CLONING OF THE RAT-BRAIN CDNA-ENCODING FOR THE SLC-1 G-PROTEIN-COUPLED RECEPTOR REVEALS THE PRESENCE OF AN INTRON IN THE GENE

In order to isolate new G protein-coupled receptors expressed in the c erebral cortex, a set of degenerate oligonucleotides corresponding to the third and seventh transmembrane segment were synthetized. Their us e in PCR on rat brain cortex mRNA amplified several cDNA fragments. On e of them, a 526 bp sequence, encoded for what was at that time an unk nown G protein-coupled receptor. An oligonucleotide derived from the s equence was then used as a probe to isolate the receptor cDNA from a r at brain cDNA library. It encodes for a 353aa protein with seven trans membrane segments, three consensus N-glycosylation sites at the amino terminus and several potential phosphorylation sites in the intracellu lar loops. This protein shares 91% overall identity with a recently cl oned human somatostatin-like receptor of 402aa named SLC-1. This sugge sts that we have cloned the rat orthologue of the human SLC-1. However , the extracellular N-terminus of the human receptor is 49 amino acids longer and shows 50% identity with the rat one. Because the human seq uence was deduced from genomic DNA, we suspected the presence of an in tron in the gene. This was confirmed by PCR using primers spanning the intron. On the basis of the sequence of a 128 kb fragment of chromoso me 22 encompassing the SLC-1 gene, we were able to deduce a corrected amino acids sequence for the human receptor. So both rat and human SLC -1 receptors are 353aa long, with three consensus N-glycosylation site s. They share 96% identity at the amino acid level and are encoded by a gene containing one intron in the coding sequence. (C) 1998 Elsevier Science B.V.