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The Basic Residues in the Membrane-Proximal C-Terminal Tail of the Rat Melanin-Concentrating Hormone Receptor 1 Are Required for Receptor Function

Department of Pharmacology, Saitama Medical School of Medicine (M.T., Y.S., K.M.), Iruma-gun, Saitama 350-0492, Japan; Celestar Lexico-Sciences, Inc. (K.M., H.D.), Makuhari, Chiba 261-8501, Japan; and Department of Microbiology, Graduate School of Kagawa Nutrition University (M.T.), Sakado, Saitama 350-0288, Japan

Address all correspondence and requests for reprints to: Dr. Yumiko Saito, Department of Pharmacology, Saitama Medical School, 38 Moro-Hongo, Moroyama-cho, Iruma-gun, Saitama 350-0492, Japan. E-mail: yumisait{at}saitama-med.ac.jp.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that plays a key role in food intake. It acts through two G protein-coupled receptors (GPCRs), MCH1R and MCH2R, of which MCH1R is the primary regulator of food intake. We have previously reported that N-linked glycosylation of the extracellular domain of MCH1R is necessary for cell surface expression and signal transduction. We now report a role for the rat MCH1R C-terminal region. We constructed serial C-terminal truncation mutants and determined the resulting changes in protein expression, cell surface expression, ligand binding, and MCH-stimulated calcium influx. By analyzing two mutants, T317 (deletion of 36 C-terminal amino acids) and R321 (deletion of 32 C-terminal amino acids), we found that the region between Phe³¹⁸ and Arg³²¹) was responsible for signal transduction. A more detailed analysis was performed with single or multiple residue mutations. Single mutations of Arg³¹⁹, Lys³²⁰, or Arg³²¹ exhibited a decrease in the cell surface expression, whereas mutations of either Arg³¹⁹ or Lys³²⁰, but not Arg³²¹, showed a significant reduction in the calcium influx. Furthermore, simultaneous mutations of Arg³¹⁹ and Lys³²⁰ produced a pronounced decrease in the efficacy of calcium influx stimulation compared with single mutations. A computational analysis revealed a dibasic amino acid motif that is conserved among many class 1 GPCRs and may be part of the amphiphilic cytoplasmic helix 8 (an eight-cytoplasmic helix). Our results therefore provide new insights into the role of the putative helix 8 in the regulation of GPCR function.

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