Cloning and characterization of a new member of the G-protein coupled receptor EDG family.
We report here the cloning of a new member of the endothelium differentiation gene (edg) subfamily of G-protein-coupled receptors. This novel cDNA sequence was cloned from the ovine pars tuberalis using a reverse transcriptase polymerase chain reaction (RT-PCR) amplification with degenerate primers homologous to the highly conserved II and VII transmembrane domains of the G-protein coupled receptor gene family. The PCR product was random primed with 32P and used as a probe to screen a size-selected cDNA ovine pars tuberalis library, which resulted in the isolation of a single clone of 2700 bp. This novel sequence was named edg-2, because its nucleic acid sequence was 55% homologous over 501nt overlap to an orphan sequence cloned from human endothelial cells, the endothelial differentiation gene, edg-1. The highest degree of aminoacid homology (42%) occurs in the seven putative transmembrane domains, 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-terminal domain. Through Northern analysis of total RNA, low levels of at 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 fibroblasts. In addition, edg-2 transcripts (415 bp) were amplified by RT-PCR from pars tuberalis, cerebral blood vessels, hypothalamus, and retina. Serum stimulation of Chinese hamster ovary (CHO) cells expressing the edg-2 receptor resulted in increased cell proliferation, as measured by [3H]-thymidine incorporation. Edg-1 and edg-2 appear to be distinct genes that may encode protein products that bind the same or related ligand.
Masana
M I
MI
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, IL 60611, USA.
Brown
R C
RC
Pu
H
H
Gurney
M E
ME
Dubocovich
M L
ML
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D014157:Transcription Factors; D019204:GTP-Binding Proteins