College of Animal Science and Technology, Nanjing Agricultural University, China.
J Zhejiang Univ Sci B. 2012 Jul;13(7):555-66. doi: 10.1631/jzus.B1100353.
We cloned the complete coding sequences of porcine Gpr3, Gpr6, and Gpr12 genes. Further, on the basis of their high levels of sequence similarity, these genes are identified as a subfamily of G protein-coupled receptors. These putative protein sequences also showed high sequence identity with other mammalian orthologs, including several highly conserved motifs. A wide expression of the Gpr3 gene in pigs was observed through tissue distribution analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR, specially in the brain, pituitary, fat, liver and oocyte, where its strong expression was observed. The Gpr3 gene was found to be located on chromosome 6 and a single exon coded for the entire open-reading frame. Expression of porcine Gpr3 in HEK293 cells resulted in constitutive activation of adenylate cyclase (AC) similar in amplitude to that produced by fully stimulated G(s)-coupled receptors. Moreover, sphingosine 1-phosphate (S1P) could increase AC activation via the constitutively active Gpr3 receptor. When a Gpr3-green fluorescent protein (GFP) construct was expressed in HEK293 cells, GFP-labeled Gpr3 protein was shown to be localized in the plasmalemma and subcellular membranes. After S1P treatment, agonist-mediated internalization could be visualized by confocal microscopy. In short, our findings suggest the porcine Gpr3, Gpr6, and Gpr12 genes as a subfamily of G protein-coupled receptors, and porcine Gpr3 was a constitutively active G protein-coupled receptor. Constitutive activation of AC and agonist-mediated internalization of Gpr3 receptor could be modulated by the S1P, suggesting that S1P might act as an activator for porcine Gpr3 receptor.
我们克隆了猪 Gpr3、Gpr6 和 Gpr12 基因的完整编码序列。此外，基于它们的高度序列相似性，这些基因被鉴定为 G 蛋白偶联受体的一个亚家族。这些假定的蛋白质序列也与其他哺乳动物同源物具有很高的序列同一性，包括几个高度保守的基序。通过逆转录聚合酶链反应 (RT-PCR) 和实时 PCR 的组织分布分析观察到 Gpr3 基因在猪中的广泛表达，特别是在大脑、垂体、脂肪、肝脏和卵母细胞中，其表达较强。Gpr3 基因被发现位于 6 号染色体上，单个外显子编码整个开放阅读框。在 HEK293 细胞中表达猪 Gpr3 导致环腺苷酸 (AC) 的组成型激活，其幅度与完全刺激的 G(s)-偶联受体产生的激活相似。此外，鞘氨醇 1-磷酸 (S1P) 可以通过组成型激活的 Gpr3 受体增加 AC 的激活。当 Gpr3-绿色荧光蛋白 (GFP) 构建体在 HEK293 细胞中表达时，GFP 标记的 Gpr3 蛋白被显示位于质膜和亚细胞膜上。在用 S1P 处理后，通过共焦显微镜可以可视化激动剂介导的内化。简而言之，我们的研究结果表明猪 Gpr3、Gpr6 和 Gpr12 基因作为 G 蛋白偶联受体的一个亚家族，而猪 Gpr3 是一种组成型激活的 G 蛋白偶联受体。AC 的组成型激活和 Gpr3 受体的激动剂介导的内化可以被 S1P 调节，这表明 S1P 可能作为猪 Gpr3 受体的激活剂。
