Received for publication May 11, 2004.
Revised August 26, 2004.
Accepted for publication August 26, 2004.

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

Abstract

The neuropeptide, neuromedin U (NmU), shows considerable structural conservation across many 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. Recently, human NmU-25 (hNmU-25) and other NmU analogs were 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 CNS respectively. Here, we have characterized signaling mediated by hNmU-R1 and hNmU-R2 expressed as recombinant proteins in HEK293 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 sub-nanomolar potency for each. Activation of phospholipase C is sustained but despite this capacity for prolonged receptor activation, repetitive application of hNmU-25 does not result in 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.

Key words: Neuropeptides, Gi family, Gq/11 family, Phospholipase C's, IP3/DAG, Calcium (G Protein Coupled Signals), G protein regulation, Ca imaging, Fluorescence techniques

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