CLONING AND CHARACTERIZATION OF A NEW MEMBER OF THE G-PROTEIN COUPLEDRECEPTOR EDG FAMILY

We report here the cloning of a new member of the endothelium differen tiation gene (edg) subfamily of G-protein-coupled receptors. This nove l cDNA sequence was cloned from the ovine pars tuberalis using a rever se transcriptase polymerase chain reaction (RT-PCR) amplification with degenerate primers homologous to the highly conserved II and VII tran smembrane domains of the G-protein coupled receptor gene family. The P CR product was random primed with P-32 and used as a probe to screen a size-selected cDNA ovine pars tuberalis library, which resulted in th e isolation of a single clone of 2700 bp. This novel sequence was name d edg-2, because its nucleic acid sequence was 55% homologous over 501 nt overlap to an orphan sequence cloned from human endothelial cells, the endothelial differentiation gene, edg-1. The highest degree of ami noacid homology (42%) occurs in the seven putative transmembrane domai ns, particularly between the transmembrane domains III and VI (53% and 64%, respectively). The intervening hydrophilic domains are short and there are numerous putative phosphorylation sites for Ser/Thr-protein kinases in the second and third intracellular loop and in the COOH-te rminal domain. Through Northern analysis of total RNA, low levels of a t least four transcripts of 2.3, 2.5, 3.2 and 4 kb were found in sheep cerebral cortex and a 4.2 kb transcript was observed in NIH/3T3 fibro blasts. In addition, edg-2 transcripts (415 bp) were amplified by RT-P CR from pars tuberalis, cerebral blood vessels, hypothalamus, and reti na. Serum stimulation of Chinese hamster ovary (CHO) cells expressing the edg-2 receptor resulted in increased cell proliferation, as measur ed by [H-3]-thymidine incorporation. Edg-1 and edg-2 appear to be dist inct genes that may encode protein products that bind the same or rela ted ligand.