Sphingosine 1-phosphate acts as an activator for the porcine Gpr3 of constitutively active G protein-coupled receptors

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.