Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176
Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176
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DOI：
10.1038/s41598-020-61370-y
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发表时间：
2020-03-10
影响因子：
4.6
通讯作者：
Doi, Masao
中科院分区：
文献类型：
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作者：
Wang, Tianyu;Nakagawa, Shumpei;Doi, Masao
G蛋白偶联受体（GPCR）是具有多种治疗应用的重要药物靶点。然而，仍有一百多个孤儿GPCR，其蛋白质功能和生化特征仍未确定。 Gpr176 编码 A 类孤儿 GPCR，它在小鼠下丘脑的生物钟调节中发挥作用，并且还与人类乳腺癌转录反应有关。在这里，我们显示 Gpr176 是 N-糖基化的。对小鼠下丘脑提取物进行肽-N-糖苷酶处理表明内源性 Gpr176 发生 N-糖基化。使用异源表达系统，我们发现 N-糖基化发生在 Gpr176 N 末端区域的四个保守天冬酰胺残基处。由于这些残基突变导致的 N-糖基化缺陷降低了 Gpr176 的蛋白表达。在分子功能水平上，Gpr176 具有组成型、不依赖激动剂的活性，导致 cAMP 合成减少。尽管 N-糖基化缺陷并没有损害这种内在活性，但由此导致的蛋白质表达减少伴随着细胞中 cAMP 抑制活性的减弱。我们还证明人 GPR176 是 N-糖基化的。重要的是，人 GPR176 保守 N-糖基化位点（rs1473415441；rs761894953）的错义变异影响 N-糖基化，从而减弱细胞中的蛋白质表达和 cAMP 抑制活性。我们表明，N-糖基化是功能性 Gpr176/GPR176 蛋白有效表达的先决条件。
G-protein-coupled receptors (GPCRs) are important drug targets with diverse therapeutic applications. However, there are still more than a hundred orphan GPCRs, whose protein functions and biochemical features remain unidentified. Gpr176 encodes a class-A orphan GPCR that has a role in circadian clock regulation in mouse hypothalamus and is also implicated in human breast cancer transcriptional response. Here we show that Gpr176 is N-glycosylated. Peptide-N-glycosidase treatment of mouse hypothalamus extracts revealed that endogenous Gpr176 undergoes N-glycosylation. Using a heterologous expression system, we show that N-glycosylation occurs at four conserved asparagine residues in the N-terminal region of Gpr176. Deficient N-glycosylation due to mutation of these residues reduced the protein expression of Gpr176. At the molecular function level, Gpr176 has constitutive, agonist-independent activity that leads to reduced cAMP synthesis. Although deficient N-glycosylation did not compromise this intrinsic activity, the resultant reduction in protein expression was accompanied by attenuation of cAMP-repressive activity in the cells. We also demonstrate that human GPR176 is N-glycosylated. Importantly, missense variations in the conserved N-glycosylation sites of human GPR176 (rs1473415441; rs761894953) affected N-glycosylation and thereby attenuated protein expression and cAMP-repressive activity in the cells. We show that N-glycosylation is a prerequisite for the efficient protein expression of functional Gpr176/GPR176.