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...

The gut endocrine system is emerging as a central player in the control of appetite and glucose homeostasis, and as a rich source of peptides with therapeutic potential in the field of diabetes and obesity. In this study we have explored the physiology of insulin-like peptide 5 (Insl5), which we identified as a product of colonic enteroendocrine L-cells, better known for their secretion of glucagon-like peptide-1 and peptideYY. i.p. Insl5 increased food intake in wild-type mice but not mice lacking the cognate receptor Rxfp4. Plasma Insl5 levels were elevated by fasting or prolonged calorie restriction, and declined with feeding. We conclude that Insl5 is an orexigenic hormone released from colonic L-cells, which promotes appetite during conditions of energy deprivation.

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.