RT Journal Article SR Electronic T1 Edg2 Receptor Functionality: Giα1 Coexpression and Fusion Protein Studies JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 407 OP 412 DO 10.1124/mol.58.2.407 VO 58 IS 2 A1 McAllister, George A1 Stanton, Josephine A. A1 Salim, Kamran A1 Handford, Emma J. A1 Beer, Margaret S. YR 2000 UL http://molpharm.aspetjournals.org/content/58/2/407.abstract AB Recombinant receptor cell lines are widely used in G-protein-coupled receptor selectivity studies. To unequivocally interpret the results of such studies, it is essential that the host cell line does not endogenously express the receptor of interest and in addition is unresponsive to the receptor's natural ligand. Here we describe an approach to overcome such difficulties associated with orphan receptors or, as in the present case, receptors whose endogenous ligand ubiquitously affects mammalian cells. The functional heterologous assay system described is for the hEdg2 receptor, which uses lysophosphatidic acid as its endogenous ligand. Once activated, this receptor mediates its effects via multiple secondary messenger pathways, including a Gi-coupled pathway. We have transiently expressed a pertussis toxin-insensitive hEdg2 receptor-ratGiα1 fusion protein into human embryonic kidney cells and have monitored the ability of compounds to stimulate [35S]GTPγS binding in membranes prepared from these cells after pretreatment with toxin. Because the assay conditions used favor Gi-mediated responses and because endogenous Giα subunits are rendered inactive, the response measured is, by definition, fusion protein-mediated. Consequently, we have developed an assay that monitors definitively Edg2 receptor-mediated responses in a mammalian cell line. A limited structure activity relationship study suggests that the lysophospholipid carbon chain has a role in receptor activation and in addition indicates that certain modifications to the phosphate group are tolerated.