Selective activation of a chimeric Gi1/sG protein α subunit by the human IP prostanoid receptor:: Analysis using agonist stimulation of high affinity GTPase activity and [35S]Guanosine-5′-O-(3-thio)triphosphate binding

A FLAG-tagged form of the human IP prostanoid receptor was expressed stably in HEK 293 cells. This bound [H-3]iloprost with high affinity and stimulated cAMP production when exposed to agonist. Iloprost produced weak stimulation of GTPase activity and [S-35]guanosine-5'-O-(3-thio)triphosphate binding in membranes of these cells. Pretreatment of cells with pertussis toxin did not modify iloprost-mediated stimulation, but this was blocked by cholera toxin. The effects of iloprost were not increased by coexpression of either G(s alpha) or G(i1 alpha). In contrast, coexpression of a chimeric G protein alpha subunit in which the carboxyl-terminal six amino acids of G(i1 alpha) were altered to those of G(s alpha) resulted in robust stimulation by iloprost. Because the chimeric G protein alpha subunit (G(i1)/G(s6 alpha)) is not a substrate for either pertussis or cholera toxin, pretreatment of cells coexpressing the IP prostanoid receptor and G(i1)/G(s6 alpha) with a mixture of these toxins resulted in resolution of the signal derived from activation of the chimeric G protein. Agonist-stimulated [S-35]guanosine-5'-O-(3-thio)triphosphate binding and GTPase activity assays are the most commonly used strategies to examine interactions between G protein-coupled receptors and G proteins. These usually are not appropriate for receptors such as the IP prostanoid receptor that interact with G proteins with low rates of guanine nucleotide exchange and hydrolysis. Chimeric G proteins such as G(i1)/G(s)6 alpha that allow appropriate receptor contacts to be converted to the higher nucleotide turnover rates typical of the G(i) family G proteins can overcome this and offer a novel means to examine agonist function at such receptors.