Steven W. Sutton

Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

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 35S-labeled guanosine 5′-O-(3-thiotriphosphate) (GTPγS) incorporation in cells over-expressing 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 GTPγS binding and inhibits cAMP accumulation in GPCR135 overexpressing cells with EC50 values of 0.25 and 0.35 nM, respectively. 125I-Relaxin-3 binds GPCR135 at high affinity with a Kd 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.

Identification of relaxin-3/INSL7 as a ligand for GPCR142.

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 35S-GTPγS incorporation in response to relaxin-3. However, whereas GPCR135 signaling could be converted to calcium mobilization using a Gqi5 or Gα16 G-proteins, GPCR142 was only capable of functioning in the presence of Gα16. In the accompanying article (Liu, C., Eriste, E., Sutton, S., Chen, J., Roland, B., Kuei, C., Farmer, N., Jörnvall, 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.

Oxysterols direct B-cell migration through EBI2.

EBI2 (also called GPR183) is an orphan G-protein-coupled receptor that is highly expressed in spleen and upregulated upon Epstein–Barr-virus infection. Recent studies indicated that this receptor controls follicular B-cell migration and T-cell-dependent antibody production. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol metabolism. The biological effects of oxysterols have largely been credited to the activation of nuclear hormone receptors. Here we isolate oxysterols from porcine spleen extracts and show that they are endogenous ligands for EBI2. The most potent ligand and activator is 7α,25-dihydroxycholesterol (OHC), with a dissociation constant of 450u2009pM for EBI2. In vitro, 7α,25-OHC stimulated the migration of EBI2-expressing mouse B and T cells with half-maximum effective concentration values around 500 pM, but had no effect on EBI2-deficient cells. In vivo, EBI2-deficient B cells or normal B cells desensitized by 7α,25-OHC pre-treatment showed reduced homing to follicular areas of the spleen. Blocking the synthesis of 7α,25-OHC in vivo with clotrimazole, a CYP7B1 inhibitor, reduced the content of 7α,25-OHC in the mouse spleen and promoted the migration of adoptively transferred pre-activated B cells to the T/B boundary (the boundary between the T-zone and B-zone in the spleen follicle), mimicking the phenotype of pre-activated B cells from EBI2-deficient mice. Our results show an unexpected causal link between EBI2, an orphan G-protein-coupled receptor controlling B-cell migration, and the known immunological effects of certain oxysterols, thus uncovering a previously unknown role for this class of molecules.

Inhibition of fatty acid amide hydrolase produces analgesia by multiple mechanisms

British Journal of Pharmacology (2006) 148, 114. doi:10.1038/sj.bjp.0706773

R3(BΔ23–27)R/I5 Chimeric Peptide, a Selective Antagonist for GPCR135 and GPCR142 over Relaxin Receptor LGR7 IN VITRO AND IN VIVO CHARACTERIZATION

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(BΔ23–27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly23 to Arg, a truncation at the C terminus (Gly24-Trp27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(BΔ23–27)R/I5 binds to human GPCR135 (IC50 = 0.67 nm) and GPCR142 (IC50 = 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(BΔ23–27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC50 = 0.25 nm, pA2 = 9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(BΔ23–27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(BΔ23–27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.

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

G-protein-coupled receptor 135 (GPCR135), a former orphan GPCR also known as SALPR, has recently been shown to be modulated by relaxin-3 (R3). In addition to GPCR135, R3 has been shown to be an agonist for GPCR142 (which is a pseudogene in the rat) and to activate LGR7, which is primarily the receptor for relaxin-1/2. The interaction of R3 with LGR7 has confounded the autoradiographic study of the GPCR135 distribution in the rat CNS due to significant expression of LGR7 in the brain. R3/I5, a chimera of the B-chain of R3 bonded to the A-chain of INSL-5, is a specific GPCR135 agonist which is highly selective for GPCR135 over LGR7. [125I]R3/I5 specifically binds to sites on rat brain sections with a pharmacology matching results from membrane preparations of recombinant GPCR135 receptors. Autoradiographic studies show the GPCR135 receptor density is most prominent in areas such as the olfactory bulb, sensory cortex, amygdala, thalamus, paraventricular nucleus, supraoptic nucleus, inferior and superior colliculus. The GPCR135 mRNA distribution generally overlaps the pattern of GPCR135 binding sites shown by autoradiography using [125I]R3/I5. The nucleus incertus, which has been implicated in the extrapituitary actions of corticotropin-releasing hormone, is the primary source of R3 in the rat central nervous system and expresses GPCR135 receptors. These binding autoradiography and in situ hybridization data suggest that GPCR135 plays an important role in the central processing of sensory signals in rats, are consistent with a putative role for R3/GPCR135 as modulators of stress responses, and confirm the identity of R3 as the central nervous system ligand for GPCR135.

Distribution of relaxin-3 and RXFP3 within arousal, stress, affective, and cognitive circuits of mouse brain.

Relaxin‐3 (RLN3) and its native receptor, relaxin family peptide 3 receptor (RXFP3), constitute a newly identified neuropeptide system enriched in mammalian brain. The distribution of RLN3/RXFP3 networks in rat brain and recent experimental studies suggest a role for this system in modulation of arousal, stress, metabolism, and cognition. In order to facilitate exploration of the biology of RLN3/RXFP3 in complementary murine models, this study mapped the neuroanatomical distribution of the RLN3/RXFP3 system in mouse brain. Adult, male wildtype and RLN3 knock‐out (KO)/LacZ knock‐in (KI) mice were used to map the central distribution of RLN3 gene expression and RLN3‐like immunoreactivity (‐LI). The distribution of RXFP3 mRNA and protein was determined using [35S]‐oligonucleotide probes and a radiolabeled RXFP3‐selective agonist ([125I]‐R3/I5), respectively. High densities of neurons expressing RLN3 mRNA, RLN3‐associated β‐galactosidase activity and RLN3‐LI were detected in the nucleus incertus (or nucleus O), while smaller populations of positive neurons were observed in the pontine raphé, the periaqueductal gray and a region adjacent to the lateral substantia nigra. RLN3‐LI was observed in nerve fibers/terminals in nucleus incertus and broadly throughout the pons, midbrain, hypothalamus, thalamus, septum, hippocampus, and neocortex, but was absent in RLN3 KO/LacZ KI mice. This RLN3 neural network overlapped the regional distribution of RXFP3 mRNA and [125I]‐R3/I5 binding sites in wildtype and RLN3 KO/LacZ KI mice. These findings provide further evidence for the conserved nature of RLN3/RXFP3 systems in mammalian brain and the ability of RLN3/RXFP3 signaling to modulate “behavioral state” and an array of circuits involved in arousal, stress responses, affective state, and cognition. J. Comp. Neurol. 518:4016–4045, 2010.

Modulation of hippocampal theta oscillations and spatial memory by relaxin-3 neurons of the nucleus incertus

Hippocampal theta rhythm is thought to underlie learning and memory, and it is well established that pacemaker neurons in medial septum (MS) modulate theta activity. Recent studies in the rat demonstrated that brainstem-generated theta rhythm occurs through a multisynaptic pathway via the nucleus incertus (NI), which is the primary source of the neuropeptide relaxin-3 (RLN3). Therefore, this study examined the possible contribution of RLN3 to MS activity, and associated hippocampal theta activity and spatial memory. In anesthetized and conscious rats, we identified the ability of intraseptal RLN3 signaling to modulate neuronal activity in the MS and hippocampus and promote hippocampal theta rhythm. Behavioral studies in a spontaneous alternation task indicated that endogenous RLN3 signaling within MS promoted spatial memory and exploratory activity significantly increased c-Fos immunoreactivity in RLN3-producing NI neurons. Anatomical studies demonstrated axons/terminals from NI/RLN3 neurons make close contact with septal GABAergic (and cholinergic) neurons, including those that project to the hippocampus. In summary, RLN3 neurons of the NI can modulate spatial memory and underlying hippocampal theta activity through axonal projections to pacemaker neurons of the MS. NI/RLN3 neurons are highly responsive to stress and express corticotropin-releasing factor type-1 receptors, suggesting that the effects observed could be an important component of memory processing associated with stress responses.

Relaxin family peptide receptors--from orphans to therapeutic targets.

The relaxin family peptides have distinct expression profiles and physiological functions. Several of them are the cognate ligands for 4 G-protein-coupled relaxin family peptide receptors (RXFPs; formerly LGR7, LGR8, GPCR135, GPCR142). The relaxin/RXFP1 system has roles in reproductive physiology but is also involved in fibrosis, wound healing and responses to infarction. Relaxin has a potential use in congestive heart failure where fibrosis plays an important role in organ failure. The INSL3/RXFP2 system has biological roles in reproductive biology that may have limited therapeutic potential. However, the recently characterized relaxin-3/RXFP3 system is important in stress/anxiety and body composition. RXFP3 receptor antagonists are potentially novel anti-anxiety and anti-obesity drugs.

Metabolic and neuroendocrine responses to RXFP3 modulation in the central nervous system.

Neuroanatomical studies have shown relaxin‐3 neurons, primarily found in the rodent nucleus incertus (NI), project widely into a large number of areas expressing the relaxin‐3 receptor (RXFP3), and these data suggest relaxin‐3/RXFP3 signaling modulates sensory, emotional, and neuroendocrine processing. The similar distribution of this receptor–ligand pair in the rat, mouse, and monkey brain suggests that experimental findings obtained in lower species will translate to higher species. A role for relaxin‐3 and RXFP3 in modulating stress responses is strongly suggested by the expression of corticotropin‐releasing factor R1 (CRF‐R1) by NI cells, increased relaxin‐3 expression in the NI after stress or CRF injection, and hormonal responses to intracerebroventricular (i.c.v.) relaxin‐3 injection. Recent data are consistent with a further role for this ligand–receptor pair in modulating memory. In addition, relaxin‐3 has been reported to modulate feeding and body weight control. Acute or chronic central (i.c.v. or intraparaventricular) injections of relaxin‐3 have shown a consistent stimulatory effect on food consumption while relaxin was inactive, suggesting the phagic effect of relaxin‐3 is mediated by RXFP3. We have confirmed the role of RXFP3 in modulating feeding and body weight by using a selective RXFP3 agonist (R3/I5) and antagonist [R3(Δ23–27)R/I5], collecting feeding, body weight, hormone, and body composition data. In addition, we have preliminary body weight and magnetic resonance imaging data from relaxin‐3 knockout mice, which on a 129S5:B6 background are smaller and leaner than congenic controls. These data suggest relaxin‐3, acting through RXFP3, is involved in coordinating stress, learning and memory, and feeding responses as predicted on the basis of neuroanatomy.