Vol. 54, Issue 2, 249-257, August 1998

Selective Activation of a Chimeric G_i1/G_s G Protein  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

Chee Wai Fong, Daljit S. Bahia, Stephen Rees, and Graeme Milligan

Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, Scotland (C.W.F., D.S.B., G.M.) and Receptor Systems Unit, Glaxo Wellcome Research and Development, Stevenage, Hertfordshire, SG1 2NY, England (S.R.)

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_s or G_i1. In contrast, coexpression of a chimeric G protein  subunit in which the carboxyl-terminal six amino acids of G_i1 were altered to those of G_s resulted in robust stimulation by iloprost. Because the chimeric G protein  subunit (G_i1/G_s6) is not a substrate for either pertussis or cholera toxin, pretreatment of cells coexpressing the IP prostanoid receptor and G_i1/G_s6 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_s6 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.