GIT Receptor

GIT Receptor Dass S. Vinay1 and Byoung S. Kwon2,3,* 1

Department of General Surgery, University of Michigan Medical School, 1516 MSRB I, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA 2 The Immunomodulation Research Center, University of Ulsan, Ulsan, Korea 3 Department of Ophthalmology, LSUMC, 2020 Gravier Street Suite B, New Orleans, LA 70112, USA * corresponding author tel: 504-412-1200 ex 1379, fax: 504-412-1315, e-mail: [email protected] DOI: 10.1006/rwcy.2000.16015.

SUMMARY

BACKGROUND

The TNF and TNF receptor gene superfamilies control a variety of distinct physiological functions such as cell proliferation, differentiation, and survival, etc. A newly emerging member this family with strong role in T cell homeostasis is GITR (glucocorticoidinduced tumor necrosis factor receptor) (Nocentini et al., 1998). A majority of glucocorticoid hormones are known to induce apoptosis (Nocentini et al., 1998). It is surprising to note that these hormones also protect the cells from undergoing apoptosis under the influence of discreet stimuli (Riccardi et al., 1999). These events are thought to involve the participation of GITR and GILZ genes by controlling events like activation of NFB and expression of Fas/ FasL molecules (Riccardi et al., 1999). Recently, a human homolog of murine GITR was discovered (Kwon et al., 1999; Gurney et al., 1999). The human receptor was called AITR (activation-induced TNFR member) (Kwon et al., 1999) and hGITR (Gurney et al., 1999). Its ligand was cloned and called AITRL (Kwon et al., 1999) and hGITRL (Gurney et al., 1999). Within the cytoplasmic domain, the AITR shares a striking homology with 4-1BB and CD27 (Kwon et al., 1999). AITR associates with TRAF1 (TNF receptor-associated factor 1), TRAF2, and TRAF3, and induces NFB activation via TRAF2 (Kwon et al., 1999). AITRL was expressed in endothelial cells (Kwon et al., 1999). Expression of GITR appears to be activation dependent as stimulation of T lymphocytes by anti-CD3 mAb, Con A, or phorbol 12-myristate 13-acetate plus Ca-ionophore treatment readily upregulates its levels (Nocentini et al., 1997).

Optimal induction of T cell activation is incomplete without the cognate interaction between T cells and antigen presenting cell (APC)-derived cell surface molecules (Schwarz, 1990). A number of APC-derived cell surface determinants have been shown to possess the ability to potentiate/desensitize T cell effector functions. Although it is considered that B7/CD28 is central to this pathway, studies of CD28-deficient mice (Shahnian et al., 1993) have shown that this may not be the limiting factor in immune regulation. Recent studies indicate that several ligand/receptor pairs, beyond the B7/CD28 pathway, also have the ability to initiate and propel the ongoing immune reaction. Among these are three members of the expanding TNFR family: 4-1BB, Ox40, and GITR.

Discovery The glucocorticoid-induced tumor necrosis factor receptor (GITR) family-related gene was cloned first from dexamethasone-treated murine T cell hybridoma (3DO) cells by differential display technique.

Alternative names Published data from our laboratory provide evidence that a novel 25 kDa protein named activationinducible protein of the TNF receptor (AITR) is the human homolog of the murine GITR (Kwon et al., 1999). The AITR has 55% identity with murine GITR at the amino acid level. It is shown to activate

1756 Dass S. Vinay and Byoung S. Kwon by transducing signals through TRAF2-mediated mechanism. The expression of AITR is inducible by PMA and ionomycin, anti-CD3 plus anti-CD28 monoclonal antibodies. It is detected as a 1.25 kb mRNA in lymph nodes, PBLs and weakly in the spleen and colorectal adenocarcinoma cell line (SW 480) (Kwon et al., 1999).

Structure GITR is a 228 amino acid type I transmembrane protein characterized by three cysteine pseudorepeats in the extracellular domain. It is similar to 4-1BB in the intracellular domain. The full-length GITR cDNA revealed a 1005 bp long sequence. Northern blot analysis suggested that GITR mRNA is about 1.1 kb long.

Main activities and pathophysiological roles Evidence accumulated thus far suggests that GITR expression confers resistance to TCR/CD3-induced apoptosis of transfected T cells. This resistance is independent of Fas triggering. However, modulation of GITR expression does not seem to modify the sensitivity to apoptotic stimuli other than TCR triggering (Nocentini et al., 1997).

GENE

Accession numbers GenBank: Murine GITR: U82534

Sequence Full cDNA from a T lymphocyte cDNA library revealed a 1005 bp long sequence. Northern analysis of GITR mRNA was found to be 1.1 kb long. Nucleotide sequencing of the three cDNA clones showed the presence of a single 684 bp ORF beginning at nucleotide position 46 and extending to a TGA termination codon at position 730. The putative initiation codon at position 46 is surrounded by a sequence (AGCACTATGG). The termination codon is followed by a 30 UTR of 276 bp. A canonical polyadenylylation signal is present from 18 bp 50 to the poly (A) tail.

PROTEIN

Sequence See Figure 1.

Description of protein The molecule putatively encoded by the GITR mRNA is a cysteine-rich protein of 228 amino acids. The two hydrophobic regions are present, probably representing the signal peptide and the transmembrane domain. A cleavage site for the signal peptide can be found between GLY (at position ÿ1) and Gln (at position 1), despite the unusual presence of Asp at position ÿ3. The transmembrane domain is located between positions 135 and 157 of the mature protein. Given this, GITR can be categorized as a type I membrane protein. The molecular weight of the predicted native protein is 25,334, consistent with that obtained by in vitro translation of the cloned cDNA. The predicted molecular weight of the putative mature protein before further posttranslational modifications is 23,321.

Relevant homologies and species differences The GITR amino acid sequence exhibits marked homologies with 4-1BB, a member of the TNF/ NGFR family. The GITR cytoplasmic domain spans amino acids 158±209 of the mature protein. It has a striking homology with the cytoplasmic domains of murine and human 4-1BB and CD27 (Figure 1) but does not show any significant homology with other members of the TNF/NGFR family (Gravestein et al., 1993). This similarity defines a new intracellular motif that could identify a subfamily of the TNF/NGFR family, including GITR, 4-1BB, and CD27.

Cell types and tissues expressing the receptor GITR is not detectable in freshly derived lymphoid tissues (including thymocytes, spleen, and lymph node T cells), liver, kidney, and brain and T cell hybridoma 3D0. However, low levels of GITR mRNA were detected by competitive RT-PCR in T cell hybridoma, thymocytes, spleen, and lymph node T cells.

GIT Receptor 1757 Figure 1 (a) Deduced amino acid sequence of AITR. The potential signal sequence is indicated as bold characters and the transmembrane region is indicated in boxes. (b) Comparison of the amino acid sequence of AITR with the murine GITR. Bold Cs within three cysteine pseudorepeat motifs indicate cysteine residues found in the extracellular domain of the TNFR superfamily members. Conserved acidic amino acid clusters of the cytoplasmic domain are indicated in boxes.

A 1 61 121 181

MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRD YPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTF SGGHEGHCKPWTDCTQFGFLTVFPGNKTHVAVCVPGSPPAEPLGWLTVVLLAVAACVLLL TSAQLGLHIWQLRKTQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

B

AITR GITR

➯ cysteine pseudorepeat #1 MAQHGAMGAFRALCGLALLCALSLGQRPT-GGPGCGPGRLLLGTGTDARCCRVHTTRCCRD M------GAWAMLYGVSMLCVLDLGQPSVVEEPGCGPGKVQNGSGNNTRCCSLYA-----cysteine pseudorepeat #2 ➯➯ ➯➯ YPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTF -PGKEDCPKERCICVTPEYHCGDPQCKICKHYPCQPGQRVESQGDIVFGFRCVACAMGTF cysteine pseudorepeat #3 ➯ SGGHEGHCKPWTDCTQFGFLTVFPGNKTHVAVCVPGSPPAEPLGWLTVVLLAVAACVLLL SAGRDGHCRLWTNCSQFGFLTMFPGNKTHNAVCIPEPLPTEQYGHLTVIFLVMAACIFFL

AITR GITR

TSAQLGLHIWQLRKTQL-------LLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV TTVQLGLHIWQLRRQHMCPRETQPFAEVQLSAEDACSFQFREEERGEQT-EEKCHLGGRWP

➯

➯

AITR GITR

Regulation of receptor expression GITR expression in T cells was found to increase 4- to 8-fold upon treatment with immobilized antiCD3, Con A, PMA+ Ca2‡ ionophore. However, the induction kinetics was slow (no increase before 6 hours) (Nocentini et al., 1997).

60 48

➯

AITR GITR

120 109 180 169 234 229

apoptosis appears to be specific, as other apoptotic signals (Fas triggering, dexamethasone treatment, and UV irradiation) were not modulated by GITR transfection, indicating that GITR is a new member of TNF/NGFR family involved in the regulation of TCR-mediated cell death.

References BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors To date, known GITR-induced biological effects are restricted to offering resistance to TCR/CD3-induced apoptosis. The protection toward TCR/CD3-induced

Gravestein, L. A., Blom, B., Nolten, L. A., de Vries, E., van der Horst, G., Ossendorp, F., Borst, J., and Loenen, W. A. M. (1993). Cloning and expression of murine CD27: comparison with 4-1BB, another lymphocyte-specific member of the nerve growth factor receptor family. Eur. J. Immunol. 23, 943±950. Gurney, A. L., Marsters, S. A., Huang, R. M., Pitti, R. M., Mark, D. T., Baldwin, D. T., Gray, A. M., Dowd, A. D., Brush, A. D., Heldens, A. D., Schow, A. D., Goddard, A. D., Wood, W. I., Baker, K. P., Godowski, P. J., and Ashkenazi, A. (1999). Identification of a new member of the tumor necrosis factor family and its receptor, a human ortholog of mouse GITR. Curr. Biol. 9, 215±218. Kwon, B., Yu, K. Y., Ni, J., Yu, G. L., Jang, I.-K., Kim, Y.-J., Xing, L., Lium D., Wang, S. X., and Kwon, B. S. (1999). Identification of a novel activation-inducible protein of the

1758 Dass S. Vinay and Byoung S. Kwon tumor necrosis factor receptor superfamily and its ligand. J. Biol. Chem. 274, 6056±6061. Nocentini, G., Giunchi, L., Ronchetti, S., Krausz, L. T., Bartoli, A., Moaca, R., Migliorati, G., and Riccardi, C. (1997). A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis. Proc. Natl Acad. Sci. USA 94, 6216±6221. Nocentini, G., Giunchi, L., Ronchetti, S., Bartoli, A., Migliorati, G., and Riccardi, C. (1998). Glucocorticoids: regulation of gene expression and apoptosis. J. Chemother. 10, 187±191. Riccardi, C., Cifone, M. G., and Migliorati, G. (1999). Glucocorticoid hormone-induced modulation of gene expression and regulation of T-cell death: role of GITR and GILZ, two dexamethasone-induced genes. Cell Death Differ. 6, 1182±1189. Schwarz, R. H. (1990). A cell culture model for T lymphocyte clonal anergy. Science 248, 1349±1356.

Shahnian, A., Pieffer, K., Lee, K. P., Kundig, T. M., Kishihara, K., Wakeham, A., Kawai, K., Ohashi, P. M., Thompson, C. B., and Mak, T. W. (1993). Differential T cell costimulatory requirements in CD28-deficient mice. Science 261, 609±612.

ACKNOWLEDGEMENTS Authors are grateful to Dr Byoung Suk Kwon for sharing certain unpublished research data on the human homolog of murine GITR, the AITR. SRC funds to IRC from the Korean Ministry of Science and Technology and NIH Grants (AI28125 and DE12156) are greatly appreciated.