Comparative analysis of three murine G-protein coupled receptors activated by sphingosine-1-phosphate

Abstract

The cloning and analysis of the first identified lysophosphatidic acid (LPA) receptor gene, lp(A1) (also referred to as vzg-1 or edg-2), led us to identify homologous murine genes that might also encode receptors for related lysophospholipid ligands. Three murine genomic clones (designated lp(B1), lp(B2), and lp(B3)) were isolated, corresponding to human/rat Edg-1, rat H218/AGR16, and human edg-3, respectively. Based on the amino acid similarities of their predicted proteins (44-52% identical), the three lp(B) genes could be grouped into a separate G-protein coupled receptor subfamily, distinct from that containing the LPA receptor genes lp(A1) and lp(A2). Unlike lp(A1) and lp(A2), which contain multiple coding exons, all lp(B) members contained a single coding exon. Heterologous expression of individual lp(B) members in a hepatoma cell line (RH7777), followed by 35S-GTPγS incorporation assays demonstrated that each of the three LP(B) receptors conferred sphingosine-1-phosphate-dependent, but not lysophosphatidic acid- dependent, G-protein activation. Northern blot and in situ hybridization analyses revealed overlapping as well as distinct expression patterns in both embryonic and adult tissues. This comparative characterization of multiple sphingosine-1-phosphate receptor genes and their spatiotemporal expression patterns will aid in understanding the biological roles of this enlarging lysophospholipid receptor family.