Abstract
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 Gs-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.
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Project supported by the National High-Tech R&D Program (863) of China (No. 2006AA10Z136) and a Grant-in-Aid for Innovative Training of Doctoral Students in Jiangsu Province of China (No. CXLX11-0701)
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Zhang, Bl., Li, Y., Ding, Jh. et al. Sphingosine 1-phosphate acts as an activator for the porcine Gpr3 of constitutively active G protein-coupled receptors. J. Zhejiang Univ. Sci. B 13, 555–566 (2012). https://doi.org/10.1631/jzus.B1100353
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DOI: https://doi.org/10.1631/jzus.B1100353
Key words
- G protein-coupled receptor
- Constitutive activity
- Sphingosine 1-phosphate
- Receptor internalization
- Porcine Gpr3
- Molecular cloning