Melanin-concentrating hormone receptor and signaling system in central nervous system

黑色素浓缩激素（melanin - concentration hormone， MCH）是一种下丘脑神经肽，在食物摄入中起着关键作用。它通过两种G蛋白偶联受体（gpcr） MCH1R和MCH2R起作用，其中MCH1R是主要的食物摄入调节因子。我们之前报道过MCH1R胞外结构域的n -链糖基化是细胞表面表达和信号转导所必需的。我们现在报道了大鼠MCH1R c端区域的作用。我们构建了一系列c端截断突变体，并测定了由此引起的蛋白质表达、细胞表面表达、配体结合和mch刺激的钙内流的变化。通过分析两个突变体ΔT317（缺失36个c端氨基酸）和ΔR321（缺失32个c端氨基酸），我们发现Phe^<318>和Arg^<321>之间的区域负责信号转导。对单个或多个残基突变进行了更详细的分析。Arg^<319>、Lys^<320>或Arg^<321>的单突变均显著降低了细胞表面表达，而Arg^<319>或Lys^<320>的单突变均显著降低了钙的内流，而Arg^<321>的单突变则不显著。与单一突变相比，Arg^<319>和Lys^<320>同时突变导致钙内流刺激的效果明显下降。计算分析揭示了在许多1类gpcr中保守的双碱性氨基酸基序，可能是两亲性细胞质螺旋8的一部分。因此，我们的研究结果为推测的螺旋8在GPCR功能调节中的作用提供了新的见解。此外，我们还研究了mch诱导的内化途径的机制，这对受体反应的脱敏或调节很重要。流式细胞术定量分析表明，MCH1R通过PKC-、β-阻滞蛋白2-和动力蛋白i依赖通路经历mch诱导的快速内化过程，其中一部分c端尾部在内化过程中起重要作用。少

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, ΔT317 (deletion of 36 C-terminal amino acids) and ΔR321 (deletion of 32 C-terminal amino acids), we found that the region between Phe^<318> and Arg^<321> was responsible for signal transduction. A more detailed analysis was performed with single or multiple residue mutations. Single mutations of Arg^<319>, Lys^<320> or Arg^<321> exhi … More bited a decrease in the cell surface expression, while mutations of either Arg^<319> or Lys^<320>, but not Arg^<321>, showed a significant reduction in the calcium influx. Simultaneous mutations of Arg^<319> and Lys^<320> 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 a part of the amphiphilic cytoplasmic helix 8. Our results therefore provide new insights into the role of the putative helix 8 in the regulation of GPCR function. Furthermore, we investigated the mechanism underlying the MCH-induced internalization pathway, which is important for the desensitization or regulation of the receptor response. Quantitative analysis by flow cytometry indicated that MCH1R undergoes rapid MCH-induced internalization through a PKC-, β-arrestin 2- and dynamin I-dependent pathway and that a portion of the C-terminal tail plays an important role in the internalization process. Less