Molecular cloning of a novel putative G protein-coupled receptor from rat aortic smooth muscle. Downregulation of the mRNA level by the cyclic AMP messenger pathway

Abstract

A cDNA clone encoding a novel putative G protein-coupled receptor was isolated from rat aortic vascular smooth muscle cell cDNA library by the polymerase chain reaction (PCR) using degenerate oligonucleotide primers and subsequent hybridization screening with a cloned PCR product. Sequence analysis of this clone (AGR9) shows that it encodes a 483 amino acid protein with seven streches of hydrophobic amino acids, which are presumed to represent transmembrane domains. In addition, AGR9 protein exhibits several structural features characteristic of the G protein-coupled receptor family, which include the existence of potential N-linked glycosylation sites in the amino-terminal region, phosphorylation sites by serine/threonine kinases in the intracellular regions, and a number of well-conserved residues among most of the G protein-coupled receptors. Northern blot analysis indicates abundant expression of a major 3.9 kb AGR9 mRNA in brain, lung, heart, stomach, intestine, cultured rat aortic smooth muscle cells and cardiac myocytes. Treatment of rat aortic smooth muscle cells with the adenylyl cyclase activator forskolin causes a marked and transient decrease in the steady-state level of AGR9 mRNA. Dibutyryl cyclic AMP and the β-adrenergic agonist isoproterenol mimick the effect of forskolin. The ligand for AGR9 receptor has yet to be identified, however, these results suggest the existence of a novel G protein-coupled receptor expressed in the cardiovascular, central nervous and digestive systems.