Identification of an EDG7 Variant, HOFNH30, a G-Protein- Coupled Receptor for Lysophosphatidic Acid Laura Rydelek Fitzgerald,* George M. Dytko,* Henry M. Sarau,† Ishrat Jahan Mannan,* Catherine Ellis,‡ Pamela A. Lane,‡ Kong B. Tan,§ Paul R. Murdock,§ , ** Shelagh Wilson, ¶, ** Derk J. Bergsma,‡ Robert S. Ames,‡ James J. Foley,† David A. Campbell, , ** Lynnette McMillan,§ Nicholas Evans, ¶, ** Nabil A. Elshourbagy,‡ Heather Minehart,§ and Ping Tsui‡ ,1 *Department of Renal Biology, †Department of Pulmonary Biology, and ‡Department of Molecular Biology, §Gene Expression Sciences, ¶ Vascular Biology, and Genetic Technologies, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania 19406; and **New Frontiers Science Park, Southern Way, Harlow, Essex, United Kingdom Received May 23, 2000 We have identified a cDNA, designated HOFNH30, which encodes a 354 amino acid G-protein-coupled re- ceptor (GPCR). This receptor has 96% amino acid iden- tity to the Jurkat-T cell-derived EDG7 and could be a splice variant. RT-PCR analysis demonstrated that HOFNH30 mRNA is expressed in placenta whereas EDG7 mRNA shows highest expression in prostate. The HOFNH30 gene is localized to human chromosome 1p22.3-1p31.1. When HOFNH30 was expressed in RBL- 2H3 cells, LPA and phosphatidic acid (PA) induced a calcium mobilization response with EC 50 values of 13 nM and 3 M, respectively. LPA also induced phos- phorylation of mitogen-activated protein kinase (p42 MAPK and p44 MAPK ) in HOFNH30-transfected but not vector-transfected RBL-2H3 cells. In the present study, we have identified a novel variant from the EDG receptor family, a GPCR for which LPA is a high- affinity endogenous ligand. © 2000 Academic Press Key Words: phospholipid; FLIPR; seven-trans- membrane. The phospholipid, lysophosphatidic acid (LPA), has been shown to produce a wide array of biological re- sponses including proliferation of a variety of cell types, contraction of smooth muscle and retraction of neurites (1, 2). Many of these effects appear to be produced by extracellular LPA interacting with a fam- ily of seven-transmembrane GPCRs. Three such recep- tors belonging to the EDG subfamily of GPCRs, EDG-2, -4, and -7, were identified as LPA receptors (3–7). The EDG family of receptors are so named because the first member was discovered as an immediate-early gene that was induced during differentiation of endothelial cells and termed endothelial differentiation gene-1 (EDG-1) (8). Later EDG-1 was shown to respond to sphingosine-1-phosphate (S-1-P) (9, 10). Two homolo- gous receptors, EDG-3 and -5, have also been cloned and respond to S-1-P (11, 12). The ligand for EDG-6 has not yet been established (13). In the present study, we have identified a human cDNA, designated HOFNH30, that may be a splice variant of the recently described EDG7 clone (7, 14). HOFNH30 has a predicted amino acid sequence of a seven-transmembrane GPCR with 50, 46, and 96% identity to EDG-2, -4, and -7, respectively. Expression profiling using RT-PCR demonstrated that HOFNH30 mRNA is expressed in placenta whereas EDG7 mRNA shows highest expression in prostate. In RBL-2H3 cells expressing HOFNH30, LPA and high concentrations of phosphatidic acid mediate increases in intracellular calcium. Further, we show that LPA can mediate phos- phorylation of p42 MAPK and p44 MAPK in cells overex- pressing HOFNH30. METHODS Materials. LPA (L--lysophosphatidic acid, oleoyl; oleoyl-sn- glycero-3-phosphate), PA (L--phosphatidic acid; 1,2-diacyl-sn- glycero-3-phosphate), and other lipids were obtained from Sigma (St Louis, MO). Cloning. A novel expressed sequence tag (EST), termed HOFNH30, was identified from a human ovarian tumor cDNA li- brary by searching a private database (Human Genome Sciences, Rockville, MD). Plasmid containing HOFNH30 was resequenced and found to be truncated at the 5'-end of the open reading frame (ORF). 5'-RACE (rapid amplification of cDNA ends) was performed with Marathon-ready human ovary and placenta cDNA (Clontech, Palo Alto, CA). Amplification was performed with a gene-specific primer 1 To whom correspondence should be addressed at Mail code UE0548, Department of Molecular Biology, 709 Swedeland Road, King of Prussia, PA 19406. Fax: (610) 270-5093. E-mail: Ping_Tsui- 1@SBPHRD.com. Biochemical and Biophysical Research Communications 273, 805– 810 (2000) doi:10.1006/bbrc.2000.2943, available online at http://www.idealibrary.com on 805 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.