Characterization of SEQ-9 an Orphan G protein-Coupled Receptor
Current research in pharmaceuticals is primarily focused on cell signaling pathways. G-proteins (GTP binding proteins) are a particularly favorite drug target because of their ubiquitous expression and the vast number of agonists that bind to them. Due to these factors they are implicated in a whole array of physiological diseases and disorders. SEQ-9, a novel G-protein coupled receptor (GPCR), was identified from a proprietary human genomic database and is characterized by common motifs that are shared among GPCRs. The goal of the project was to clone a novel full-length GPCR ID'd from a proprietary database. The function of the resulting construct was characterized by assays commonly used to study GPCR function in cell signaling pathways. A BLAST search indicated that SEQ-9 was the human homolog of the rat EDG-8, sharing a 87.7% identity at the amino acid level. Edg-8 has been shown to have SIP (Spingosine-I-Phosphate) as its ligand therefore, this information raised the possibility of SIP as a ligand for SEQ-9. An adenylyl cyclase assay showed that SIP induced an inhibition of forskolin-stimulated intracellular cyclic AMP levels suggesting coupling through a G.-coupled signaling pathway. A FLIPR () assay was also performed to monitor levels of calcium within the cells. A four to five-fold increase in calcium mobilization was seen when the cells were stimulated with SIP, suggesting coupling through a Gq signaling pathway. These experiments strongly suggest that SIP is a ligand for SEQ-9, and that this GPCR couples through both the Gi and Gq signaling pathways.
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