriate inhibition of orexigenic hypothalamic arcuate nucleus neurons independently of leptin receptor/STAT3 signaling. J Neurosci 2007; 27:69-74. 81. Björnholm M, Münzberg H, Leshan R et al. Mice lacking inhibitory leptin receptor signals are lean with normal endocrine function. J Clin Invest 2007; 117:1354-1360. 82. Cota D, Proulx K, Smith KA et al. Hypothalamic mTOR signaling regulates food intake. Science 2006; 312:927-930. 83. Wauman J, De Smet AS, Catteeuw D et al. Insulin Receptor Substrate 4 Couples the Leptin Receptor to Multiple Signaling Pathways. Mol Endocrinol 2008; 22(4):965-977. 84. Huang W, Dedousis N, Bhatt BA et al. Impaired activation of phosphatidylinositol 3-kinase by leptin is a novel mechanism of hepatic leptin resistance in diet-induced obesity. J Biol Chem 2004; 279:21695-21700.


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85. Anderwald C, Müller G, Koca G et al. Short-term leptin-dependent inhibition of hepatic gluconeogenesis is mediated by insulin receptor substrate-2. Mol Endocrinol 2002; 16:1612-1628. 86. Kitamura T, Kitamura Y, Kuroda S et al. Insulin-induced phosphorylation and activation of cyclic nucleotide phosphodiesterase 3B by the serine-threonine kinase Akt. Mol Cell Biol 1999; 19:6286-6296. 87. Szanto I, Kahn CR. Selective interaction between leptin and insulin signaling pathways in a hepatic cell line. Proc Natl Acad Sci USA. 2000; 97:2355-2360. 88. Kellerer M, Koch M, Metzinger E et al. Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 ( JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways. Diabetologia 1997; 40:1358-1362. 89. Xu AW, Kaelin CB, Takeda K et al. PI3K integrates the action of insulin and leptin on hypothalamic neurons. J Clin Invest 2005; 115:951–958. 90. Benomar Y, Roy AF, Aubourg A et al. Cross down-regulation of leptin and insulin receptor expression and signalling in a human neuronal cell line. Biochem J 2005; 388:929-939. 91. Ning K, Miller LC, Laidlaw HA et al. A novel leptin signalling pathway via PTEN inhibition in hypothalamic cell lines and pancreatic beta-cells. EMBO J 2006; 25: 2377-2387. 92. Zhao AZ, Shinohara MM, Huang D et al. Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes. J Biol Chem 2000; 275:11348-11354. 93. Zhao AZ, Bornfeldt KE, Beavo JA. Leptin inhibits insulin secretion by activation of phosphodiesterase 3B. J Clin Invest 1998; 102:869-873. 94. Zhao AZ, Huan JN, Gupta S et al. A phosphatidylinositol 3-kinase phosphodiesterase 3B-cyclic AMP pathway in hypothalamic action of leptin on feeding. Nat Neurosci 2002; 5:727-728. 95. Niswender KD, Morton GJ, Stearns WH et al. Intracellular signalling. Key enzyme in leptin-induced anorexia. Nature 2002; 413:794-795. 96. Morrison CD, Morton GJ, Niswender KD et al. Leptin inhibits hypothalamic Npy and Agrp gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling. Am J Physiol Endocrinol Metab 2005; 289:E1051-1057. 97. Kohno D, Nakata M, Maekawa F et al. Leptin suppresses ghrelin-induced activation of neuropeptide Y neurons in the arcuate nucleus via phosphatidylinositol 3-kinase- and phosphodiesterase 3-mediated pathway. Endocrinology 2007; 148:2251-2263. 98. Choudhury AI, Heffron H, Smith MA. The role of insulin receptor substrate 2 in hypothalamic and beta cell function. J Clin Invest 2005; 115:940-950. 99. Maures TJ, Kurzer JH, Carter-Su C. SH2B1 (SH2-B) and JAK2: a multifunctional adaptor protein and kinase made for each other. Trends Endocrinol Metab 2007; 18:38-45. 100. Li Z, Zhou Y, Carter-Su C et al. SH2B1 enhances leptin signaling by both Janus kinase 2 Tyr813 phosphorylation-dependent and -independent mechanisms. Mol Endocrinol 2007; 21:2270-2281. 101. Duan C, Li M, Rui L. SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin. J Biol Chem 2004; 279:43684-43691. 102. Ren D, Zhou Y, Morris D et al. Neuronal SH2B1 is essential for controlling energy and glucose homeostasis. J Clin Invest 2007; 117:397-406. 103. Spanswick D, Smith MA, Groppi VE et al. Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels. Nature 1997; 390:521-525. 104. Kieffer TJ, Heller RS, Leech CA et al. Leptin suppression of insulin secretion by the activation of ATP-sensitive K channels in pancreatic beta-cells. Diabetes 1997; 46(6):1087-1093. 105. Plum L, Ma X, Hampel B et al. Enhanced PIP3 signaling in POMC neurons causes KATP channel activation and leads to diet-sensitive obesity. J Clin Invest 2006; 116:1886-1901. 106. Harvey J, McKay NG, Walker KS et al. Essential role of phosphoinositide 3-kinase in leptin-induced K(ATP) channel activation in the rat CRI-G1 insulinoma cell line. J Biol Chem 2000; 275:4660-4669. 107. MacGregor GG, Dong K, Vanoye CG et al. Nucleotides and phospholipids compete for binding to the C terminus of KATP channels. Proc Natl Acad Sci USA 2002; 99:2726-2731. 108. Claret M, Smith MA, Batterham RL et al. AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons. J Clin Invest 2007; 117:2325-2336. 109. Minokoshi Y, Kim YB, Peroni OD et al. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 2002; 415:339-343. 110. Uotani S, Abe T, Yamaguchi Y. Leptin activates AMP-activated protein kinase in hepatic cells via a JAK2-dependent pathway. Biochem Biophys Res Commun 2006; 351:171-175. 111. Unger RH. The hyperleptinemia of obesity-regulator of caloric surpluses. Cell 2004; 117:145-146. 112. Misra P. AMP activated protein kinase: a next generation target for total metabolic control. Expert Opin Ther Targets 2008; 12:91-100.

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113. Leclerc I, Woltersdorf WW, da Silva Xavier G et al. Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion. Am J Physiol Endocrinol Metab 2004; 286:E1023-1031. 114. Minokoshi Y, Alquier T, Furukawa N et al. AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 2004; 428:569-574. 115. Andersson U, Filipsson K, Abbott CR et al. AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem 2004; 279:12005-12008. 116. Claret M, Smith MA, Batterham RL et al. AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons. J Clin Invest 2007; 117:2325-2336. 117. La Cava A, Matarese G. The weight of leptin in immunity. Nat Rev Immunol 2004; 4:371-379. 118. Peelman F, Iserentant H, Eyckerman S et al. Leptin, immune responses and autoimmune disease. Perspectives on the use of leptin antagonists. Curr Pharm Des 2005; 11:539-548. 119. Gertler A. Development of leptin antagonists and their potential use in experimental biology and medicine. Trends Endocrinol Metab 2006; 17:372-378. 120. Elinav E, Gertler A. Use of leptin antagonists as anti-inflammatory and anti-fibrotic reagents. In: Gertler A, ed. Leptin and Leptin Antagonists. Austin: Landes Bioscience, 2009; 133-140. 121. Matarese G, DeRosa V. Use of anti-leptin or anti-leptin receptor antibodies as blockers of immune response. In: Gertler A, ed. Leptin and Leptin Antagonists, Austin: Landes Bioscience, 2009; 126-132. 122. Laubner K, Kieffer TJ, Lam NT et al. Inhibition of preproinsulin gene expression by leptin induction of suppressor of cytokine signaling 3 in pancreatic beta-cells. Diabetes. 2005; 54:3410-3417. 123. Hosoi T, Okuma Y, Nomura Y. Leptin regulates interleukin-1beta expression in the brain via the STAT3-independent mechanisms. Brain Res 2002; 949:139-146. 124. Pinteaux E, Inoue W, Schmidt L et al. Leptin induces interleukin-1beta release from rat microglial cells through a caspase 1 independent mechanism. J Neurochem 2007; 102:826-833. 125. Dreyer MG, Juge-Aubry CE, Gabay C et al. Leptin activates the promoter of the interleukin-1 receptor antagonist through p42/44 mitogen-activated protein kinase and a composite nuclear factor kappa B/ PU.1 binding site. Biochem J 2003; 370:591-599. 126. Lin S, Saxena NK, Ding X et al. Leptin increases tissue inhibitor of metalloproteinase I (TIMP-1) gene expression by a specificity protein 1/signal transducer and activator of transcription 3 mechanism. Mol Endocrinol 2006; 20:3376-3388. 127. Broekaert D, Eyckerman S, Lavens D et al. Comparison of leptin- and interleukin-6-regulated expression of the rPAP gene family: evidence for differential co-regulatory signals. Eur Cytokine Netw 2002; 13:78-85. 128. Gong L, Yao F, Hockman K et al. Signal transducer and activator of transcription-3 is required in hypothalamic agoutirelated protein/neuropeptide Y neurons for normal energy homeostasis. Endocrinol 2008; 149:3346-3354. 129. Jiang L, Li Z, Rui L. Leptin stimulates both JAK2-dependent and JAK2-independent signaling pathways. J Biol Chem 2008; 283:28066-28073.

Chapter 2

Insights in the Activated LR Complex and the Rational Design of Antagonists Frank Peelman, Lennart Zabeau and Jan Tavernier*

Introduction

T

he hormone leptin plays an important role in the control of body weight. Leptin is mainly produced and secreted by adipocytes as a 16 kDa nonglycosylated polypeptide and plasma leptin levels positively correlate with body fat energy stores.1,2 To a lesser extent, leptin is also expressed in other tissues such as the epithelium of the stomach, placenta, skeletal muscle and brain.3,4 Spontaneous loss of function mutations in the leptin encoding ob gene (for example in ob/ ob mice) give rise to a complex syndrome that includes morbid obesity, hypothermia, infertility, hyperglycemia, decreased insulin sensitivity and hyperlipidemia.5 Leptin turned out to be a quite pleiotropic cytokine and its effects are not restricted to energy homeostasis, but also include neuroendocrine function,6 angiogenesis,7 bone formation,8 reproduction9 and immune responses.10 Leptin mediates its effects by binding and activation of the leptin receptor (LR), encoded by the db gene.11 Loss of function mutations in the db gene lead to a phenotype that is comparable to that of the ob/ob mouse. The LR is a single-membrane spanning class I cytokine receptor. Like all members of the class I cytokine receptor family, the receptor has no intrinsic kinase activity and uses cytoplasmic-associated Janus kinase 2 ( JAK2) for intracellular signalling. In a generally accepted model, leptin-binding leads to formation of an activated receptor complex, allowing JAK2 cross-phosphorylation. JAK2 then rapidly phosphorylates several tyrosine residues in the cytosolic domain of the receptor (in the case of the mouse LR, tyrosines at positions 985, 1077 and 1138). Phosphorylated tyrosines 1077 and 1138 bind STAT5 (signal transducer and activator of transcription 5), while tyrosine 1138 further recruits STAT1 and STAT3.12,13 Although other STATs can be recruited, STAT3:STAT3 dimers are the most dominant after leptin stimulation. Once recruited, STATs themselves become a substrate for JAKs and homo- or heterodimerize upon phosphorylation, translocate to the nucleus and modulate transcription of target genes. Other signalling pathways activated by the LR include MAPK14 and phosphoinositide 3 kinase pathways.15 Thus far, six LR isoforms have been identified (LRa-f ): one long form (LRb or LRlo) and four short forms (LRa,c,d,f ) are generated by alternative splicing. A sixth, soluble form (LRe) is a result of ectodomain shedding and/or alternative splicing in respectively men and mice. High expression of LRlo, the major signalling isoform, is observed in certain nuclei of the hypothalamus,16 a region of the brain involved in the regulation of body weight. Expression could also be shown in several other cell types including liver, pancreas, lung, kidney, adipose tissues, endothelial cells and cells of the immune system, thereby forming the basis of several peripheral biological functions of leptin. *Corresponding Author: Jan Tavernier—Department of Medical Protein Research, VIB, and Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. Email: [email protected]


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Leptin as a Disease-Promoting Factor: Rationale for Leptin Antagonists

It has become clear over the last few years that leptin plays a role in both innate and adaptive immunity (reviewed in ref. 10). In innate immunity, leptin promotes secretion of inflammatory cyto-kines and the activation of macrophages, neutrophils and natural killer cells. Functions in adaptive immunity include thymic homeostasis, naïve CD4+ cell proliferation and promotion of T helper 1 (TH1) responses. Moreover, leptin can act as a negative signal for the expansion of CD4+CD25high regulatory T-cells (TRegs), a T-cell subpopulation known to dampen immune reactions.17 Leptin is involved in the onset and/or progression of several T-cell controlled autoimmune diseases, like Crohn’s disease,18 rheumatoid arthritis,19 multiple sclerosis20 and autoimmune hepatitis.21 Leptin or LR deficiency can protect against onset of experimentally induced diseases in rodents. In leptin deficient animals, leptin administration results in a switch from TH2 to TH1 controlled responses. Furthermore, administration to wild type mice worsens the clinical manifestations in these models for autoimmune diseases. In some of these diseases, in situ production of the cytokine could be shown in active inflammatory lesions, thereby representing a significant local source of leptin. Overweight is a risk factor for postmenopausal breast cancer. The LR is expressed on breast cancer cells and promotes their growth in vitro.22 Cleary et al crossed MMTV-TGF-alpha mice, which develop mammary tumors, with ob/ob or db/db mice.23 In the MMTV-TGF-alpha female mice, tumor incidence increases with increased body weight and vice versa. However, both the obese MMTV-TGF-alpha/Lep(ob)Lep(ob) and MMTV-TGF-alpha/Lep(db)Lep(db) female mice do not develop mammary tumors, strongly supporting the idea that leptin is a necessary factor for mammary tumor development. The involvement of leptin in immune diseases and breast cancer provided a rationale for the development of leptin antagonists. Different strategies can be used to reduce leptin’s activities. Anti-leptin antibodies or soluble LR that scavenge free leptin in circulation24 and blocking antibodies against the LR.25 Another approach is the use of leptin mutants or synthetic peptides derived from leptin that block LR activation.26-28 In this chapter, we discuss recent insights in the mechanism of LR activation and how these led to the development of leptin antagonists. We summarize how these insights can be used to guide the optimization of leptin antagonists.

Structure of Leptin and Its Receptor Homology with the IL-6 and G-CSF Receptor Systems

The structure of leptin revealed by crystallography showed that leptin is a four helix-bundle cytokine: 4 α-helices are arranged in a typical up-up-down-down fold.29 The leptin structure shows the highest similarity with the long chain α-helical cytokines of the interleukin-6 (IL-6) family, including IL-6, leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM) and with granulocyte-colony stimulating factor (G-CSF). To a lesser extent, it also resembles the other long chain α-helical cytokines, such as growth hormone (GH) and prolactin. The LR belongs to the class I cytokine receptor family, which typically contains a so-called cytokine receptor homology (CRH) domain in its extracellular domain. This CRH structure consists of two barrel-like domains, each around 100 amino acids in length, which resemble the fibronectin type III (FN III) fold. Two conserved disulfide bridges are found in the N-terminal domain, while a WSXWS motif is characteristic for the C-terminal part. The LR contains two such CRH domains, CRH1 and CRH2, which are separated by an immunoglobulin-like (Ig) domain and followed by two membrane proximal FN III domains (Fig. 1). Based on sequence similarity and overall architecture of the ectodomains, the LR is most related to the G-CSF receptor and the glycoprotein 130 (gp130) family receptors, including gp130, LIF and OSM receptors. Unique to the LR is the presence of an additional N-terminal CRH module and two, instead of three, FN III domains (Fig. 1). The N-terminal CRH domain seems to be preceded by an additional domain of

Insights in the Activated LR Complex and the Rational Design of Antagonists

17

Figure 1. Topology of the LR, compared with the topology of the erythropoietin (Epo) receptor, the gp130 family receptors and the G-CSF receptor.

approximately 100 residues, which shows no clear sequence homology to other domains. The disulfide pattern and secondary structure suggest that it might be a degenerated Ig-like domain.30 The CRH2 domain is the main high-affinity leptin binding site.31-33 The other domains do not show detectable leptin binding when expressed in vitro. All domains are necessary for receptor activation, except for CRH1, deletion of which reduces the full receptor activation capacity by about 50%.31,33,34

Evidence for Receptor Oligomerisation and Higher Order Clustering LR Oligomerisation in the Absence of Ligand

Many cytokine receptors exist as inactive, preformed complexes on the cellular surface. Examples include the receptors for Epo,35-37, GR38 and IL-6.39 There is a growing body of evidence that also the LR appears as ligand-independent oligomers: purified soluble extracellular LR domain from baculovirus-infected insect cells behaves as dimers in SDS-PAGE and gelfiltration experiments.40,41 This clustering could also be demonstrated with membrane-bound receptors.42,43 White and coworkers extended these findings and showed that LR long and short homo-oligomerize in the absence of ligand, while hetero-oligomerisation between both isoforms was only observed in the presence of leptin.42 This may help to explain why the long form is able to signal in the presence of an excess signal-deficient short forms as seen in many tissues. A quantitative bioluminescence resonance energy transfer (BRET) approach illustrated that in living cells 60% of the LR exists as constitutive dimers.44 Using a series of LR deletion and cysteine to serine mutants, we recently demonstrated that this clustering most likely involves disulphide bridges between residues of the CRH2 domain.43

LR Becomes Activated Upon Higher Order Clustering

We examined the requirements for leptin signalling in more detail with a complementation-of-signalling strategy.33 Here, the LR was made signalling deficient in two ways: in the LR-F3 mutant all cytoplasmic tyrosines were mutated to phenylalanines, while in the LR ∆box1 mutant

18


two prolines necessary for JAK activation were replaced by alanines. While these mutants are both unable to signal via the JAK/STAT pathway, a clear STAT3-dependent signal is generated upon leptin stimulation when they are co-expressed in cells. Assuming that JAK/STAT signalling requires at least two JAK kinases and one tyrosine residue, the complementation can only be explained by the presence of at least three LR chains in the leptin:LR complex. The complementation of signalling is completely lost when the extracellular domains of the mutants are replaced by that of the strict homodimeric EpoR, suggesting that the higher order clustering is determined by the extracellular domains of the LR.

Three Binding Sites in Leptin

4-Helix bundle cytokines activate their receptors by contacting two or more receptor subunits through multiple binding sites in the cytokine. This orientates the extracellular domains of the receptor chains in the right position for receptor activation. Epo and GH bind to their receptors through two binding sites.45,46 Binding site I is found at the fourth helix (helix D) and contacts with the CRH domain of a first receptor. Binding site II is formed by the surfaces of the anti-parallel first and third helix (helices A and C) and binds the CRH domain of a second receptor. The homodimeric Epo and GH receptors thus use the same CRH binding epitope to bind to two totally different binding sites in their cytokine ligand.45,46 Cytokines of the IL-6 family and G-CSF contain a third receptor binding site at the N-terminus of helix D.47,48 This binding site III binds to an Ig-like domain in the receptor. In the IL-6 receptor complex, IL-6 uses three binding sites: binding site I binds to the CRH domain of the IL-6Rα. Binding site II binds to the CRH domain of a first gp130 chain, leading to a heterotrimeric IL-6:IL-6Rα:gp130 complex. Two trimers subsequently form a hexamer, in which binding site III of IL-6 contacts the immunoglobulin-like domain of a second gp130 chain (Fig. 2). The G-CSF:G-CSF receptor system does not have a binding site I or an α-receptor chain. This receptor complex is 2:2 tetramer. Binding site II of G-CSF binds to the CRH domain of a first G-CSF receptor chain, while binding site III binds to the Ig-like domain of the second G-CSF receptor chain (Fig. 3). Structural superposition of the leptin crystal structure with other four helix bundle cytokines was used to identify the position of possible binding sites I, II or III in leptin. Residues in these areas were mutated and the leptin mutants were tested in LR activation assays and in an assay that determines their binding to CRH2. A predicted binding site II is found in the middle of helices A and C (for a schematic representation of the secondary structures within leptin, see Fig. 4). Mutations in this site show a clearly decreased affinity for the CRH2 domain, suggesting that binding site II interacts with this domain. Surprisingly, the mutants did not show a large decrease

Figure 2. The 2:2:2 IL-6:IL-6Rα:gp130 complex.


19

Figure 3. The 2:2 G-CSF:G-CSF receptor complex.

in EC50 value or maximal LR activation. This finding is unexpected and suggests that the loss of affinity for CRH2 might be compensated by other leptin:receptor interactions. The contribution of such additional interactions to high affinity binding might explain why several studies report a slightly higher affinity of leptin for the full-length LR than for CRH2 (summarized in ref. 49). The predicted binding site III is found around the N-terminus of helix D and contains residues in the A-B and C-D loops. Several mutations in binding site III led to a strong decrease in the maximal LR activation. Binding site I is found in the helical face of helix D and the A-B loop. Mutations in binding site I in this study had a less pronounced effect, with moderately decreased maximal receptor activation potential. While mutations in binding sites I and III both decrease maximal receptor activation potential, they do not affect binding to CRH2 or the EC50 value for LR activation. Niv-Spector et al used a different approach to identify binding site III in leptin.27 In the viral IL-6:gp130 complex, the binding site III interaction involves a hydrophobic strand at the N-terminus of the gp130 Ig-like domain that interacts with a hydrophobic strand in the viral IL-6. Using hydrophobic cluster analysis, similar hydrophobic strands were predicted at residues 39 to 42 in leptin and at residues 325-328 in the LR Ig-like domain. Mutations of two or more residues to alanine in these predicted strands in leptin abolished LR activation capacity. These do not affect the secondary structure of the protein, or binding to the LR or the isolated CRH2 domain. Similarly, mutation of residues 325-328 to alanines in the predicted strand at the N-terminus of the Ig-like domain drastically reduces LR activation. Based upon the mutagenesis of leptin and analogy with other receptor systems, we propose the following interaction scheme for leptin/receptor complex: Binding site II in leptin interacts with the CRH2 domain (Fig. 5). Binding site III interacts with the Ig-like domain of a second LR chain. Since no α-receptor chain is necessary for leptin, it might interact with a third or fourth LR chain. These predicted interactions of leptin and its

Figure 4. Schematic representation of the secondary structures within the leptin molecule. Boxes represent helices, lines the connecting loops. Numbers are the positions of the beginning and end of the helices.

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Figure 5. The hexameric 2:4 leptin:LR complex.

receptor were further investigated by homology modelling and mutagenesis of the LR domains and modelling of the leptin-receptor complex.

Models of the CRH2-Leptin Complex

Several homology models of leptin bound to CRH2 have been presented.32,50,51 Hiroike et al presented a model of a 2:2 leptin/CRH2 complex based upon a crystal structure of a 2:2 complex of G-CSF with the G-CSF receptor CRH.50 In this model, each leptin molecule contacts two different CRH2 domains via a major and minor binding site.50 However, the minor site in the G-CSF later turned out to be an artefact of crystallization. 48 Nonetheless, the major interface in the model corresponds with the interface proposed in the models described below. Sandowski et al built a homology model for the complex of human leptin with the CRH2 domain, based on the crystal structures of the gp130 CRH domain and of GH bound to the CRH of its receptor.32 This model was later used to model the CRH2 of the chicken LR.52 We built a homology model of the mouse leptin:CRH2 domain using a superposition of the structures of several cytokine:CRH complexes as a guide for the alignment and the structure of the G-CSF:G-CSF receptor complex as template.51 The leptin CRH2 interface model resembles in many aspects the model of Hiroike et al.50 The recombinant chicken CRH2 domain, expressed in E. coli was extensively mutated by Niv-Spector et al and the leptin binding properties of the mutants were tested in vitro.52 We made mutants of the recombinant mouse CRH2 domain, expressed in COS-1 cells and tested the effect of the mutations on leptin binding.51 The same mutations were introduced in the full-length LR and their effect on LR JAK/STAT signalling was tested. Both mutagenesis studies demonstrate the importance of a region of four consecutive hydrophobic residues in CRH2: 501-IFFL-504 in mouse LR CRH2, 504-VFLL-507 in chicken LR CRH2 (Fig. 6). Mutations at these residues affect leptin binding. In all homology models, the region forms a central part of the interaction surface with leptin. In the mouse leptin:CRH2 homology model, the four hydrophobic residues become buried upon leptin binding (Fig. 6). The residues make contact with L13 and L86 in mouse leptin. L86 mutants have lower affinity for CRH2 and the L86S mutant has a drastically increased EC50 value for LR activation.51 Leptin residues that were predicted to be part of binding site II by structural superposition with other cytokines,28 all interact with the CRH2 domain in the model: D9,


21

Figure 6. Model for the leptin:CRH2 complex, with indication of the mutations in leptin (yellow) or CRH2 (blue) that affect the KD of the interaction. A color version of this image is available at www.landesbioscience.com/curie.

T12, L13, K15, T16 in helix A, N78, N82, D85, L86 in helix C. Mutations of these residues lead to lower affinity for CRH2 in in vitro binding assays28,51 (Fig. 6).

Models for the Ig-Like and CRH1 Domains

In the gp130 receptor family and the G-CSF receptor, the Ig-like domain interacts with binding site III in the cytokine ligand. We built homology models for the Ig-like domain and pinpointed possible binding site III interaction residues by alignment/superposition with Ig-like domains of gp130 family receptors and the G-CSF receptor. The residues were mutated in the mouse LR and the effects of the mutations on LR activation were tested. Several of these mutations have a drastic effect on the maximal response to leptin stimulation, without affecting the EC50 value, similar to the effect seen with the leptin binding site III mutants. These mutants form a continuous cluster on the surface of the Ig-like domain, at a position that superposes with the binding site III-interacting region in the Ig-like domain of the IL-6 family and G-CSF receptors (Fig. 7). We therefore propose that this cluster of residues interacts with leptin binding site III. The area of the cluster in the Ig-like domain has a positive electrostatic surface potential, that is probably compatible with the negative electrostatic surface potential found in our predicted binding site III in leptin, which is situated around the N-terminus of helix D (Fig. 7). The Study of Niv-Spector et al predicts binding site III around residue 39-42 in leptin, in contrast with our study, which places binding site III around the N-terminus of helix D. The two predicted binding sites III are actually quite distant from each other in the crystal structure. When the leptin crystal structure is superposed onto the G-CSF molecule in the crystal structure of the G-CSF receptor complex, residues 39-42 do not come near the Ig-like domain. Similar structural

22


superposition of the leptin crystal structure onto the IL-6 molecule in the crystal structure of the extracellular part of the IL-6 receptor complex, brings residues 39-42 at binding site I, in contact with the IL-6Rα. In contrast, the region surrounding the N-terminus of helix D approaches the Ig-like domains in both superpositions. We therefore propose that residues 39-42 are part of binding site I and that mutations at these position affect LR activation by affecting binding site I. Niv-Spector et al used hydrophobic cluster analysis to predict a hydrophobic strand at the N-terminus of the Ig-like domain (residues 325-VFTT-328) that might be involved in binding site III interactions and this in analogy with the virus Il-6/gp130 complex. Mutation of residues 325-328 to four alanines abolishes LR activation. However, homology modelling of the CRH1 domain demonstrates that these residues are probably an integral part of the structure of CRH1 and that most of the hydrophobic residues in the strand are buried inside CRH1. It is therefore unlikely that residues 325-328 are part of binding site III. Moreover, deletion of the entire CRH1 domain, including residues 325-328 only leads to 50% reduction of the maximal LR activation.34 The role of the CRH1 domain remains elusive. A Q269P mutation in the CRH1 domain causes the obesity in the fa/fa rat, with defective and partially constitutive LR signalling.53-55 In our model for CRH1, the Q269P mutation leads to severe steric clashes between the introduced proline residue and the first tryptophan of the CRH1 WSXWS motif. This probably affects the stability or the correct folding of this domain. It cannot be excluded that the CRH1 domain might be more important at lower, physiologically relevant LR expression levels and that some effects of CRH1 deletion are not detected at the high LR expression levels in in vitro overexpression systems.

Homology Model for a Hexameric 2:4 Leptin:LR Complex

Based upon mutagenesis data for leptin and its receptor, we proposed a hexameric model for the leptin:LR complex. 2:2 tetramer and 2:4 hexamer leptin:LR complexes were built using the crystal structure of the IL-6 receptor complex as a guide for modelling.34 In the tetramer model, the leptin binding site II:CRH2 interaction is modelled as described above. Binding site III is situated around the N-terminus of helix D and interacts with the Ig-like domain of a second LR chain. Binding site III and the Ig domain might attract each other by an opposing electrostatic surface potential. The binding site II and III interactions would allow the formation of a 2:2 leptin:LR tetramer complex, as found for the G-CSF receptor complex. However, the tetramer model seems to contradict some previous findings: 1. The LR can oligomerize via disulfide bridges. The tetramer model does not allow disulfide bridge formations between LR chains. Disulfide bridges cannot be introduced by simply moving or rotating the receptor chains or addition of models of the FN III domains. 2. Our JAK/STAT complementation assay suggests that the leptin:LR complex must contain more than two receptor chains.33 3. Mutations at positions 39-42 in leptin can have a very strong effect on LR activation capacity. F41 belongs to our predicted binding site I and other mutations in this putative binding site I also affect LR activation. None of the residues 39-42 has an interaction partner in the tetramer model. These three issues can be resolved by considering a hexamer leptin:LR complex (Fig. 8). In the IL-6 receptor complex, binding site I interacts with the CRH domain of the IL-6Rα. We created a hexameric 2:4 leptin:LR model complex by putting CRH2 of the LR at the positions of the IL-6Rα CRH. F41 and binding site I residues in leptin now all interact with the additional CRH2 domains.

Homology Models for the Fibronectin Type III Domains

FN III domains have no detectable affinity for leptin, but are absolutely essential for signalling.43 When these domains are expressed as soluble proteins, they appear as disulfide linked oligomers on SDS-PAGE. The LR contains two conserved cysteines, on positions 672 and 751. Mutation


23 Figure 7, left. A) Model for the Ig-like domain of the LR, with indication of the mutations that affect LR activation (top). The area around the cluster has a positive electrostatic surface potential (blue) (bottom).34 B) Model for mouse leptin with indication of the binding site III mutations that affect LR activation (top). The area around S120 and T121 (circled) has a negative surface potential (red) (bottom).

Figure 8, above. A 2:4 hexameric model for the leptin:LR complex.34 Side view, with indication of the mutations in binding sites II that affect binding to CRH2 (purple), or mutations in binding site I (orange) or III (red) that affect maximal LR activation.34,52

Figure 9, right. Homology model of the tandem FN III domains of the mouse LR. Two cysteines are exposed at the surface and potentially capable of inter-chain disulfide bridges.

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of C751 to serine has limited effect on ligand binding and receptor activation, the C672S mutant exhibits a marked reduction in STAT3-dependent signalling. The double mutant is completely devoid of biological activity, although leptin binding remains unaffected. The FN III domains connect the leptin interacting CRH2 and Ig-like domains with the transmembrane domain and thus may act as levers that communicate LR rearrangements induced by leptin binding to the transmembrane and intracellular domains. A receptor variant with an extracellular domain consisting of only the FN III domains shows a marked increase in ligand-independent signalling. This illustrates that these domains can position the intracellular domains in such a way that JAK activation and thus signalling are possible. Homology models for the LR FN III domains were built using the crystal structure of type III repeats 7-10 of fibronectin56 as a template. In Figure 9, the FN III domains are illustrated. The cysteine residues are found on the surface, accessible for possible inter-chain disulphide formation.

Mechanism of LR Activation

The following models for LR activation can be proposed: Leptin first binds to the CRH2 domain of a first LR via its binding site II. After this first high affinity binding step, two models are possible: Model 1: The bound leptin molecule binds to a second LR chain via CRH2:binding site I interactions (Fig. 10). These trimeric complexes subsequently interact with a third LR chain or another trimer complex via binding site III and the Ig-like domain: Model 2: The dimeric leptin:LR complexes form tetramers via interactions of binding site III and the Ig-like domain (Fig. 11). Subsequently, leptin binding site I interacts with the CRH2 domain of additional LR chains: These models are in line with the presence of three binding sites, the oligomeric nature of the LR and the JAK/STAT complementation assay, but remain hypothetical at present. It is also not

Figure 10. Model 1.

Figure 11. Model 2.


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clear why the LR has developed such a complicated activation process, when other homomeric receptors, such as the Epo, GH and G-CSF receptors work by much simpler mechanisms. The LR exists as preformed oligomers, possibly linked by disulphide bridges. Leptin binding could lead to a spatial reorganisation of receptor chains in the preformed complex, resulting in correct positioning and activation of the cytoplasmic associated JAK kinases. This hypothesis is supported by BRET experiments. In cells expressing short LR forms fused to luciferase and YFP, leptin treatment resulted in a marked enhancement in energy transfer signals, possibly reflecting specific conformational changes.44

Development of Leptin-Based Antagonists

Insight into the binding sites of leptin and its interaction with the LR has led to opportunities for the development for leptin antagonists. Mutations that affect the initial binding step via site II can affect the EC50 value for LR activation, as shown by the L86S leptin mutant.51 Mutations that affect the next interaction steps via binding sites I or III do not affect the EC50 value but affect the maximal LR activation capacity and can even lead to mutants that avidly bind to the receptor without activating it, as is the case for the S120A/T121A mutant and for mutations at position 39-42. Such mutants are potential LR antagonists: the leptin mutant will bind to CRH2 without subsequent LR activation and will block binding of wild type leptin by competitive binding. Several such mutants have been proposed as leptin antagonists (for an overview, see ref. 49). A first antagonistic leptin mutant is the R128Q human leptin mutant, developed by Verploegen et al.57 The mutant binds normally to the LR, but fails to trigger a proliferative response in LR expressing Ba/F-3 cells. R128Q leptin induces weight gain in mice. However, when the R128Q mutation is introduced in leptin of other species, such as sheep or chicken, it does not always result in an antagonist and sometimes even in a weak agonist.58 Surprisingly, injection of the R128Q mutant in rats resulted in a strong dose-dependent decrease in food intake.59 The human leptin mutant R128Q leptin is therefore not a suitable tool for investigating the physiological actions of leptin. R128 is not part of any of the three binding sites, but is largely buried in the leptin structure. The effects of the R128Q mutation are probably indirect, possibly via binding site I or III. The S120A/T121A mutant was tested in our mutagenesis study of leptin binding site III.28 The mutant showed no LR activation in a JAK/STAT signalling-based luciferase assay in Hek293T cells, while its binding to the CRH2 domain was unaffected. The mutant acted as an inhibitor of wild type human and mouse leptin in the JAK/STAT signalling assay. When injected in mice, it showed a clear induction of weight gain, suggesting that the S120A/T121A mutant is an antagonist in vitro and in vivo. Both the human and the mouse S120A/T121A mutant can inhibit mouse or human LR activation. While the R128Q mutant shows LR activation at higher concentrations in an in vitro JAK/STAT signalling assay, this is not the case for comparable concentrations of the S120A/T121A mutant. Niv-spector et al found that the mutations at residues 39-42 in human and ovine leptin led to leptin mutants that were unable to activate the LR, while retaining normal secondary structure and LR binding.27 These mutants potently antagonize leptin-induced proliferation of Ba/F-3 stably expressing the LR. In a similar way, mouse and rat leptin can be transformed into potent antagonists by introduction of the 39-42 mutations.27 While all the 39-41 mutants are devoid of agonistic activity, the S120A/T121A mutant shows some low agonistic effect in the very sensitive Ba/F-3 proliferation assay.27 Our homology model of a hexameric LR complex suggests that residues 39-42 might be part of a binding site I. This would mean that mutations in binding site I, as well as mutations in binding site III (S120A/T121A) both can block receptor activation and lead to antagonistic leptin molecules. In fact, any molecule that avidly binds to CRH2 without activating the receptor will potentially be an antagonist. This is supported by the work of Gonzalez and Leavis.26 These authors showed that the synthetic peptide LPA-2, corresponding to helix C of human leptin (residues 70-95) is sufficient for high affinity (0,6.10–10 M) binding to the LR. The peptide antagonizes LR activation in vitro and in vivo: intrauterine injection of the peptide reduced the number of implantation sites

26


and uterine horns with implanted embryos60 and local injection of LPA-2 in mammary fat pads blocked mammary tumor growth.61

Optimization of Leptin-Based Antagonists

The aforementioned antagonists all work by binding to CRH2 and blocking the binding of leptin. Full antagonism requires that almost all receptors are blocked. The antagonists are therefore used at concentrations that exceed their KD or their IC50 for antagonsism. In the bloodstream, this translates in a requirement for µg/ml concentrations of antagonist. Unfortunately, leptin has a short circulation half-life, with reported values ranging from 5.4 minutes in rats to 25 minutes in humans.62-64 The same most likely holds true for the antagonistic leptin mutants. Several options exist for extending the half-life of leptin (or leptin mutants): an antibody against leptin can be co-injected with the antagonist thereby drastically increasing its half-life in circulation.57 Another option is the use of fusion proteins, where the antagonist is coupled to a molecule that has a long circulation half-life, such as albumin or the constant chains of Ig. In the leptin:LR complexes, the leptin N-and C-termini point away from the complex, allowing the fusion to other proteins. A fusion protein of leptin S120A/T121A to mouse albumin retains its antagonistic properties, while a fusion protein of leptin S120A/T121A to the Fc portion of a mouse IgG1 becomes slightly agonistic, possibly by the bridging effect of the Fc molecule (Peelman et al, unpublished results). The most favourable solution for extending the half-life of leptin antagonists might be the pegylation of the leptin mutants. Covalent modification with high molecular weight polyethylene glycol (PEG) chains is a very efficient method for improving the pharmacokinetics of biomolocules65 and has been shown to increase the half-life of wild-type leptin.66,67 A branched polyethylene glycol (PEG) N- hydroxysuccinimide (NHS), molecular weight 40 kDa, was used for pegylation. This pegylation reagent covalently binds to amines and leads to very efficient PEGylaytion (>30% of leptin PEGylated), with one or two PEG molecules per labelled leptin;68 our unpublished results). Unfortunately, pegylation of leptin antagonist mutants drastically decreases their antagonistic potency by more than six fold;68 (our unpublished results). A reason for this decrease in efficiency might be that modification of certain lysine residues blocks the interaction with the LR. K5 and K15 for example are part of the predicted leptin:CRH2 interaction interface and modification of these residues would almost certainly decrease binding to CRH2. A solution might be the specific deletion of certain lysines in leptin. Homology models of the leptin:receptor complex are useful guidelines for rational choices for such mutagenesis. Another way to increase the potency of the antagonists would be the increase of the affinity for the receptor. The leptin residues that are important for binding to CRH2 have been thoroughly mapped by mutagenesis studies. This information can be used to guide directed evolution, e.g., through degenerated primers as a strategy to improve the affinity for the CRH2 domain.

Concluding Remarks

The disease promoting role of leptin in animal models for autoimmune diseases and breast cancer has raised interest in leptin antagonists. Injection of anti-leptin antibodies or soluble LR antagonizes leptin’s action by reducing the bioavailable leptin. An alternative approach might be the use of neutralising anti-LR antibodies that block activation. With the exception of CRH1, every extracellular domain of the LR is indispensible for LR activation. Blocking antibodies can thus be targeted against the FN III domains, the CRH2 domain or the Ig-like domains. The study of the leptin:LR interaction has led to the development of binding site I or III leptin mutants, that bind but do not activate the LR and thus work as competitive inhibitors. However, the leptin mutants have very short half-lives in circulation, so modifications, such as pegylation, hyperglycosylation or coupling to a partner with high half-life will be needed to increase their efficiency. The new insights into leptin interaction with its receptor can be used to optimize such modifications. If leptin antagonism turns out to have therapeutic potential, the effect of leptin antagonists on body weight control, glucose metabolism, bone formation and other processes that are regulated by leptin are a concern. Many of these functions are regulated centrally in the hypothalamus. It


27

is at present unclear whether it is feasible to make leptin antagonists that do not have access to targets in the hypothalamus. Leptin is transported through the blood brain barrier by an unknown transporter, possibly involving megalin or the short form of the LR. However, leptin responsive neurons that express the LR or show STAT3 activation can be labelled by BBB impermeable fluorescent tracers.69,70 ARC neurons might therefore make direct contact with the blood-circulation by projections through the BBB. If this scenario is true, it may be almost impossible to discriminate between central and peripheral functions and avoid weight gain while treating leptin-involved autoimmune diseases. A lot of unknowns remain to be solved before leptin antagonists can be considered to be of possible therapeutic value. Leptin antagonists form a new tool that will provide new insights, both in the role of leptin in disease and in the mechanism of leptin.

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24. De Rosa V, Procaccini C, La Cava A et al. Leptin neutralization interferes with pathogenic T-cell autoreactivity in autoimmune encephalomyelitis. J Clin Invest 2006; 116(2):447-455. 25. Fazeli M, Zarkesh-Esfahani H, Wu Z et al. Identification of a monoclonal antibody against the leptin receptor that acts as an antagonist and blocks human monocyte and T-cell activation. J Immunol Methods 2006; 312(1-2):190-200. 26. Gonzalez RR, Leavis PC. A peptide derived from the human leptin molecule is a potent inhibitor of the leptin receptor function in rabbit endometrial cells. Endocrine 2003; 21(2):185-195. 27. Niv-Spector L, Gonen-Berger D, Gourdou I et al. Identification of the hydrophobic strand in the A-B loop of leptin as major binding site III: Implications for large-scale preparation of potent recombinant human and ovine leptin antagonists. Biochem J 2005; 391(Pt 2):221-230. 28. Peelman F, Van Beneden K, Zabeau L et al. Mapping of the leptin binding sites and design of a leptin antagonist. J Biol Chem 2004; 279(39):41038-41046. 29. Zhang F, Basinski MB, Beals JM et al. Crystal structure of the obese protein leptin-E100. Nature 1997; 387(6629):206-209. 30. Haniu M, Arakawa T, Bures EJ et al. Human leptin receptor. Determination of disulfide structure and N-glycosylation sites of the extracellular domain. J Biol Chem 1998; 273(44):28691-28699. 31. Fong TM, Huang RR, Tota MR et al. Localization of leptin binding domain in the leptin receptor. Mol Pharmacol 1998; 53(2):234-240. 32. Sandowski Y, Raver N, Gussakovsky EE et al. Subcloning, expression, purification and characterization of recombinant human leptin-binding domain. J Biol Chem 2002; 277(48):46304-46309. 33. Zabeau L, Defeau D, Van der Heyden J et al. Functional analysis of leptin receptor activation using a Janus kinase/signal transducer and activator of transcription complementation assay. Mol Endocrinol 2004; 18(1):150-161. 34. Peelman F, Iserentant H, De Smet AS et al. Mapping of binding site III in the leptin receptor and modeling of a hexameric leptin receptor complex. J Biol Chem 2006; 281(22):15496-15504. 35. Constantinescu SN, Keren T, Socolovsky M et al. Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain. Proc Natl Acad Sci USA 2001; 98(8):4379-4384. 36. Livnah O, Stura EA, Middleton SA et al. Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation. Science 1999; 283(5404):987-990. 37. Remy I, Wilson IA, Michnick SW. Erythropoietin receptor activation by a ligand-induced conformation change. Science 1999; 283(5404):990-993. 38. Frank SJ. Receptor dimerization in GH and erythropoietin action—It takes two to tango, but how? Endocrinology 2002; 143(1):2-10. 39. Schuster B, Meinert W, Rose-John S et al. The human interleukin-6 (IL-6) receptor exists as a preformed dimer in the plasma membrane. FEBS Letters 2003; 538(1-3):113-116. 40. Devos R, Guisez Y, Van der Heyden J et al. Ligand-independent dimerization of the extracellular domain of the leptin receptor and determination of the stoichiometry of leptin binding. J Biol Chem 1997; 272(29):18304-18310. 41. Rock FL, Peterson D, Weig BC et al. Binding of leptin to the soluble ectodomain of recombinant leptin receptor: a kinetic analysis by surface plasmon resonance. Horm Metab Res 1996; 28(12):748-750. 42. White DW, Tartaglia LA. Evidence for ligand-independent homo-oligomerization of leptin receptor (OB-R) isoforms: A proposed mechanism permitting productive long-form signaling in the presence of excess short-form expression. J Cell Biochem 1999; 73(2):278-288. 43. Zabeau L, Defeau D, Iserentant H et al. Leptin receptor activation depends on critical cysteine residues in its fibronectin type III subdomains. J Biol Chem 2005; 280(24):22632-22640. 44. Couturier C, Jockers R. Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers. J Biol Chem 2003; 278(29):26604-26611. 45. de Vos AM, Ultsch M, Kossiakoff AA. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 1992; 255(5042):306-312. 46. Syed RS, Reid SW, Li C et al. Efficiency of signalling through cytokine receptors depends critically on receptor orientation. Nature 1998; 395(6701):511-516. 47. Boulanger MJ, Chow DC, Brevnova EE et al. Hexameric structure and assembly of the interleukin-6/ IL-6 alpha-receptor/gp130 complex. Science 2003; 300(5628):2101-2104. 48. Layton JE, Hall NE. The interaction of G-CSF with its receptor. Frontiers in Bioscience 2006; 11:3181-3189. 49. Gertler A. Development of leptin antagonists and their potential use in experimental biology and medicine. Trends Endocrinol Metab 2006; 17(9):372-378. 50. Hiroike T, Higo J, Jingami H et al. Homology modeling of human leptin/leptin receptor complex. Biochem Biophys Res Comm 2000; 275(1):154-158.


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51. Iserentant H, Peelman F, Defeau D et al. Mapping of the interface between leptin and the leptin receptor CRH2 domain. J Cell Sci 2005; 118(Pt 11):2519-2527. 52. Niv-Spector L, Raver N, Friedman-Einat M et al. Mapping leptin-interacting sites in recombinant leptin-binding domain (LBD) subcloned from chicken leptin receptor. Biochem J 2005; 390(Pt 2):475-484. 53. da Silva BA, Bjorbaek C, Uotani S et al. Functional properties of leptin receptor isoforms containing the gln→pro extracellular domain mutation of the fatty rat. Endocrinology 1998; 139(9):3681-3690. 54. Iida M, Murakami T, Ishida K et al. Substitution at codon 269 (glutamine→proline) of the leptin receptor (OB-R) cDNA is the only mutation found in the Zucker fatty (fa/fa) rat. Biochem Biophys Res Comm 1996; 224(2):597-604. 55. White DW, Wang DW, Chua SC et al. Constitutive and impaired signaling of leptin receptors containing the Gln Pro extracellular domain fatty mutation. Proc Natl Acad Sci USA 1997; 94(20):10657-10662. 56. Leahy DJ, Aukhil I, Erickson HP. 2.0 A crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region. Cell 1996; 84(1):155-164. 57. Verploegen SA, Plaetinck G, Devos R et al. A human leptin mutant induces weight gain in normal mice. FEBS Letters 1997; 405(2):237-240. 58. Raver N, Vardy E, Livnah O et al. Comparison of R128Q mutations in human, ovine and chicken leptins. Gen Comp Endocrinol 2002; 126(1):52-58. 59. Brunner L, Whitebread S, Leconte I et al. A peptide leptin antagonist reduces food intake in rodents. Int J Obes Relat Metab Disord 1999; 23(5):463-469. 60. Ramos MP, Rueda BR, Leavis PC et al. Leptin serves as an upstream activator of an obligatory signaling cascade in the embryo-implantation process. Endocrinology 2005; 146(2):694-701. 61. Gonzalez RR, Cherfils S, Escobar M et al. Leptin signaling promotes the growth of mammary tumors and increases the expression of vascular endothelial growth factor (VEGF) and its receptor type two (VEGF-R2). J Biol Chem 2006; 281(36):26320-26328. 62. Klein S, Coppack SW, Mohamed-Ali V et al. Adipose tissue leptin production and plasma leptin kinetics in humans. Diabetes 1996; 45(7):984-987. 63. Vila R, Adan C, Rafecas I et al. Plasma leptin turnover rates in lean and obese Zucker rats. Endocrinology 1998; 139(11):4466-4469. 64. Zeng J, Patterson BW, Klein S et al. Whole body leptin kinetics and renal metabolism in vivo. The Am J Physiol 1997; 273(6 Pt 1):E1102-1106. 65. Hamidi M, Azadi A, Rafiei P. Pharmacokinetic consequences of pegylation. Drug Delivery 2006; 13(6):399-409. 66. Hukshorn CJ, Menheere PP, Westerterp-Plantenga MS et al. The effect of pegylated human recombinant leptin (PEG-OB) on neuroendocrine adaptations to semi-starvation in overweight men. EEur J Endocrinol 2003; 148(6):649-655. 67. Hukshorn CJ, Saris WH, Westerterp-Plantenga MS et al. Weekly subcutaneous pegylated recombinant native human leptin (PEG-OB) administration in obese men. J Clin Endocrinol Metab 2000; 85(11):4003-4009. 68. Solomon G, Niv-Spector L, Gonen-Berger D et al. Preparation of leptin antagonists by site-directed mutagenesis of human, ovine, rat and mouse leptin's site III: Implications on blocking undesired leptin action in vivo. Annals NY Acad Sci 2006; 1091:531-539. 69. Cheunsuang O, Morris R. Astrocytes in the arcuate nucleus and median eminence that take up a fluorescent dye from the circulation express leptin receptors and neuropeptide Y Y1 receptors. Glia 2005; 52(3):228-233. 70. Faouzi M, Leshan R, Bjornholm M et al. Differential accessibility of circulating leptin to individual hypothalamic sites. Endocrinology 2007; 148(11):5414-5423.

Chapter 3

Study of Leptin:

Leptin Receptor Interaction by FRET and BRET Julie Dam, Cyril Couturier, Patty Chen and Ralf Jockers*

Abstract

U

nderstanding the molecular mechanism of the leptin:leptin receptor (OB-R) interaction and the OB-R activation process is crucial for the development of drugs that target OB-Rs. Recently developed resonance energy transfer (RET)-based assays participated significantly in the establishment of the current model of OB-R activation. According to this model, OB-Rs exist as preformed homodimers in the basal state. Leptin binding induces a ligand-induced conformational change within these dimers, which triggers receptor activation by facilitating the transphosphorylation of receptor-associated janus kinase 2. The concomitant formation of tetrameric complexes (dimers of dimers) has also been suggested but still remains to be firmly established. RET-based techniques also hold great potential for the screening of small molecular weight compounds targeting the OB-R.

Introduction

Leptin is a member of the cytokine family having a four α-helical bundle structure. It is a well-known anorexigenic hormone secreted mainly into the bloodstream by adipose tissue and controls food intake and energy homeostasis primarily by acting at the hypothalamic arcuate nucleus (ARC). Mutations leading to a functional defect in either leptin or its receptor (OB-R) result in a complex syndrome that includes morbid obesity. Besides the adipostatic function, leptin also plays a direct role in the peripheral system regulating metabolism, hematopoiesis, immunity and reproduction. Among the six different types of OB-R, which result from alternative splicing or proteolysis, two main isoforms were mainly studied. The long functional isoform OB-Rb, mainly expressed in the ARC, displays a full intracellular domain (302 residues) with docking sites for the Janus Tyrosine Kinase ( JAK2) and Signal Transducer and Activator of Transcription 3 (STAT3). The short isoform OB-Ra is ubiquitously expressed and has a truncated intracellular domain (34 residues) lacking the STAT3 binding site but is still able to interact and activate JAK2. Whereas leptin functions have been broadly documented, the molecular mechanism of OB-R activation remains elusive. Nevertheless, the structure of several other cytokines and growth factors were investigated extensively during the last decade, in order to define the manner in which these molecules interact with receptors and to reveal the mechanism of signal transduction across the membrane. The crystal structure of leptin/OB-R is unavailable but the successful use of biochemical, biophysical and molecular modeling techniques brought new insights into the leptin activation mechanism that will be presented in this article.

*Corresponding Author: Ralf Jockers—Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Department of Cell Biology, Paris, France. Email: [email protected]


Study of Leptin: Leptin Receptor Interaction by FRET and BRET

31

Activation Mechanism of OB-R Studied with Biochemical Methods Lessons from Other Cytokine Receptors

For over a decade, the model of growth hormone (GH)-induced receptor dimerization has served as a dogma for cytokine receptor activation.1 This model was supported by the crystal structure of the 2:1 complex between the purified extracellular domain of the GH receptor (GHR) and the GH2 whereas the complex with GH antagonists revealed a 1:1 stoichiometry favoring the notion that the unbound receptor is a monomer.3,4 Moreover, the model was corroborated by the observation that GH but not the antagonist induces the formation of covalently disulfide-linked dimers.5,6 However, the GH-induced dimerization paradigm was then challenged by several studies demonstrating by co-immunoprecipitation that the GHR exists at the plasma membrane as dimer in the absence of ligand,7 that dimerization itself is insufficient for GHR activation8,9 and that a GH antagonist can bind to receptor dimers at the cell surface.10,11 More recently, the crystal structure of unliganded GHR being a dimer, contributed to establish a model of GHR activation involving a slight rotation of subunits within a dimeric receptor upon ligand binding.12 In the case of the erythropoietin receptor (EpoR), crystallographic data from the extracellular domain is also available confirming its dimeric form in the resting state. EpoR adopts distinct dimeric configurations dependent on being unliganded,13 Epo-bound14 or bound to agonistic15 or antagonistic16 peptides. The open scissors-like configuration of the preformed dimer is envisioned to keep the cytoplasmic domain apart in an inactive state and ligand occupancy would bring the extracellular and cytoplasmic domains into close proximity to allow signaling. These data were further confirmed by fragment complementation assay.17 Concerning more complex Cytokine receptors like Granulocyte Colony-Stimulating factor (G-CSF) or Interleukin-6 (IL-6) type cytokines and their receptors gp130, Leukemia Inhibitory Factor Receptor (LIFR), Ciliary Neurotrophic Factor Receptor (CNTFR) and Oncostatin M Receptor (OSMR), the oligomeric state of the activated complex seems to be of higher order. From studies of the GH/GHR complex, it was generally admitted that cytokines were recognized by their receptors at two sites equivalent to site I and site II of GH. This dogma was not valid for IL-6 type cytokines where three distinct receptor binding sites (I-II-III) have been clearly demonstrated by binding and mutagenesis studies.18-21 The organization of three binding epitopes suggested the formation of higher order complexes.22 The stoichiometry of the signaling complex is different between G-CSFR and IL-6R. G-CSF was shown to form a 2:2 tetrameric complex mediated by binding site II and III.23-25 The IL-6 receptor complex was shown to form a 2:2:2 hexameric assembly composed of two IL-6 ligands in complex with two gp130 chains and two specific IL-6Rα chains at three different binding interfaces.26 On the other hand, without the IL6Rα chain, the viral homolog of IL-6 and gp-130 molecules form a tetrameric 2:2 structure consisting of two sets of 1:1 complexes from vIL-6 and human Ig–CRH domains of gp130.27

The Leptin/OB-R System Studied with Biochemical Methods

OB-R is a member of the class I cytokine receptor family with a larger N-terminal extracellular domain than GHR and EpoR. The extracellular domain consists of more domains than necessary for ligand binding. These extra domains are probably involved in receptor activation and signal transmission into the cell. The extracellular domain is composed of two so-called cytokine receptor homology (CRH) domains, a membrane distal domain CRH1 and a proximal domain CRH2. These domains are separated by an immunoglobulin (Ig) domain and followed by two fibronectin like (FNIII) domains. Binding studies with recombinant OB-R subdomains and molecular modeling of the leptin/OB-R complex indicated the existence of three different binding sites similar to the IL6-system.28-34 Indeed, Leptin and OB-R show the highest structural similarity to G-CSF and to the IL-6 family. Similarly to these cytokines, several observations suggested that OB-R exists as a dimer. Cross-linking of OB-R and leptin revealed western blot bands with apparent molecular weights corresponding to monomers, dimers and higher order oligomers.35 Dimers were detected even with the soluble receptor composed of the whole OB-R extracellular domain but truncated of its transmembrane and intracellular domain.36 Further evidence for ligand-independent

32

Figure 1, legend viewed on following page.


Study of Leptin: Leptin Receptor Interaction by FRET and BRET

33

homo-oligomerization of OB-R were provided by co-immunoprecipitation experiments.37,38 Moreover, dimer formation of OB-R would explain why coexpression of wild type OB-R inhibits the partially constitutive activity of the N269P receptor mutant.39 Importantly, complementation assays of two inactive receptors mutated in their functional intracellular domain (either no JAK2 interaction or no STAT3 docking site) also provided strong evidence for higher order clustering of OB-R.40 However, in vitro binding studies were insufficient to demonstrate such a complex as the leptin binding domain of OB-R appeared to be a monomer forming a stable complex with leptin in a 1:1 stoichiometric ratio, as revealed by gel-filtration experiments and SPR analysis.30 Altogether, despite major efforts to determine the activation mechanism of OB-R with biochemical methods, contradictory results were obtained and many questions remained unanswered. Most biochemical assays provided rather indirect information and were limited by the requirement of receptor solubilization and the use of receptor mutants and isolated subdomains. More recently, resonance energy transfer (RET) techniques were used to obtain more direct information of the oligomeric state of OB-R and the dynamics of its activation in living cells.

Methodological Introduction to FRET/BRET

RET techniques such as fluorescence RET (FRET) and bioluminescence RET (BRET) are methods of choice to study oligomerisation and activation of transmembrane receptors in living cells. They rely on a nonradiative energy transfer between an energy donor and an energy acceptor. To fulfill the conditions for energy transfer, the emission spectrum of the donor must overlap with the excitation spectrum of the acceptor (Fig. 1A).41,42 These approaches can be performed using genetically engineered fusion-proteins thus enabling the monitoring of protein-protein interactions and molecular rearrangements (i.e., conformational changes). One protein is fused to the donor and the other to the acceptor. If the two fusion proteins do not interact, only light emitted from the energy donor excitation can be monitored (Fig. 1B). If the two fusion proteins interact and position the energy donor and acceptor within a distance smaller than 10 nm, an additional light signal corresponding to the acceptor reemission due to the resonance energy transfer can be detected42-44 (Fig. 1B). In the FRET method both energy donor and acceptor are different variants of the green fluorescent proteins. The cyan fluorescent protein (CFP) is often used as energy donor and the yellow fluorescent protein (YFP) as energy acceptor. FRET measurements require an external excitatory light source for donor excitation. In the BRET method, the CFP Figure 1, viewed on previous page. A) Emission and excitation spectra of energy donor and acceptor. The resonance energy transfer occurs only if the donor and the acceptor display overlapping emission and excitation spectra respectively. B) Resonance Energy Transfer (RET) principle. The donor molecule emits light when it is excited by an external light source or in the presence of the luciferase substrate. Two possible events can occur depending on the orientation and distance of donor versus acceptor dipoles. If they do not interact, are further than 10 nm from each other, or are unfavorably oriented, there is no RET and the excited donor will emit at the donor emission wavelength. On the contrary, if there is interaction between donor and acceptor dipoles positioned at less than 10 nm from each other, RET will occur from the donor to the acceptor resulting in the excitation of the acceptor which then emits at the acceptor emission wavelength. C) FRET and BRET principle. In RET techniques, the proteins of interest X and Y are fused to donor or acceptor molecules. In the FRET event, the donor can be the Cyan Fluorescent Protein (CFP) which when excited at 433 nm transfers its energy by resonance to the acceptor, the Yellow Fluorescent Protein (YFP) which then emits at 530 nm. In the BRET method, the donor of energy is the enzyme Renilla luciferase (Rluc), which by oxidizing its substrate, coelenterazine, transmits part of the energy to the YFP acceptor, which reemits fluorescent light at 530 nm. D) Donor Saturation Curve. In the donor saturation assay, a donor at a constant concentration is progressively saturated by increasing concentrations of acceptor. When there are specific interactions between the donor and the acceptor, the saturation curve will reach a plateau (BRETmax). The BRET50 value (correlated to relative affinity between donor and acceptor) is defined as the acceptor/donor ratio at half-maximal BRETmax. Conversely, the saturation curve evolves linearly with low BRET signals when donor and acceptor do not interact specifically.

34


is replaced by a luciferase that generates light in the presence of its corresponding substrate. In a typical BRET experiment, the luciferase from Renilla reniformis (Rluc) is used as energy donor and YFP as energy acceptor (Fig. 1C). For a given donor/acceptor couple, RET intensity depends on the distance between the donor and acceptor (

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