Abstract

A FLAG-tagged form of the human IP prostanoid receptor was expressed stably in HEK 293 cells. This bound [³H]iloprost with high affinity and stimulated cAMP production when exposed to agonist. Iloprost produced weak stimulation of GTPase activity and [³⁵S]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_sα or G_i1α. In contrast, coexpression of a chimeric G protein α subunit in which the carboxyl-terminal six amino acids of G_i1α were altered to those of G_sα resulted in robust stimulation by iloprost. Because the chimeric G protein α subunit (G_i1/G_s6α) is not a substrate for either pertussis or cholera toxin, pretreatment of cells coexpressing the IP prostanoid receptor and G_i1/G_s6α with a mixture of these toxins resulted in resolution of the signal derived from activation of the chimeric G protein. Agonist-stimulated [³⁵S]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_s6α 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.