Habara Makoto, Mori Nobuko, Okada Yuki, Kawasumi Koh, Nakao Nobuhiro, Tanaka Yoshikazu, Arai Toshiro, Yamamoto Ichiro
Department of Basic Veterinary Medicine, School of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 1808602, Japan.
Department of Basic Veterinary Medicine, School of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 1808602, Japan; Laboratory of Molecular Biomedicine for Pathogenesis, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1130033, Japan.
Gen Comp Endocrinol. 2018 May 15;261:31-39. doi: 10.1016/j.ygcen.2018.01.020. Epub 2018 Jan 31.
Melanocortin 4 receptor (MC4R), which is a member of the G protein-coupled receptor (GPCR) family, mediates regulation of energy homeostasis upon the binding of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system (CNS). Melanocortin 2 receptor accessory protein 2 (MRAP2) modulates the function of MC4R. We performed cDNA cloning of cat MC4R and MRAP2 and characterized their amino acid sequences, mRNA expression patterns in cat tissues, protein-protein interactions, and functions. We found high sequence homology (>88%) with other mammalian MC4R and MRAP2 encoding 332 and 206 amino acid residues, respectively. Reverse transcription-polymerase chain reaction analysis revealed that cat MC4R and MRAP2 mRNA were expressed highly in the CNS. In CHO-K1 cells transfected with cat MC4R, stimulation with α-MSH increased intracellular cyclic adenosine monophosphate (cAMP) concentration in a dose-dependent manner. Furthermore, the presence of MRAP2 enhanced the cat MC4R-mediated cAMP production. These results suggested that cat MC4R acts as a neuronal mediator in the CNS and that its function is modulated by MRAP2. In addition, our NanoBiT study showed the dynamics of their interactions in living cells; stimulation with α-MSH slightly affected the interaction between MC4R and MRAP2, and did not affect MC4R homodimerization, suggesting that they interact in the basal state and that structural change of MC4R by activation may affect the interaction between MC4R and MRAP2.
促黑素皮质素4受体（MC4R）是G蛋白偶联受体（GPCR）家族的成员，在中枢神经系统（CNS）中，α-促黑素细胞激素（α-MSH）与之结合后介导能量稳态的调节。促黑素皮质素2受体辅助蛋白2（MRAP2）调节MC4R的功能。我们进行了猫MC4R和MRAP2的cDNA克隆，并对它们的氨基酸序列、在猫组织中的mRNA表达模式、蛋白质-蛋白质相互作用及功能进行了表征。我们发现，猫的MC4R和MRAP2与其他哺乳动物的相应蛋白具有较高的序列同源性（>88%），分别编码332和206个氨基酸残基。逆转录-聚合酶链反应分析显示，猫的MC4R和MRAP2 mRNA在中枢神经系统中高表达。在转染了猫MC4R的CHO-K1细胞中，用α-MSH刺激可使细胞内环磷酸腺苷（cAMP）浓度呈剂量依赖性增加。此外，MRAP2的存在增强了猫MC4R介导的cAMP产生。这些结果表明，猫MC4R在中枢神经系统中作为神经元介质发挥作用，其功能受MRAP2调节。此外，我们的纳米荧光互补研究显示了它们在活细胞中的相互作用动态；用α-MSH刺激对MC4R和MRAP2之间的相互作用影响轻微，且不影响MC4R同二聚化，这表明它们在基础状态下相互作用，MC4R激活后的结构变化可能影响MC4R与MRAP2之间的相互作用。
