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Signaling and Ligand Binding by Recombinant Neuromedin U Receptors: Evidence for Dual Coupling to G_q/11 and G_i and an Irreversible Ligand-Receptor Interaction

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom (P.J.B.; G.B.W.); and 7TMR Assay Development and Compound Profiling, GlaxoSmithKline, New Frontiers Science Park, Harlow, United Kingdom (P.G.S., A.W.)

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 G_q/11 and G_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.

Address correspondence to: Dr. Gary. B. Willars, Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, University Rd., LE1 9HN UK. E-mail: gbw2{at}le.ac.uk

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