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Identification of domains directing specificity of coupling to G-proteins for the melanocortin MC3 and MC4 receptors

Authors
 Chung Sub Kim  ;  Soo Hyun Lee  ;  Ryang Yeo Kim  ;  Byung Jin Kim  ;  Song Zhe Li  ;  In Hye Lee  ;  Eun Jin Lee  ;  Sung Kil Lim  ;  Yun Soo Bae  ;  Weontae Lee  ;  Ja Hyun Baik 
Citation
 Journal of Biological Chemistry, Vol.277(35) : 31310-31317, 2002 
Journal Title
 Journal of Biological Chemistry 
ISSN
 0021-9258 
Issue Date
2002
Abstract
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 cAMP- responsive 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 Arg220 to Ala and Thr232 to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg220 and Thr232, 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 α-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 melanocortin receptors in the regulation of energy homeostasis (14, 15). In previous studies, we and others have demonstrated that heterologously expressed MC3R and MC4R are coupled to the cAMP pathway. We analyzed several α-MSH analogues upon stimulation of MC3R and MC4R using a CRE (cAMP responsive element)-mediated reporter gene transcription activity assay (16), and were able to show that both MC3R and MC4R, expressed in human cell line HEK 293T, stimulate transcription when stimulated using different analogues of melanocortin at different levels. Our previous studies have shown that MC3R and MC4R may have differential efficiencies and/or modes of signaling in terms of G-protein coupling, in addition to their specific ligand-receptor interactions, which can specify subtype-specific signaling pathwaysin vivo (16). The role of the third intracellular (i3) loop in G-protein coupling specificity has been investigated extensively for many seven-transmembrane domain receptors, including adrenergic, serotonergic, muscarinic, and dopaminergic receptors (17-21). For example, swapping experiments performed upon two different G protein-coupled receptors demonstrated the importance of this loop in selective coupling to specific G-protein/effector systems (22, 23). To identify the role(s) of the third intracellular loop of the MC3R and MC4R receptors in terms of G-protein coupling specificity and receptor activation, several chimeras were constructed and characterized. We used the CRE-luciferase reporter gene assay to score the efficacy of receptor-G proteins coupling (24, 25). In parallel, amino acid mutations were generated in the third intracytoplasmic loop of MC4R to identify the residues that play a role in G-protein coupling. These mutant receptors were examined in terms of their abilities to bind melanocortin receptor-specific ligands and with respect to signal transduction at the cAMP level.
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DOI
10.1074/jbc.M112085200
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Internal Medicine (내과학교실) > 1. Journal Papers
Yonsei Authors
Lim, Sung Kil(임승길)
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URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/143489
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