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Divergent Signaling Capacities of the Long and Short Isoforms of the Leptin Receptor

1997; Elsevier BV; Volume: 272; Issue: 51 Linguagem: Inglês

10.1074/jbc.272.51.32686

ISSN

1083-351X

Autores

Christian Bjørbæk, Shigeo Uotani, Bárbara da Silva, Jeffrey S. Flier,

Tópico(s)

Adipokines, Inflammation, and Metabolic Diseases

Resumo

Leptin receptors include a long form (OBRl) with 302 cytoplasmic residues that is presumed to mediate most or all of leptins signaling, and several short forms, including one (OBRs) that has 34 cytoplasmic residues, is widely expressed, and is presumed not to signal but to mediate transport or clearance of leptin. We studied the abilities of these two receptor isoforms to mediate signaling in transfected cells. In response to leptin, OBRl, but not OBRs, underwent tyrosine phosphorylation that was enhanced by co-expression with JAK2. In cells expressing receptors and JAK2, both OBRs and OBRl mediated leptin-dependent tyrosine phosphorylation of JAK2, and this was abolished with OBRs when the Box 1 motif was mutated. In cells expressing receptors, JAK2 and IRS-1, leptin induced tyrosine phosphorylation of IRS-1 through OBRs and OBRl. In COS cells expressing hemagglutinin-ERK1 and receptors, leptin increased ERK1 kinase activity through OBRl, with the magnitude increased by co-expression of JAK1 or JAK2, and to a lesser degree through OBRs, despite greater receptor expression. In stable Chinese hamster ovary cell lines expressing OBRs or OBRl, leptin stimulated endogenous ERK2 phosphorylation. Whereas leptin stimulated tyrosine phosphorylation of hemagglutinin-STAT3 and induction of a c-fos luciferase reporter plasmid through OBRl, OBRs was without effect in these assays. In conclusion, OBRl is capable of signaling to IRS-1 and mitogen-activated protein kinase via JAK, in addition to activating STAT pathways. Although substantially weaker than OBRl, OBRs is capable of mediating signal transduction via JAK, but these activities are of as yet unknown significance for leptin biology in vivo. Leptin receptors include a long form (OBRl) with 302 cytoplasmic residues that is presumed to mediate most or all of leptins signaling, and several short forms, including one (OBRs) that has 34 cytoplasmic residues, is widely expressed, and is presumed not to signal but to mediate transport or clearance of leptin. We studied the abilities of these two receptor isoforms to mediate signaling in transfected cells. In response to leptin, OBRl, but not OBRs, underwent tyrosine phosphorylation that was enhanced by co-expression with JAK2. In cells expressing receptors and JAK2, both OBRs and OBRl mediated leptin-dependent tyrosine phosphorylation of JAK2, and this was abolished with OBRs when the Box 1 motif was mutated. In cells expressing receptors, JAK2 and IRS-1, leptin induced tyrosine phosphorylation of IRS-1 through OBRs and OBRl. In COS cells expressing hemagglutinin-ERK1 and receptors, leptin increased ERK1 kinase activity through OBRl, with the magnitude increased by co-expression of JAK1 or JAK2, and to a lesser degree through OBRs, despite greater receptor expression. In stable Chinese hamster ovary cell lines expressing OBRs or OBRl, leptin stimulated endogenous ERK2 phosphorylation. Whereas leptin stimulated tyrosine phosphorylation of hemagglutinin-STAT3 and induction of a c-fos luciferase reporter plasmid through OBRl, OBRs was without effect in these assays. In conclusion, OBRl is capable of signaling to IRS-1 and mitogen-activated protein kinase via JAK, in addition to activating STAT pathways. Although substantially weaker than OBRl, OBRs is capable of mediating signal transduction via JAK, but these activities are of as yet unknown significance for leptin biology in vivo. Leptin, the 16-kDa protein product of the ob gene (1Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11807) Google Scholar), is an adipocyte-derived hormone that influences energy homeostasis through effects on energy intake and expenditure (2Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4256) Google Scholar, 3Campfield L.A. Smith F.J. Guisez Y. Devos R. Burn P. Science. 1995; 269: 546-549Crossref PubMed Scopus (3072) Google Scholar, 4Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3884) Google Scholar), the secretion and action of metabolic hormones (5Weigle D.S. Bukowski T.R. Foster D.C. Holderman S. Kramer J.M. Lasser G. Lofton-Day C.E. Prunkard D.E. Raymond C. Kuijper J.L. J. Clin. Invest. 1995; 96: 2065-2070Crossref PubMed Scopus (447) Google Scholar, 6Schwartz M.W. Baskin D.G. Bukowski T.R. Kuijper J.L. Foster D. Lasser G. Prunkard D.E. Porte Jr., D. Woods S.C. Seeley R.J. Weigle D.S. Diabetes. 1996; 45: 531-535Crossref PubMed Google Scholar, 7Stephens T.W. Basinski M. Bristow P.K. Bue-Valleskey J.M. Burgett S.G. Craft L. Hale J. Hoffmann J. Hsiung H.M. Kriauciunas A. MacKellar W. Rosteck Jr., P.R. Schoner B. Smith D. Tinsley F.C. Zhang X.-Y. Heiman M. Nature. 1995; 377: 530-532Crossref PubMed Scopus (1478) Google Scholar, 8Chen G. Koyama K. Yuan X. Lee Y. Zhou Y.-T. O'Doherty R. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14795-14799Crossref PubMed Scopus (285) Google Scholar), and neuroendocrine function (9Ahima R.S. Prabakaran D. Mantzoros C. Qu D. Lowell B. Maratos-Flier E. Flier J.S. Nature. 1996; 382: 250-252Crossref PubMed Scopus (2693) Google Scholar). Leptin acts through receptors that bear strong sequence homology to the class I cytokine receptor family (10Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3238) Google Scholar). Mutations in the leptin receptor gene occur in db/db mice (11Chen H. Chatlat O. Tartaglia L.A. Woolf E.A. Weng X. Ellis S.J. Lakey N.D. Culpepper J. Moore K.J. Breitbart R.E. Duyk G.M. Tepper R.I. Morgenstern J.P. Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1944) Google Scholar, 12Lee G.-H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2112) Google Scholar), and fa/fa (13Phillips M.S. Liu Q. Hammond H.A. Dugan V. Hey P.J. Caskey C.J. Hess J.F. Nat. Genet. 1996; 13: 18-19Crossref PubMed Scopus (760) Google Scholar) and Koletsky (14Takaya K. Ogawa Y. Hiraoka J. Hosoda K. Yamori Y. Nakao K. Koletsky R.J. Nat. Genet. 1996; 14: 130-131Crossref PubMed Scopus (181) Google Scholar) rats, producing severe obesity with resistance to endogenous and exogenous leptin (6Schwartz M.W. Baskin D.G. Bukowski T.R. Kuijper J.L. Foster D. Lasser G. Prunkard D.E. Porte Jr., D. Woods S.C. Seeley R.J. Weigle D.S. Diabetes. 1996; 45: 531-535Crossref PubMed Google Scholar, 7Stephens T.W. Basinski M. Bristow P.K. Bue-Valleskey J.M. Burgett S.G. Craft L. Hale J. Hoffmann J. Hsiung H.M. Kriauciunas A. MacKellar W. Rosteck Jr., P.R. Schoner B. Smith D. Tinsley F.C. Zhang X.-Y. Heiman M. Nature. 1995; 377: 530-532Crossref PubMed Scopus (1478) Google Scholar, 15Cusin I. Rohner-Jeanrenaud F. Stricker-Krongrad A. Jeanrenaud B. Diabetes. 1996; 45: 1446-1450Crossref PubMed Google Scholar). At least five isoforms of the leptin receptor are generated by alternative mRNA splicing (12Lee G.-H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2112) Google Scholar), but signaling is thought to occur through the “long isoform” (OBRl) 1The abbreviations used are: OBRl, long form of leptin receptor; OBRs, short form of leptin receptor; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; SIE, Sis-inducible element; Luc, luciferase; β-gal, β-galactosidase; TK, thymidine kinase; GHR, growth hormone receptor; STAT, signal transducer activation of transcription; JAK, Janus kinase; IRS, insulin-like receptor substrate; CHO, Chinese hamster ovary; EGF, epidermal growth factor; MBP, myelin basic protein; HA, hemagglutinin; CMV, cytomegalovirus; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; MAP, mitogen-activated protein kinase. (10Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3238) Google Scholar, 12Lee G.-H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2112) Google Scholar, 16Tartaglia L.A. J. Biol. Chem. 1997; 272: 6093-6096Abstract Full Text Full Text PDF PubMed Scopus (1191) Google Scholar, 17White D.W. Kuropatwinski K.K. Devos R. Baumann H. Tartaglia L.A. J. Biol. Chem. 1997; 272: 4065-4071Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 18Ghilardi N. Skoda R.C. Mol. Endocrinol. 1997; 11: 393-399Crossref PubMed Scopus (270) Google Scholar). OBRl is predicted to have a long intracellular domain that is truncated as a result of the db mutation (11Chen H. Chatlat O. Tartaglia L.A. Woolf E.A. Weng X. Ellis S.J. Lakey N.D. Culpepper J. Moore K.J. Breitbart R.E. Duyk G.M. Tepper R.I. Morgenstern J.P. Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1944) Google Scholar, 12Lee G.-H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2112) Google Scholar). This truncated receptor is identical to the abundant, so called “short isoform” (OBRs) of the leptin receptor. OBRs is highly expressed in kidney, lungs, and choroid plexus, while OBRl is most abundant in the hypothalamus (10Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3238) Google Scholar, 12Lee G.-H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2112) Google Scholar, 19Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M.H. Skoda R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6231-6235Crossref PubMed Scopus (737) Google Scholar, 20Luoh S.M. Di Marco F. Levin N. Armanini M. Xie M.H. Nelson C. Bennett G.L. Wiliams M. Spencer S.A. Gourney A. de Sauvage F.J. J. Mol. Endocrinol. 1997; 18: 77-85Crossref PubMed Scopus (75) Google Scholar), but may be expressed at lower levels elsewhere (19Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M.H. Skoda R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6231-6235Crossref PubMed Scopus (737) Google Scholar, 21Hoggard N. Mercer J.G. Rayner D.V. Moar K. Trayburn P. Williams L.M. Biochem. Biophys. Res. Commun. 1997; 232: 383-387Crossref PubMed Scopus (271) Google Scholar, 22Dyer C.J. Simmons J.M. Matteri R.L. Keisler D.H. Domest. Anim. Endocrinol. 1997; 14: 119-128Crossref PubMed Scopus (176) Google Scholar). As with other members of the cytokine receptor family, the long form of OBR can activate JAK and STAT proteins in cultured cells transfected with leptin receptor expression vectors (18Ghilardi N. Skoda R.C. Mol. Endocrinol. 1997; 11: 393-399Crossref PubMed Scopus (270) Google Scholar, 19Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M.H. Skoda R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6231-6235Crossref PubMed Scopus (737) Google Scholar, 23Baumann H. Morella K.K. White D.W. Dembski M. Bailon P.S. Kim H. Lai C.-F. Tartaglia L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8374-8378Crossref PubMed Scopus (764) Google Scholar), and STAT proteins in hypothalami after in vivo leptin injection in normal, but not db/db, mice (24Vaisse C. Halaas J.L. Horvath C.M. Darnell Jr., J.E. Stoffel M. Friedman J.M. Nat. Genet. 1996; 14: 95-97Crossref PubMed Scopus (953) Google Scholar). Recent data suggest that leptin may also have direct effects on tissues outside the central nervous system, including effects on metabolism, reproduction, and hematopoiesis (25Shimabukuro M. Koyama K. Chen G. Wang M.Y. Trieu F. Lee Y. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4637-4641Crossref PubMed Scopus (622) Google Scholar, 26Muller G. Ertl J. Gerl M. Preibisch G. J. Biol. Chem. 1997; 272: 10585-10893Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 27Zachow R.J. Magoffin D.A. Endocrinology. 1997; 138: 847-850Crossref PubMed Scopus (273) Google Scholar, 28Mikhail A.A. Beck E.X. Shafer A. Barut B. Gbur J.S. Zupancic T.J. Schweitzer A.C. Cioffi J.A. Lacaud G. Ouyang B. Keller G. Snodgrass H.R. Blood. 1997; 89: 1507-1512Crossref PubMed Google Scholar); however, it is not clear which isoforms of the leptin receptor are involved in these actions. Recent studies show that leptin receptors undergo homo-oligomerization upon ligand binding (17White D.W. Kuropatwinski K.K. Devos R. Baumann H. Tartaglia L.A. J. Biol. Chem. 1997; 272: 4065-4071Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 29Nakashima K. Narazaki M. Taga T. FEBS Lett. 1997; 403: 79-82Crossref PubMed Scopus (75) Google Scholar), suggesting that leptin receptor signaling does not involve additional signal-transducing subunits. Leptin receptors thus belong to the growth hormone receptor subfamily, which includes GHR, erythropoietin receptor, prolactin receptor, and granulocyte colony-stimulating factor receptor, all of which are activated by homo-dimerization (30Heldin C.-H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1445) Google Scholar). In contrast, signaling by other members of the class I cytokine receptors family are induced through formation of hetero-oligomeric complexes with molecules structurally related to cytokine receptors (30Heldin C.-H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1445) Google Scholar). At least five different isoforms of the leptin receptor are predicted to exist (12Lee G.-H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2112) Google Scholar). The long form (OBRl) is predicted to have 302 cytoplasmic residues. Three short forms have predicted cytoplasmic domains ranging from 32 to 40 amino acids. These four isoforms have identical extracellular and transmembrane domains. Furthermore, the membrane-proximal 29 amino acids are also identical. The additional 273, 3, 5, and 11 cytoplasmic residues, respectively, are generated by alternative splicing and are encoded by separate exons (12Lee G.-H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2112) Google Scholar, 31Fei H. Okano H.J. Li C. Lee G.-H. Zhao C. Darnell R. Friedman J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7001-7005Crossref PubMed Scopus (658) Google Scholar). Furthermore, a soluble receptor with no transmembrane region has also been predicted (12Lee G.-H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2112) Google Scholar). The common 29 amino acid residues in the four membrane-bound leptin receptor isoforms all contain a “Box 1” motif, which is highly conserved among most members of the cytokine receptor family (32Murakami M. Narazaki M. Hibi M. Yawata H. Yasukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (492) Google Scholar). This motif is usually located within the first 20 cytoplasmic residues of the receptors. A less conserved “Box 2” motif is also found in a number of receptors of the family. This motif is usually located between the first ∼50–60 amino acids of the cytoplasmic domain and is found only in the long leptin receptor isoform (18Ghilardi N. Skoda R.C. Mol. Endocrinol. 1997; 11: 393-399Crossref PubMed Scopus (270) Google Scholar, 32Murakami M. Narazaki M. Hibi M. Yawata H. Yasukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (492) Google Scholar). Mutational analyses of these conserved regions in several receptors suggest that these two domains are required for interaction and activation of tyrosine kinases of the Janus kinase family and for receptor signaling function (32Murakami M. Narazaki M. Hibi M. Yawata H. Yasukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (492) Google Scholar, 33Gurney A.L. Wong S.C. Henzel W.J. de Sauvage F.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5292-5296Crossref PubMed Scopus (177) Google Scholar, 34Nicholson S.E. Novak U. Ziegler S.F. Layton J.E. Blood. 1995; 86: 3698-3704Crossref PubMed Google Scholar, 35Baumann H. Symes A.J. Comeau M.R. Morella K.K. Wang Y. Friend D. Ziegler S.F. Fink J.S. Gearing D.P. Mol. Cell. Biol. 1994; 14: 138-146Crossref PubMed Google Scholar). Since the short forms of the leptin receptor do not contain Box 2, it has been predicted that these isoforms are signaling inactive (10Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3238) Google Scholar, 12Lee G.-H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2112) Google Scholar, 16Tartaglia L.A. J. Biol. Chem. 1997; 272: 6093-6096Abstract Full Text Full Text PDF PubMed Scopus (1191) Google Scholar, 17White D.W. Kuropatwinski K.K. Devos R. Baumann H. Tartaglia L.A. J. Biol. Chem. 1997; 272: 4065-4071Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 18Ghilardi N. Skoda R.C. Mol. Endocrinol. 1997; 11: 393-399Crossref PubMed Scopus (270) Google Scholar). Indeed, OBRs has been shown to be incapable of stimulating some signaling pathways that are activated by OBRl (19Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M.H. Skoda R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6231-6235Crossref PubMed Scopus (737) Google Scholar, 23Baumann H. Morella K.K. White D.W. Dembski M. Bailon P.S. Kim H. Lai C.-F. Tartaglia L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8374-8378Crossref PubMed Scopus (764) Google Scholar). However, in some cases, cytokine receptors of the GHR subfamily with a mutated Box 2 motif and/or with only short intracellular domains have been shown to have signaling capability (23Baumann H. Morella K.K. White D.W. Dembski M. Bailon P.S. Kim H. Lai C.-F. Tartaglia L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8374-8378Crossref PubMed Scopus (764) Google Scholar, 36Wang Y.-D. Wood W.I. Mol. Endocrinol. 1995; 9: 303-311Crossref PubMed Google Scholar, 37Joneja B. Wojchowski D.M. J. Biol. Chem. 1997; 272: 11176-11184Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 38Lebrun J.-J. Ali S. Ullrich A. Kelly P.A. J. Biol. Chem. 1995; 270: 10664-10670Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 39Das R. Vonderhaar B.K. Mol. Endocrinol. 1995; 9: 1750-1759Crossref PubMed Google Scholar, 40Fukunaga R. Ishizaka-Ikeda E. Nagata S. Cell. 1993; 74: 1079-1087Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 41Sotiropoulos A. Perrot-Applanat M. Dinerstein H. Pallier A. Postel-Vinay M.C. Finidori J. Kelly P.A. Endocrinology. 1994; 135: 1292-1298Crossref PubMed Scopus (112) Google Scholar). In addition, several members of the receptor family to which leptin receptors belong can activate signaling pathways apart from those involving activation of the JAK-STAT pathway (42Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Silvennoinen O. Annu. Rev. Immunol. 1995; 13: 369-398Crossref PubMed Scopus (550) Google Scholar). We therefore studied activation of various signal transduction pathways by the short and the long leptin receptor isoforms after transfection into cultured mammalian cell lines. We found that OBRl underwent rapid tyrosine phosphorylation upon leptin treatment, that this was increased by co-expression of JAK2, and that both OBRs and OBRl had the ability to activate JAK2 and IRS-1 tyrosine phosphorylation in a leptin-dependent manner in transiently transfected cell lines. Both isoforms were also able to induce leptin-dependent activation of MAPK in both transiently transfected cells and in stable CHO cell lines. It is clear that OBRl more robustly activate these pathways than does OBRs. In contrast, only the long form of the leptin receptor had the ability to activate STAT3 tyrosine phosphorylation and to stimulated c-fos gene transcription. Our results show that under some conditions, the short form of the leptin receptor clearly has signaling capability and that these require Box 1, but not Box 2. These results substantially extend our knowledge of the signaling potential of two key leptin receptor isoforms. Recombinant mouse and human leptin, as well as125I-labeled human leptin, were obtained from Eli Lilly (Indianapolis, IN). Crystalline porcine insulin was a gift from Dr. R. Chance, Eli Lilly. Epidermal growth factor (EGF) and myelin basic protein (MBP) were from Sigma. All reagents for cell culture and transfection were purchased from Life Technologies Inc. Monoclonal antibody 12CA5 (anti-hemagglutinin peptide (HA)) was from Babco (Emeryville, CA). Ribosomal S6 substrate peptide, monoclonal phosphotyrosine 4G10 antibody, anti-rat C-terminal IRS-1 antibody, anti-JAK1 and -JAK2 antibodies, and the anti-NT/CT MAPK antibody were all from Upstate Biotechnology, Inc. (Lake Placid, NY). An anti-JAK-PAN antibody was a gift from Dr. Dwayne L. Barber (Ontario Cancer Institute). An anti-active ERK2 antibody was purchased from Promega. The STAT3 antibody and the phosphospecific STAT3 antibody were purchased from New England Biolabs (Beverly, MA). The leptin receptor antibody was a kind gift from Dr. Radek Skoda (University of Basel, Switzerland). An expression vector containing an N-terminal HA epitope-tagged RSK1 cDNA was generously provided by Dr. Joseph Avruch (Massachusetts General Hospital, Boston, MA). A cDNA expression vector encoding N-terminal epitope-tagged ERK1 (HA-ERK1) was a gift from Dr. Jonathan Chernoff (Fox Chase Cancer Center, Philadelphia, PA). The murine HA-STAT3 expression construct was provided by Dr. John Blenis (Harvard Medical School, Boston, MA). The human insulin receptor and rat IRS-1 cDNA expression vectors, and IRS-1 antibodies for Western blotting (raised against whole protein) were kind gifts from Dr. Morris White (Joslin Diabetes Center, Boston, MA). JAK1 and JAK2 cDNA expression vectors were gifts from Dr. Rikiro Fukunaga (Osaka University, Osaka, Japan) and from Dr. Linda Winston (The Salk Institute). The c-fos luciferase reporter plasmid (encompassing 711 base pairs upstream of the transcriptional start site of the fos promoter) was kindly given by Dr. Ralf Jankneckt (The Salk Institute). A vector containing 109 base pairs of the thymidine kinase (TK) promoter cloned upstream of the luciferase reporter gene (TK109-luc) was a gift from Dr. T. Nagaya (Nagoya University, Japan). The CMV-β-gal reporter construct was fromCLONTECH (Palo Alto, CA). The human leptin receptor long form cDNA (hOBRl) was isolated by PCR using DNA from a human total fetal brain cDNA library (Stratagene, La Jolla, CA) as template. The DNA was isolated from >2 × 106 plaques by standard techniques. The PCR reaction was carried out using Pfu DNA polymerase (Stratagene) and primers corresponding to bases 194–218 (amino acids 1–8) and to bases 3679–3704 (extending 14 bases into the 3′-untranslated region) of the hOBRl cDNA (GenBank accession no. U43168). The 5′ end of both PCR primers also contained suitable DNA restriction enzyme recognition sequences for final cloning into a mammalian expression vector (pcDNA3, Invitrogen, San Diego, CA). Furthermore, the 5′ end of the upstream PCR primers also contained a consensus Kozak sequence (CCACC) immediately 5′ to the initiation ATG codon. The mouse leptin receptor short form cDNA (mOBRs) was generated by RT-PCR from mouse brain total RNA (isolated from C57BL mice), using primers corresponding to bases 61–85 (amino acids 1–8) and to bases 2736–2760 (extending 18 base pairs into the 3′-untranslated region, GenBank accession no. U42467). The primers also encompassed enzyme restriction sequences for final cloning into the mammalian expression vector pcDNA3.1/Zeo(−) (Invitrogen). The mouse leptin receptor long form cDNA (mOBRl) was also generated by RT-PCR from mouse brain total RNA, using primers corresponding to bases 61–85 (amino acids 1–8) and to bases 3543–3567 (extending 21 base pairs into the 3′-untranslated region, GenBank accession no. U46135), and cloned into pcDNA3.1/Zeo(−). The entire coding regions of all three leptin receptor constructs were sequenced using standard double-stranded plasmid procedures. Several PCR-induced sequence errors were detected in all clones. By subcloning of restriction fragments with the correct sequence from different clones, expression vectors encompassing the full-length coding region of the different isoforms were assembled. A Box 1 mutant (Pro876-Asp-Pro878 → Ser-Asp-Ser) of mOBRs (OBRs Box 1(mt)) was also generated using the site-directed mutagenesis kit from CLONTECH. The entire coding region of this construct was sequenced using standard double-stranded plasmid techniques. The TK109-luc reporter plasmid was linearized at a unique SalI site just upstream of the TK promoter by restriction enzyme digestion and dephosphorylated using alkaline phosphatase as described by the manufacturer (Boerhinger Mannheim). Two 5′-phosphorylated primers encompassing complementary sequences of two direct copies of the m67/SIE sequence (5′-TCGACATTTCCCGTAAATCCATTTCCCGTAAATC-3′ and 5′-TCGAGATTTACGGGAAATGGATTTACGGGAAATG-3′) (43Wagner B.J. Hayes T.E. Hoban C.J. Cochran B.H. EMBO J. 1990; 9: 4477-4484Crossref PubMed Scopus (554) Google Scholar) were ligated in presence of the linearized vector and subsequently transformed into Escherichia coli using standard techniques. To identify positive clones, DNA from several independent colonies were subjected to sequencing using standard double-stranded plasmid protocols. COS-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM, low glucose) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 10 μg/ml streptomycin at 37 °C in 5% CO2. CHO cells were grown in Ham's F-12 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 10 μg/ml streptomycin. 293 cells were grown in DMEM (high glucose) and as COS-1 cells, except that plates were coated with gelatin. PC12 cells were grown in DMEM (high glucose) supplemented with 10% fetal calf serum, 5% horse serum, 100 units/ml penicillin, and 10 μg/ml streptomycin at 37 °C in 5% CO2. For leptin binding assays, COS-1 cells were grown in 24-well plates and transfected using 1.6 μl of LipofectAMINE and 200 ng of DNA per well according to the manufacturer's protocol. For the luciferase, β-galactosidase, and kinase assays, cells were grown in six-well plates and transfected using 10 μl of LipofectAMINE and 1.0 μg of each plasmid DNA. In all experiments including JAK cDNA, the amounts of transfected JAK cDNA were one-tenth of the total amount of DNA transfected if nothing else indicated. For Western blotting experiments, cells were grown in 10-cm dishes if nothing else is noted, and transfected using 50 μl of LipofectAMINE and a total of 20 μg of plasmid DNA, if nothing else is indicated. All cells were serum-starved for 12–15 h prior to stimulation with hormones. Cells were harvested 48 h after transfection for the luciferase and β-galactosidase assays and lysed in 500 μl of lysis buffer A (25 mm glycylglycine, 15 mm MgSO4, 4 mm EGTA with 1% Triton X-100 and 2 mmdithiothreitol). For Western blotting experiments and kinase assays, cells were harvested 72 h after transfection by aspirating the medium, rinsing in ice-cold phosphate-buffered saline, and scraping into 1000 μl of ice-cold lysis buffer B (1% Nonidet P-40, 0.5% Triton X-100, 10% glycerol, 150 mm NaCl, 2 mmNa3VO4, 20 mm NaF, 1 mmphenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 50 mm Tris-HCl, pH 7.4). The lysate was clarified by centrifugation at 23,000 × g for 15 min, and the supernatant was immunoprecipitated as described below. CHO cells were transfected with mOBRs or mOBRl expression vectors as described above, and Zeocin (Invitrogen)-resistant clones were isolated over a period of 2 weeks. Leptin receptor-expressing clones (CHO-OBRs and CHO-OBRl) were identified by125I-leptin binding experiments as described below. Transfections were carried out in triplicates in 24-well tissue culture plates as described above. Forty-eight hours after transfection, cells were serum-deprived for 15 h at 37 °C, 5% CO2 and then incubated in 200 μl of binding buffer (DMEM, 0.1% bovine serum albumin) with 1 × 105 cpm of human125I-leptin, with or without unlabeled human leptin (100 nm final concentration) and incubated at room temperature for 30 min. Cells were then washed three times in 1 ml of binding buffer, lysed in 500 μl of lysis buffer C (1% Nonidet P-40, 0.5% Triton X-100, 1 n NaOH), and finally subjected to measurement of bound 125I-leptin in a γ counter. CHO-OBRs and CHO-OBRl cells were grown to confluence and starved for 15 h before experiments. Immunoprecipitations were performed at 4 °C by incubating clarified cell extracts with the OBR, 12CA5, or IRS-1 (Upstate Biotechnology, Inc.) antibodies and protein A-agarose beads (1:15 dilution of a 50% slurry in 1% Nonidet P-40, 0.5% Triton X-100, 10% glycer

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