Abstract
Relaxin-3 (R3) is the most recently identified member of the insulin superfamily, which is composed of peptides with diverse sequences held together by characteristic disulfide links connecting A and B peptide chains. R3 has nearly exclusive expression in the brainstem. It was demonstrated to be an additional ligand for the relaxin receptor LGR7, which is a class-C hormone receptor type G-protein coupled receptor (GPCR). We recently identified R3 as a ligand for two orphan G-protein coupled receptors, GPCR135 (aka SALPR) and GPCR142 (aka GPR100), which are class-A GPCRs and typical neuropeptide receptors. The predominant brain expression for both R3 and GPCR135, coupled with their high affinity interaction, strongly suggests that R3 is the endogenous ligand for GPCR135. Both R3 and GPCR135 from different species are highly conserved from genetic sequences to in vitro pharmacology. In contrast, GPCR142 is a pseudogene in rats, and the mouse gene is less conserved with human GPCR142, suggesting that GPCR142 may have a diminished role as a receptor for R3 in rodents. Further studies of GPCR142 in monkeys, cows, and pigs demonstrate that GPCR142 in those species shares high homology to the human GPCR142, and that it behaves similarly to the human receptor in vitro. This suggests that GPCR142 has conserved functions in these non-rodent species, including humans. In addition, the tissue expression pattern of GPCR142, primarily in peripheral tissue, is drastically different from R3, suggesting that GPCR142 may have an endogenous ligand other than R3. Sequence analysis among insulin/relaxin family members shows that insulin-like peptide 5 (INSL5) is the closest member to R3. Pharmacological characterization shows that INSL5 is a specific agonist for GPCR142, but not for GPCR135. Specifically, INSL5 binds to and activates GPCR142 at high affinity. Although INSL5 binds to GPCR135 at low affinity, it does not activate GPCR135. INSL5 mRNA is primarily expressed in the periphery, and its expression pattern overlaps with that of GPCR142, consistent with INSL5 being the endogenous ligand for GPCR142. Endogenous ligands and receptors tend to co-evolve. Consequently, INSL5, like GPCR142, is a pseudogene in rats, which further implies that INSL5/GPCR142 is an endogenous ligand/receptor pair. R3 can activate GPCR135, GPCR142, and LGR7. Therefore, in vivo administration of R3 could potentially activate all three receptors, which complicates the functional studies of GPCR135. By substituting the A chain of R3 with the A chain of INSL5, we devised a chimeric peptide (R3/I5), which is about 1000-fold more selective for GPCR135 and GPCR142, than for LGR7. C-terminal truncation of this chimeric peptide resulted in a potent antagonist [R3(BΔ23-27)R/I5] for GPCR135 and GPCR142, with no affinity for LGR7. The selective agonist and antagonist pair is particularly helpful for in vivo studies of GPCR135 in rats lacking GPCR142. R3 is highly expressed in the nucleus incertus, a region of the brain stem, which has been known to send afferent connections to different brain regions. [125I]R3/I5 is a radioligand that has an improved signal/noise ratio compared to [125I]R3. Autoradiographic distribution of GPCR135 binding sites using [125I]R3/I5 in rat brain shows that GPCR135 receptor is prominent in many regions, including olfactory bulb, amygdala, thalamus, somatosensory cortex, and superior colliculus, which have been reported to have connections to the nucleus incertus. Different brain regions serve different functions. The expression pattern of R3 and GPCR135 in the brain suggests multiple functions of R3 and GPCR135. The high level expression of R3 in the brainstem co-localizes with the expression of corticotrophin releasing factor receptor 1 (CRF1), suggesting a potential role of R3/GPCR135 in stress response. Water-restraint stress-induced R3 mRNA expression in the brain stem seems to support this hypothesis. In addition, recent studies have shown that acute and chronic intracerebroventricular (i.c.v.) administration of R3 induces feeding in rats. More specifically, i.c.v. injection of R3/I5 (GPCR135 selective agonist) stimulates feeding in rats, an effect that can be blocked by the GPCR135-selective antagonist R3(BΔ23-27)/I5, thus confirming the involvement of R3 and GPCR135 in feeding. The availability of those pharmacological tools should greatly facilitate future studies of the physiology of GPCR135 and GPCR142.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsPreview
Unable to display preview. Download preview PDF.
References
Adham IM, Burkhardt E, Benahmed M, Engel W (1993) Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells. J Biol Chem 268:26668–26672
Bani D, Maurizi M, Bigazzi M (1995) Relaxin reduces the number of circulating platelets and depresses platelet release from megakaryocytes: studies in rats. Platelets 6:330–335
Bathgate RA, Samuel CS, Burazin TC, Layfield S, Claasz AA, Reytomas IG, Dawson NF, Zhao C, Bond C, Summers RJ, Parry LJ, Wade JD, Tregear GW (2002) Human relaxin gene 3 (H3) and the equivalent mouse relaxin (M3) gene. Novel members of the relaxin peptide family. J Biol Chem 277:1148–1157
Bell GI, Merryweather JP, Sanchez-Pescador R, Stempien MM, Priestley L, Scott J, Rall LB (1984) Sequence of a cDNA clone encoding human preproinsulin-like growth factor II. Nature 310:775–777
Bell GI, Pictet RL, Rutter WJ, Cordell B, Tischer E, Goodman HM (1980) Sequence of the human insulin gene. Nature 284:26–32
Bogatcheva NV, Truong A, Feng S, Engel W, Adham IM, Agoulnik AI (2003) GREAT/LGR8 is the only receptor for insulin-like 3 peptide. Mol Endocrinol 17:2639–2646
Bullesbach EE, Schwabe C (1999) Tryptophan B27 in the relaxin-like factor (RLF) is crucial for RLF receptor-binding. Biochemistry 38:3073–3078
Bullesbach EE, Schwabe C (2005) LGR8 signal activation by the relaxin-like factor. J Biol Chem 280:14586–14590
Bullesbach EE, Yang S, Schwabe C (1992) The receptor-binding site of human relaxin II. A dual prong-binding mechanism. J Biol Chem 267:22957–22960
Burazin TC, Bathgate RA, Macris M, Layfield S, Gundlach AL, Tregear GW (2002) Restricted, but abundant, expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain. J Neurochem 82:1553–1557
Casten GG, Gilmore HR, Houghton FE, Samuels SS (1960) A new approach to the management of obliterative peripheral arterial disease. Angiology 11:408–414
Chalmers DT, Lovenberg TW, De Souza EB (1995) Localization of novel corticotropin-releasing factor receptor (CRF2) mRNA expression to specific subcortical nuclei in rat brain: comparison with CRF1 receptor mRNA expression. J Neurosci 15:6340–6350
Chen J, Kuei C, Sutton SW, Bonaventure P, Nepomuceno D, Eriste E, Sillard R, Lovenberg TW, Liu C (2005) Pharmacological Characterization of Relaxin-3/INSL7 Receptors GPCR135 and GPCR142 from Different Mammalian Species. J Pharmacol Exp Ther 312:83–95
Conklin BR, Farfel Z, Lustig KD, Julius D, Bourne HR (1993) Substitution of three amino acids switches receptor specificity of Gqα to that of Giα. Nature 363:274–280
Fredriksson R, Hoglund PJ, Gloriam DE, Lagerstrom MC, Schioth HB (2003) Seven evolutionarily conserved human rhodopsin G protein-coupled receptors lacking close relatives. FEBS Lett 554:381–388
Goto M, Swanson LW, Canteras NS (2001) Connections of the nucleus incertus. J Comp Neurol 438:86–122
Gudermann T, Birnbaumer M, Birnbaumer L (1992) Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells. J Biol Chem 267:4479–4488
Hida T, Takahashi E, Shikata K, Hirohashi T, Sawai T, Seiki T, Tanaka H, Kawai T, Ito O, Arai T, Yokoi A, Hirakawa T, Ogura H, Nagasu T, Miyamoto N, Kuromitsu J (2006) Chronic intracerebroventricular administration of relaxin-3 increases body weight in rats. J Recept Signal Transduct Res 26:147–158
Hsu SY (2003) New insights into the evolution of the relaxin-LGR signaling system. Trends Endocrinol Metab 14:303–309
Hsu SY (2003) Relaxin signaling in reproductive tissues. Mol Cell Endocrinol 202:165–170
Hsu SY, Nakabayashi K, Nishi S, Kumagai J, Kudo M, Sherwood OD, Hsueh AJ (2002) Activation of orphan receptors by the hormone relaxin. Science 295:671–674
Hsu SY, Semyonov J, Park JI, Chang CL (2005) Evolution of the signaling system in relaxin-family peptides. Ann NY Acad Sci 1041:520–529
Hudson P, Haley J, John M, Cronk M, Crawford R, Haralambidis J, Tregear G, Shine J, Niall H (1983) Structure of a genomic clone encoding biologically active human relaxin. Nature 301:628–631
Hudson P, John M, Crawford R, Haralambidis J, Scanlon D, Gorman J, Tregear G, Shine J, Niall H (1984) Relaxin gene expression in human ovaries and the predicted structure of a human preprorelaxin by analysis of cDNA clones. EMBO J 3:2333–2339
Koman A, Cazaubon S, Couraud PO, Ullrich A, Strosberg AD (1996) Molecular characterization and in vitro biological activity of placentin, a new member of the insulin gene family. J Biol Chem 271:20238–20241
Krajnc-Franken MA, van Disseldorp AJ, Koenders JE, Mosselman S, van Duin M, Gossen JA (2004) Impaired nipple development and parturition in LGR7 knockout mice. Mol Cell Biol 24:687–696
Kuei C, Sutton S, Bonaventure P, Pudiak C, Shelton J, Zhu J, Nepomuceno D, Wu J, Chen J, Kamme F, Seierstad M, Hack MD, Bathgate RAD, Hossain MA, Jwade JD, Atack J, Lovenberg TW, Liu C (2007) R3(BΔ23–27)R/I5 chimeric peptide, a selective antagonist for GPCR135 and GPCR142 over LGR7: in vitro and in vivo characterization. J Biol Chem 282:25425–25435
Kumagai J, Hsu SY, Matsumi H, Roh JS, Fu P, Wade JD, Bathgate RA, Hsueh AJ (2002) INSL3/Leydig insulin-like peptide activates the LGR8 receptor important in testis descent. J Biol Chem 277:31283–31286
Laugwitz KL, Allgeier A, Offermanns S, Spicher K, Van Sande J, Dumont JE, Schultz G (1996) The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families. Proc Natl Acad Sci USA 93:116–120
Liu C, Chen J, Kuei C, Sutton S, Nepomuceno D, Bonaventure P, Lovenberg TW (2005) Relaxin-3/insulin-like peptide 5 chimeric peptide, a selective ligand for G protein-coupled receptor (GPCR)135 and GPCR142 over leucine-rich repeat-containing G protein-coupled receptor 7. Mol Pharmacol 67:231–240
Liu C, Chen J, Sutton S, Roland B, Kuei C, Farmer N, Sillard R, Lovenberg TW (2003) identification of relaxin-3/INSL7 as a ligand for GPCR142. J Biol Chem 278:50765–50770
Liu C, Eriste E, Sutton S, Chen J, Roland B, Kuei C, Farmer N, Jornvall H, Sillard R, Lovenberg TW (2003) Identification of relaxin-3/INSL7 as an endogenous ligand for the orphan G-protein-coupled receptor GPCR135. J Biol Chem 278:50754–50764
Liu C, Kuei C, Sutton S, Chen J, Bonaventure P, Wu J, Nepomuceno D, Kamme F, Tran DT, Zhu J, Wilkinson T, Bathgate R, Eriste E, Sillard R, Lovenberg TW (2005) INSL5 is a high affinity specific agonist for GPCR142 (GPR100). J Biol Chem 280:292–300
Lok S, Johnston DS, Conklin D, Lofton-Day CE, Adams RL, Jelmberg AC, Whitmore TE, Schrader S, Griswold MD, Jaspers SR (2000) Identification of INSL6, a new member of the insulin family that is expressed in the testis of the human and rat. Biol Reprod 62:1593–1599
Ma S, Bonaventure P, Ferraro T, Shen PJ, Burazin TC, Bathgate RA, Liu C, Tregear GW, Sutton SW, Gundlach AL (2007) Relaxin-3 in GABA projection neurons of nucleus incertus suggests widespread influence on forebrain circuits via G-protein-coupled receptor-135 in the rat. Neuroscience 144:165–190
Matsumoto M, Kamohara M, Sugimoto T, Hidaka K, Takasaki J, Saito T, Okada M, Yamaguchi T, Furuichi K (2000) The novel G-protein coupled receptor SALPR shares sequence similarity with somatostatin and angiotensin receptors. Gene 248:183–189
McGowan BM, Stanley SA, Smith KL, Minnion JS, Donovan J, Thompson EL, Patterson M, Connolly MM, Abbott CR, Small CJ, Gardiner JV, Ghatei MA, Bloom SR (2006) Effects of acute and chronic relaxin-3 on food intake and energy expenditure in rats. Regul Pept 136:72–77
McGowan BM, Stanley SA, Smith KL, White NE, Connolly MM, Thompson EL, Gardiner JV, Murphy KG, Ghatei MA, Bloom SR (2005) Central relaxin-3 administration causes hyperphagia in male Wistar rats. Endocrinology 146:3295–3300
Nagayama Y, Kaufman KD, Seto P, Rapoport B (1989) Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor Biochem. Biophys Res Commun 165:1184–1190
Osheroff PL, Ho WH (1993) Expression of relaxin mRNA and relaxin receptors in postnatal and adult rat brains and hearts. Localization and developmental patterns. J Biol Chem 268:15193–15199
Potter E, Sutton S, Donaldson C, Chen R, Perrin M, Lewis K, Sawchenko PE, Vale W (1994) Distribution of corticotropin-releasing factor receptor mRNA in the rat brain and pituitary. Proc Natl Acad Sci USA 91:8777–8781
Rinderknecht E, Humbel RE (1978) The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin. J Biol Chem 253:2769–2776
Sherwood OD (2004) Relaxin's physiological roles and other diverse actions. Endocr Rev 25:205–234
Sudo S, Kumagai J, Nishi S, Layfield S, Ferraro T, Bathgate R, Hsueh AJ (2003) H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the ectodomain and the exoloop 2. J Biol Chem 278:7855–7862
Sutton SW, Bonaventure P, Kuei C, Nepomuceno D, Wu J, Zhu J, Lovenberg TW, Liu C (2005) G-protein-coupled receptor (GPCR)-142 does not contribute to relaxin-3 binding in the mouse brain: further support that relaxin-3 is the physiological ligand for GPCR135. Neuroendocrinology 82:139–150
Sutton SW, Bonaventure P, Kuei C, Roland B, Chen J, Nepomuceno D, Lovenberg TW, Liu C (2004) Distribution of G-protein-coupled receptor (GPCR)135 binding sites and receptor mRNA in the rat brain suggests a role for relaxin-3 in neuroendocrine and sensory processing. Neuroendocrinology 80:298–307
Tanaka M, Iijima N, Miyamoto Y, Fukusumi S, Itoh Y, Ozawa H, Ibata Y (2005) Neurons expressing relaxin 3/INSL 7 in the nucleus incertus respond to stress. Eur J Neurosci 2:1659–1670
Ullrich A, Bell JR, Chen EY, Herrera R, Petruzzelli LM, Dull TJ, Gray A, Coussens L, Liao YC, Tsubokawa M (1985) Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature 313:756–761
Ullrich A, Gray A, Tam AW, Yang-Feng T, Tsubokawa M, Collins C, Henzel W, Le Bon T, Kathuria S, Chen E (1986) Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 5:2503–2512
Yamaguchi Y, Yoshikawa K (2001) Cutaneous wound healing: an update. J Dermatol 28:521–534
Zhao L, Roche PJ, Gunnersen JM, Hammond VE, Tregear GW, Wintour EM, Beck F (1999) Mice without a functional relaxin gene are unable to deliver milk to their pups. Endocrinology 140:445–453
Author information
Authors and Affiliations
Corresponding author
Editor information
Rights and permissions
Copyright information
© 2008 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Liu, C., Lovenberg, T.W. (2008). Relaxin-3, INSL5, and Their Receptors. In: Civelli, O., Zhou, QY. (eds) Orphan G Protein-Coupled Receptors and Novel Neuropeptides. Results and Problems in Cell Differentiation, vol 46. Springer, Berlin, Heidelberg. https://doi.org/10.1007/400_2007_055
Download citation
DOI: https://doi.org/10.1007/400_2007_055
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-540-78350-3
Online ISBN: 978-3-540-78351-0
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)