Molecular cloning and characterisation of GPR74 a novel G-protein coupled receptor closest related to the Y-receptor family

A novel gene product, GPR74, with homology to the seven transmembrane-domai n receptor superfamily, has been cloned. GPR74 has been identified from the expressed sequence tags (EST) database. Subsequent PCR amplification of th at sequence and screening of a human heart cDNA library led to the isolatio n of a 1.7-kb cDNA clone encoding a protein of 408 amino acids. GPR74 shows highest amino acid identity (33%) to the human neuropeptide Y-receptor sub type Y2. The human and mouse genes for GPR74 have been isolated and their e xon-intron structures determined. In both species the gene consists of four exons spanning around 20 kb with the exon-intron borders being 100% conser ved. Northern analysis of various human tissues reveals highest levels of m RNA expression in brain and heart. In situ hybridisation analysis of rat br ain tissue confirms this result and identifies the hippocampus and amygdala nuclei as the brain areas with particular high expression of GPR74 mRNA. F luorescence in situ hybridisation, PCR analysis on a radiation hybrid panel and interspecific mouse backcross mapping have localised the genes to huma n chromosome 4q21 and mouse chromosome 5. Expression of the human GPR74 cDN A as a GFP-fusion protein in various cell lines reveals the inability of th e recombinant receptor protein to reach the cell surface. This is consisten t with the lack of NPY specific binding in these cells and suggests that un known factors are required for a full functional receptor complex. (C) 2000 Elsevier Science B.V. All rights reserved.