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 ${}^{35}\text{S-GTPγS}$ 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.

Introduction

Lysophosphatidic acid (LPA; 1-acyl-2-sn-glycerol-3-phosphate)² and sphingosine-1-phosphate (S1P; 1-phospho-2-amino-4-trans-octadecene-1,3-diol) are bioactive lysophospholipids that can stimulate a large range of responses in various cell types. These responses include acute neurite retraction, actin stress-fiber formation, cell proliferation, Ca⁺⁺ and Cl⁻ conductance changes, smooth muscle contraction, and platelet activation (Durieux, 1995; Moolenaar et al., 1997; Spiegel et al., 1998). Though some S1P effects appear to be mediated intracellularly, it is now evident that both LPA and S1P signal through specific G-protein coupled receptors (GPCRs; Hecht et al., 1996; Fukushima et al., 1998; Lee et al., 1998b; Zondag et al., 1998). The first such GPCR identified, ventricular zone gene-1 (vzg-1, now referred to as lp_A1, and also called edg-2/mrec/Gpcr26) increased responsiveness to LPA in cell rounding and adenylate cyclase inhibition assays when overexpressed in cerebral cortical cell lines (Hecht et al., 1996). Heterologous expression of LP²_A1 in hepatoma (RH7777) and neuroblastoma (B103) cell lines which have no endogenous responses to LPA further demonstrated that this receptor is sufficient in conferring specific ${}^3\text{H-LPA}$ binding and multiple LPA-dependent responses (Fukushima et al., 1998), consistent with its initial identification as a receptor for LPA. Independent support for this identity was obtained by expression of LP_A1 in either yeast (Erickson et al., 1998) or lymphoid cells (An et al., 1997b), in each case conferring or potentiating responses to LPA.

We hypothesized that orphan receptor genes with substantial amino acid sequence similarity to LP_A1 could encode additional lysophospholipid receptors. Amino acid identities amongst members of other GPCR subfamilies which recognize the same ligand are generally around 35%. Three orphan GPCRs isolated from human or rat (Edg-1, H218/AGR16, and edg-3) shared 32–36% identity with LP_A1, and were thus candidate lysophospholipid receptors. Edg-1 was initially isolated in a subtractive screen for genes induced upon human endothelial cell differentiation (Hla and Maciag, 1990). H218 and AGR16 (names given independently to the same rat gene) were isolated by low-stringency screening with a D2 dopamine receptor probe or by degenerate PCR from rat aortic smooth muscle, respectively (Okazaki et al., 1993; MacLennan et al., 1994), while edg-3 was isolated from human genomic DNA using degenerate cannabinoid receptor primers (Yamaguchi et al., 1996).

Murine homologs of Edg-1, H218/AGR16, and edg-3 could be useful in the study of lysophospholipid receptors. First, receptor clones from the same organism can be studied comparatively in terms of sequences, expression patterns, genomic structures, and chromosomal localizations. Second, heterologous expression of receptors from the same species could allow comparative functional studies of ligand specificity and efficacy. Third, murine clones would provide necessary reagents for producing receptor-null mice. Recently, several groups have reported that non-murine receptors encoded by Edg-1, H218/AGR16, and edg-3 are S1P receptors (An et al., 1997a; Lee et al., 1998b; Zondag et al., 1998). For Edg-1, S1P induces Ca⁺⁺-dependent changes in cell morphology, induction of P-cadherin mRNA, increases MAP kinase activity, and internalization of the Edg-1 receptor (Lee et al., 1998b; Zondag et al., 1998). For the H218 and edg-3, S1P induces serum-response element (SRE) activation and changes in Ca⁺⁺ flux (An et al., 1997a). None of the previous studies showed that receptor expression conferred S1P responsiveness in the form of specific and direct G-protein activation.

Here we show that the three identified murine receptors can be grouped in a subfamily distinct from the lp_A LPA receptor genes. The coding regions of each are contained in a single exon. Each receptor confers S1P-inducible G-protein activation when heterologously expressed in a cell line that is unresponsive to S1P or LPA. Overall, expression patterns are distinct, both in the embryo and adult, although overlapping expression can be detected in some individual tissues. These three genes thus appear to encode a subfamily of differentially expressed S1P receptors.