Sphingosine-1-phosphate is a high-affinity ligand for the G protein-coupled receptor GPR6 from mouse and induces intracellular Ca2+ release by activating the sphingosine-kinase pathway

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Abstract

We identified and cloned the mouse orthologue of human GPR6 as a new member of the lysophospholipid-receptor family. Sphingosine-1-phosphate (S1P) activated GPR6, transiently expressed in frog oocytes or in Chinese hamster ovary (CHO) cells, with high specificity and nanomolar affinity. The GPR6 gene was found to be located on chromosome 10B1 and a single exon coded for the entire open-reading frame. Signal transduction of S1P was inhibited by pertussis toxin, suggesting a coupling of GPR6 to an inhibitory G protein. In CHO cells transfected with GPR6, the sphingosine-kinase pathway mediated Ca2+ mobilization from internal stores. Apoptotic cell death was induced by serum deprivation or H2O2 treatment and was prevented by S1P in GPR6-, but not in vector-transfected CHO cells. The antiapoptotic effect of S1P required activation of sphingosine kinase and was accompanied by an increase in MAP-kinase phosphorylation.

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Materials and methods

Amplification and cloning of mouse GPR6 into the dual-function vector pXOON. By searching the mouse genomic database (Ensemble) with the rat and human GPR6 sequences, we identified a mouse orthologue of GPR6. A 1153 bp fragment, covering the entire open-reading frame of mouse GPR6 and 66 additional nucleotides of the non-coding regions, was amplified by PCR from adult murine brain cDNA and genomic mouse DNA using 5-CAGGGAGCAACATCTAGCGGCGATGAA-3 as forward and 5-TGTTGGGTTTGTGGTGTTTGTGAGGGA-3

Cloning of mouse GPR6 and sequence analysis

Comparison of the nucleotide sequences of rat and human GPR6 with the mouse genomic database allowed construction of PCR primers. A single band of 1153 bp, corresponding to the expected size of the intronless coding region, was obtained by PCR from an adult murine brain cDNA library and from mouse genomic DNA. Sequence analysis revealed an open-reading frame of 1089 nucleotides encoding a protein of 363 amino acids with a calculated molecular mass of 38 kDa (Fig. 1). A hydropathy blot showed the

Discussion

We identified a mouse orthologue of the orphan G protein-coupled receptor GPR6 that shares 99% sequence identity with rat GPR6 [23] and 93% with human GPR6 [30]. Like its rat and human counterparts murine GPR6 is predominantly expressed in the brain [23], [30] and at lower levels in testis, skeletal muscle, and in 15- and 17-day-old embryos (Fig. 2A). Compared with other GPCRs, GPR6 is most related to GPR3 and GPR12 [31], sharing 55% sequence identity with them. One approach to identify

Acknowledgements

We thank Dr. Jenny Stables for providing us with the CHO/Gα16/mtAEQ cells, Dr. Thomas Jesperson for the kind gift of the pXOON vector, Agata Blaszczyk-Wewer and Ute Süsens for excellent technical assistance, and Dr. Wolfgang Hampe and Dr. Axel Methner for critically reading the manuscript.

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