The melanocortin receptors, MC3R and MC4R, are G protein-coupled receptors that are involved in regulating energy homeostasis. Using a luciferase reporter gene under the transcriptional control of a cAMPresponsive element (CRE), the coupling efficiency of the MC4R and MC3R to G-proteins was previously shown to be different. MC4R exhibited only 30 -50% of the maximum activity induced by MC3R. To assess the role of the different MC3R and MC4R domains in G-protein coupling, several chimeric MC3R/MC4R receptors were constructed. The relative luciferase activities, which were assessed after transfecting the chimeric receptors into HEK 293T cells, showed that the i3 (3rd intracellular) loop domain has an essential role in the differential signaling of MC3R and MC4R. To reveal which amino acid residue was involved in the MC4R-specific signaling in the i3 loop, a series of mutant MC4Rs was constructed. Reporter gene analysis showed that single mutations of Arg 220 to Ala and Thr 232 to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg 220 and Thr 232 , in the i3 loop of MC4R play crucial roles in G-protein coupling and the subtype-specific signaling pathways. An examination of the inositol phosphate (IP) levels in the cells transfected with either MC3R or MC4R after being exposed to the melanocortin peptides revealed significant stimulation of IP production by MC3R but no detectable increase in IP production was observed by MC4R. Furthermore, none of the MC4R mutants displayed melanocortin peptide-stimulated IP production. Overall, this study demonstrated that MC3R and MC4R have distinct signaling in either the cAMP-or the inositol phospholipid-mediated pathway with different conformational requirements.Melanocortins are peptide hormones that are derived from the precursor peptide pro-opiomelanocortin, by a series of proteolytic cleavages (1). The melanocortins are known to have a broad spectrum of physiological actions, which include the regulation of melanocyte pigmentation (2), thermoregulation (3), obesity (4), control of the cardiovascular system (5), and learning and memory (6), and have also been found to have immunomodulatory effects (7). These hormones mediate their effects through G protein-coupled receptors by stimulating adenylate cyclase (8). To date five melanocortin receptor subtypes, with different patterns of tissue expression in the brain and peripheral body, have been cloned and characterized (8 -12).It has been reported that the activation of melanocortin 4 receptor (MC4R) 1 by ␣-melanocyte-stimulating hormone (MSH) increases energy expenditure and decreases food intake. Moreover, the genetic disruption of MC4R was found to cause obesity in mice (13). Recent experiments in MC3R-null mice indicate that the inactivation of MC3R results in increased fat mass and reduced body mass, despite the fact that the animals were hypophagic and maintained normal metabolic rates (14, 15). These results suggest the nonredundancy of the MC3R and MC4R m...
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