Regulation of G protein signaling for "feeding receptor" melanin-concentrating hormone receptor 1

黑色素浓集激素（MCH）受体1（MCH 1 R）是A类G蛋白偶联受体（GPCR）。MCH系统与多种生理功能有关，包括摄食调节、能量代谢和应激诱导的反应。我们已经报道了一个二元基序在膜近端的C-末端区域的重要性MCH 1 R功能。在本项目中，我们进一步揭示了MCH 1 R（R155）细胞内环2中的Arg残基在受体功能中起着关键作用。为了从小鼠脑中纯化潜在的MCH 1 R相互作用蛋白，将N末端、第二胞内环（i2）、第三环（i3）或C末端尾表达为GST融合蛋白并固定在谷胱甘肽-琼脂糖上。MALDI-TOF-MS分析鉴定了几个蛋白质，其中一个与i2结构域优先相互作用的蛋白质属于Ser/Thr激酶家族。在用MCH 1 R和激酶瞬时转染的293 T细胞中，激动剂对MCH 1 R的刺激导致活化的激酶从细胞质募集到质膜，其中激酶与MCH 1 R共定位。一个很好的特点抑制剂激酶显着干扰MCH诱导的内化。这是第一个蛋白质-蛋白质相互作用证明改变激动剂刺激的MCH 1 R的内化。

Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) is a class A G protein-coupled receptor (GPCR). The MCH system has been linked to a variety of physiological functions, including the regulation of feeding, energy metabolism and stress-induced responses. We have already reported the importance of a dibasic motif in the membrane-proximal C-terminal region for MCH1R function. In this project, we further reveal that an Arg residue in intracellular loop 2 of MCH1R (R155) plays a critical role in receptor function. To purify potential MCH1R interacting proteins from mouse brain, the N-terminus, second intracellular loop (i2), third intracellular loop (i3) or the C terminal tail was expressed as a GST fusion protein and immobilized on glutathione-agarose. MALDI-TOF-MS analysis identified several proteins, and one of protein preferentially interacted with i2 domain was found to belong to Ser/Thr kinase family. In 293T cells that were transiently transfected with MCH1R and the kinase, stimulation of MCH1R by agonist results in the recruitment of the activated kinase from cytoplasm to the plasma membrane, in which the kinase was colocalized with MCH1R. A well-characterized inhibitor for the kinase did significantly interfere MCH-induced internalization. This is the first protein-protein interaction demonstrated to alter agonist-stimulated internalization of the MCH1R.