GPCR135, publicly known as somatostatin-and angiotensin-like peptide receptor, is expressed in the central nervous system and its cognate ligand(s) has not been identified. We have found that both rat and porcine brain extracts stimulated 35 S-labeled guanosine 5-O-(3-thiotriphosphate) (GTP␥S) incorporation in cells overexpressing GPCR135. Multiple rounds of extraction, purification, followed by N-terminal sequence analysis of the ligand from porcine brain revealed that the ligand is a product of the recently identified gene, relaxin-3 (aka insulin-7 or INSL7). Recombinant human relaxin-3 potently stimulates GTP␥S binding and inhibits cAMP accumulation in GPCR135 overexpressing cells with EC 50 values of 0.25 and 0.35 nM, respectively.125 I-Relaxin-3 binds GPCR135 at high affinity with a K d value of 0.31 nM. Relaxin-3 is the only member of the insulin/relaxin superfamily that can activate GPCR135. In situ hybridization showed that relaxin-3 mRNA is predominantly expressed in the dorsomedial ventral tegmental nucleus of the brainstem (aka nucleus incertus), as well as in discrete cells in the lateral periaqueductal gray and in the central gray nucleus. GPCR135 is expressed abundantly in the hypothalamus with discrete expression in the paraventricular nucleus of the hypothalamus and supraoptic nucleus, as well as in the cortex, septal nucleus, and preoptical area. Relaxin-3 has previously been shown to bind and activate the LGR7 relaxin receptor. However, we believe that neuroanatomical colocalization of GPCR135 and relaxin-3, coupled with a clear high affinity interaction, suggest that GPCR135 is the receptor for relaxin-3. The identification of relaxin-3 as the ligand for GPCR135 provides the framework for the discovery of a new brainstem/hypothalamus circuitry.The recent completion of the sequencing of the human genome revealed thousands of new genes. Among them are many orphan G-protein-coupled receptors (GPCRs), 1 which are identified from genomic DNA or mRNA sequences based on their predicted seven-transmembrane structures. Searching for ligands of the orphan GPCRs has because been an intense research area and has yielded numerous significant discoveries in the past decade (1-15). Identification of ligand/receptor pairs provides a basis for the understanding of the physiological roles of those GPCRs and their ligands, which can involve the central nervous, endocrine, reproductive, cardiovascular, immune, inflammatory, digestive, and metabolic systems (1-15). The identification of ligands for their receptors also provides additional opportunities to discover agonists and antagonists as innovative drugs to exert pharmacological effects by interacting with these newly identified receptors.Relaxin is a member of the insulin superfamily. The hallmark of this protein family is the presence of two peptide subunits that are arranged by three disulfide bonds (16 -19). Whereas insulin is known to play a major role in glucose metabolism and signals through the insulin receptor, a single transmembrane growth factor/tyr...

Both relaxin-3 and its receptor (GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/ INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(B⌬23-27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly 23 to Arg, a truncation at the C terminus (Gly 24 -Trp 27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(B⌬23-27)R/I5 binds to human GPCR135 (IC 50 ‫؍‬ 0.67 nM) and GPCR142 (IC 50 ‫؍‬ 2.29 nM) with high affinity and is a potent functional GPCR135 antagonist (pA2 ‫؍‬ 9.15) but is not a human LGR7 ligand. Furthermore, R3(B⌬23-27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC 50 ‫؍‬ 0.25 nM, pA2 ‫؍‬ 9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(B⌬23-27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(B⌬23-27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.Relaxin-3 (R3) 2 (1) is the most recently identified member of the insulin-relaxin peptide family. Both relaxin-3 and its receptor, GPCR135 (2), are predominantly expressed in the brain (2, 3). GPCR135, an inhibitory receptor, is expressed in many regions of the rodent brain such as the superior colliculus, sensory cortex, olfactory bulb, amygdale, and paraventricular nucleus (4 -6), suggesting potential physiological involvement in neuroendocrine and sensory processing. Recent in vivo studies have further shown that relaxin-3 and GPCR135 are involved in the stress response and in regulation of feeding. More specifically, water restraint stress or intracerebroventricular corticotrophin-releasing factor (CRF) infusion induces relaxin-3 expression in cells of the nucleus incertus, a region where CRF receptor-1 is also expressed (7), and central administration of relaxin-3 induces feeding in rat (8, 9). These findings suggest that GPCR135 and relaxin-3 may be involved in multiple physiological processes, some of which might be as yet unknown.In vitro relaxin-3 activates GPCR135 (2), GPCR142 (10), and LGR7 (11) receptors. The predominant brain expression of both relaxin-3 and GPCR135, coupled with their high affinity interaction, strongly suggests that relaxin-3 is the endogenous ligand for GPCR135 (2). Phar...

We have recently identified the insulin-like peptide relaxin-3 (aka INSL7) as the endogenous ligand for an orphan G-protein-coupled receptor, GPCR135 (aka somatostatin-and angiotensin-like peptide receptor). Analysis of possible receptors related to GPCR135 revealed a single orphan receptor, GPCR142. Thus, we tested whether GPCR142 could also respond to relaxin-3 or related insulin-like molecules. Surprisingly, GPCR142 was activated by nanomolar concentrations of relaxin-3 but was completely unresponsive to all other known insulin-like peptides. We evaluated by reverse transcriptase-PCR the expression of GPCR142 mRNA in a variety of human tissues and found expression in brain, kidney, testis, thymus, placenta, prostate, salivary gland, thyroid, and colon. In an analysis of other species, we were able to find a full-length mouse homolog of GPCR142, but were unable to detect any complete GPCR142 transcripts in rat. With respect to intracellular signaling, GPCR142 is similar to GPCR135 in that it potently inhibits adenylate cyclase and stimulates 35 S-GTP␥S incorporation in response to relaxin-3. However, whereas GPCR135 signaling could be converted to calcium mobilization using a G qi5 or G␣ 16 G-proteins, GPCR142 was only capable of functioning in the presence of G␣ 16 . In the accompanying article (Liu, C., Eriste, E., Sutton, S., Chen, J., Roland, B., Kuei, C., Farmer, N., Jö rnvall, H., Sillard, R., and Lovenberg, T. W. (2003) J. Biol. Chem. 278, 50754 -50764), we present the case that relaxin-3, which has previously been shown to bind to the relaxin receptor LGR7, is most likely the endogenous ligand for GPCR135. In this report, we show an additional receptor, GPCR142, which is also selectively activated by relaxin-3. However, the anatomical localization of GPCR142 suggests that GPCR142 may have different physiological functions.Relaxin-3 (1) is a member of the insulin superfamily, where each member consists of two peptide subunits arranged by three disulfide bridges. Recently, two leucine-rich repeat-containing G-protein-coupled receptors (LGRs), 1 LGR7 and LGR8 (2, 3), have been identified as the receptors for relaxin (3-7).LGR7 and LGR8 belong to the type III hormone receptor family (follicle stimulating hormone (FSH), luteinizing hormone (LH), and thyroid stimulating hormone (TSH) etc. that stimulate adenylate cyclase). Relaxin-3 was recently demonstrated to be an additional ligand for the relaxin receptor LGR7, but not LGR8 (8). Instead, the ligand INSL3 (9), another member of the insulin/relaxin family of peptides, has been shown to be an additional ligand for LGR8 (10).As part of a directed effort to identify ligands for orphan G-protein-coupled receptors (GPCRs), we established a systematic program to create tissue extracts as a source of possible GPCR ligands. In the accompanying article (23), we report the purification and identification of the peptide ligand, relaxin-3, as an endogenous ligand for orphan receptor GPCR135, aka SALPR (somatostatin-and angiotensin-like peptide receptor).In that report...

Insulin-like peptide 5 (INSL5) is a peptide that belongs to the relaxin/insulin family, and its receptor has not been identified. In this report, we demonstrate that INSL5 is a specific agonist for GPCR142. Human INSL5 displaces the binding of 125 I-relaxin-3 to GPCR142 with a high affinity (K i ‫؍‬ 1.5 nM). In a saturation binding assay, 125 The relaxin/insulin family peptides include insulin (1), IGF1 1 (2), IGF2 (3), relaxin (4, 5), INSL3 (6), INSL4 (7), INSL5 (8), INSL6 (9), and relaxin-3/INSL7 (10). Except for IGF1 and IGF2, which are single chain peptides, each member of the family consists of two peptide subunits (an A-chain and a B-chain) that are linked by three disulfide bonds (4 -14). Insulin, IGF1, and IGF2 are known to be involved in the regulation of glucose metabolism (15) and signal through tyrosine kinase/ growth factor receptors, which are single transmembrane receptors (16, 17). Relaxin plays multifunctional roles including uterus relaxation, reproductive tissue growth, and collagen remodeling in females (18). In addition, relaxin has been reported to play important roles in nonreproductive functions including wound healing, cardiac protection, and allergic responses (19). The receptor for relaxin has been identified recently as a leucine-rich repeat containing the G-protein-coupled receptor (LGR) LGR7 (20). Although relaxin also activates LGR8 in vitro (20), recent studies show that LGR8 is likely the endogenous receptor for INSL3 and is involved in testis descent (21,22). To date, the receptors for INSL4, INSL5, and INSL6 have not been identified. Relaxin-3 (also known as INSL7), the most recently identified member of the family, was reported to be an additional ligand for LGR7 (23). We recently identified relaxin-3 as a ligand for two orphan G-protein-coupled receptors GPCR135 (14) and GPCR142 (24). The predominant brain expression for both 14,25, 26) and GPCR135 (14,26), coupled with their high affinity interaction, strongly suggests that relaxin-3 is the endogenous ligand for GPCR135. The tissue expression pattern of GPCR142 (also known as GPR100), which is primarily in peripheral tissues (24), is drastically different from that of relaxin-3, suggesting that GPCR142 may have an endogenous ligand other than relaxin-3. Furthermore, despite the high conservation of relaxin-3 in different species, GPCR142 is less conserved in the mouse and is a pseudogene in the rat (26), suggesting that GPCR142 may have a diminished role in rodents and may function as a receptor for a different ligand (other than relaxin-3) in other mammals. Sequence analysis among insulin/relaxin family members indicates that INSL5 shares high homology to relaxin-3 (Fig. 1A), suggesting that it may be an additional ligand for GPCR135, GPCR142, LGR7, or LGR8. In this report, we demonstrate that INSL5 is an agonist for GPCR142 but not for GPCR135, LGR7, or LGR8.

Relaxin-3, the most recently identified member of relaxin/insulin family, is an agonist for leucine-rich repeat-containing G protein-coupled receptor (LGR)7, GPCR135, and GPCR142.LGR7 can be pharmacologically differentiated from GPCR135 and GPCR142 by its high affinity for relaxin. Selective ligands that specifically activate GPCR135 or GPCR142 are highly desirable for studying their functional roles. We have created chimeric peptides that consist of the B-chain of human relaxin-3 in combination with various A-chains from other members of the relaxin/insulin family. Pharmacological characterization of these chimeric peptides indicates the A-chain from relaxin-1, relaxin-2, insulin-like peptide (INSL)3, and INSL6 does not change the pharmacological properties of relaxin-3 significantly. In contrast, substitution of the relaxin-3 A-chain with the A-chain from INSL5 results in a chimeric peptide that selectively activates GPCR135 and GPCR142 over LGR7. This study demonstrates that the A-chains among some of the insulin/relaxin family members are pharmacologically exchangeable. The relaxin-3/INSL5 chimeric peptide is a potential tool to study in vivo function of GPCR135. In addition, because of the substitution of a very hydrophobic peptide (the A-chain of relaxin-3) with a very hydrophilic peptide (the A-chain from INSL5), the radiolabeled 125 I-relaxin-3/INSL5 chimera is a suitable ligand (high-affinity, low-nonspecific binding) for receptor autoradiographic studies on tissue sections. -3 (Bathgate et al., 2002), the newest member of the relaxin (Hudson et al., 1983(Hudson et al., , 1984)/insulin superfamily, has been recently reported as a ligand for two related orphan G protein-coupled receptors (GPCRs), GPCR135 (Liu et al., 2003a) and GPCR142 (Liu et al., 2003a) in addition to leucine-rich repeat-containing G protein-coupled receptor (LGR) LGR7 (Hsu et al., 2000Sudo et al., 2003). Both relaxin-3 and GPCR135 are predominantly expressed in the brain (Matsumoto et al., 2000;Bathgate et al., 2002;Burazin et al., 2002;Liu et al., 2003b) and are highly conserved among species from fish to humans Hsu et al., 2003;Liu et al., 2003b;Chen et al., 2005), suggesting that this ligand/receptor pair may play an important role in the central nervous system. The GPCR142 expression pattern is distinct from that of GPCR135 and relaxin-3 with an abundant peripheral tissue distribution in addition to expression in the brain (Liu et al., 2003a). GPCR142 is highly conserved among human, monkey, cow, and pig, but it is less conserved in the mouse, and a pseudogene in the rat despite extensive conservation of relaxin-3 genes in both rodent species (Chen et al., 2004), suggesting that GPCR142 has distinct function(s) from GPCR135 and diminished function(s) in the rodents. We recently reported that human insulin-like peptide 5 (INSL5) is a selective agonist for human GPCR142 . Recombinant human INSL5 activates human GPCR142 at high affinity but does not activate human Article, publication date, and citation information can be f...