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Termination of signalling by G-protein-coupled receptors requires inactivation of the G alpha-subunits of heterotrimeric G-proteins and the re-association of G alpha- and G betagamma-subunits. Inactivation of G alpha-subunits is achieved by the hydrolysis of bound GTP by an intrinsic GTPase activity, which is considerably enhanced by GTPase activating proteins. Regulators of G-protein signalling (RGS) proteins are a large family of GTPase activating proteins, many of which have structures indicating roles beyond GTPase activating protein activity and suggesting that the identity of the RGS protein recruited may also be critical to other aspects of signalling. There is some evidence of selective effects of RGS proteins against different G-protein-coupled receptors coupling to the same signalling pathways and growing evidence of physical interactions between RGS proteins and G-protein-coupled receptors. However, it is unclear as to how common such interactions are and the circumstances under which they are functionally relevant. Here we have examined potential selectivity of RGS2, 3 and 4 against signalling mediated by G alpha q/11-coupled muscarinic M3 receptors and gonadotropin-releasing hormone in an immortalised mouse pituitary cell line. Despite major structural differences between these two receptor types and agonist-dependent phosphorylation of the muscarinic M3- but not gonadotropin-releasing hormone receptor, signalling by both receptors was similarly inhibited by expression of either RGS2 or RGS3, whereas RGS4 has little effect. Thus, at least in these circumstances, RGS protein-dependent inhibition of signalling is not influenced by the nature of the G-protein-coupled receptor through which the signalling is mediated.

Neuromedin U (NmU) is a neuropeptide showing high levels of structural conservation across different species. Since its discovery in 1985, NmU has been implicated in numerous physiological roles, including smooth muscle contraction, energy homeostasis, stress, intestinal ion transport, pronociception, and circadian rhythm. Two G-protein-coupled receptors have been identified for NmU and cloned from humans, rats, and mice. Recombinantly expressed NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins, and NmU binds essentially irreversibly, preventing signaling to repetitive applications of NmU. However, it is unclear whether these properties reflect those of endogenously expressed NmU receptors or how these properties influence the functional consequences of NmU receptor signaling. Here, we have explored the signaling by rat NmU receptors expressed endogenously in cultured rat colonic smooth muscle cells and explore the functional consequence of this signaling by investigating the NmU-mediated contraction of ex vivo rat colonic smooth muscle preparations. We demonstrate that endogenous rat NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins. Furthermore, we show complex patterns of Ca(2+) signaling, including oscillations, and provide evidence of essentially irreversible binding of NmU to smooth muscle cells. Challenge of either circular or longitudinal rat isolated colonic smooth muscle preparations with NmU resulted in robust contractions. Stimulation was direct, and paradoxically, repetitive applications of NmU mediated repetitive contractions with no evidence of desensitization, highlighting a major discrepancy in the behavior of NmU in single cells and in intact tissues. The reason for this discrepancy is presently unknown.

The neuropeptide neuromedin U (NmU) shows considerable structural conservation across species. Within the body, it is widely distributed and in mammals has been implicated in physiological roles, including the regulation of feeding, anxiety, pain, blood flow, and smooth muscle contraction. Human NmU-25 (hNmU-25) and other NmU analogs were recently identified as ligands for two human orphan G protein-coupled receptors, subsequently named hNmU-R1 and hNmU-R2. These receptors have approximately 50% amino acid homology, and, at least in mammalian species, NmU-R1 and NmU-R2 are expressed predominantly in the periphery and central nervous system, respectively. Here, we have characterized signaling mediated by hNmU-R1 and hNmU-R2 expressed as recombinant proteins in human embryonic kidney 293 cells, particularly to define their G protein coupling and the activation and regulation of signal transduction pathways. We show that these receptors couple to both Galpha(q/11) and Galpha(i). Activation of either receptor type causes a pertussis toxin-insensitive activation of both phospholipase C and mitogen activated-protein kinase and a pertussis toxin-sensitive inhibition of adenylyl cyclase with subnanomolar potency for each. Activation of phospholipase C is sustained, but despite this capacity for prolonged receptor activation, repetitive application of hNmU-25 does not cause repetitive intracellular Ca2+ signaling by either recombinant receptors or those expressed endogenously in isolated smooth muscle cells from rat fundus. Using several strategies, we show this to be a consequence of essentially irreversible binding of hNmU-25 to its receptors and that this is followed by ligand internalization. Despite structural differences between receptors, there were no apparent differences in their activation, coupling, or regulation.