SELECTIVE ACTIVATION OF A CHIMERIC G(I1) (S)G PROTEIN ALPHA-SUBUNIT BY THE HUMAN IP PROSTANOID RECEPTOR - ANALYSIS USING AGONIST STIMULATION OF HIGH-AFFINITY GTPASE ACTIVITY AND [S-35] GUANOSINE-5'-O-(3-THIO)TRIPHOSPHATE BINDING/

A FLAG-tagged form of the human IP prostanoid receptor was expressed s tably in HEK 293 cells. This bound [H-3]iloprost with high affinity an d stimulated cAMP production when exposed to agonist. Iloprost produce d weak stimulation of GTPase activity and [S-35]guanosine-5'-O-(3-thio )triphosphate binding in membranes of these cells. Pretreatment of cel ls with pertussis toxin did not modify iloprost-mediated stimulation, but this was blocked by cholera toxin. The effects of iloprost were no t increased by coexpression of either G(s alpha) or G(i1 alpha). In co ntrast, coexpression of a chimeric G protein alpha subunit in which th e carboxyl-terminal six amino acids of G(i1 alpha) were altered to tho se of G(s alpha) resulted in robust stimulation by iloprost. Because t he chimeric G protein alpha subunit (G(i1)/G(s6 alpha)) is not a subst rate for either pertussis or cholera toxin, pretreatment of cells coex pressing the IP prostanoid receptor and G(i1)/G(s6 alpha) with a mixtu re of these toxins resulted in resolution of the signal derived from a ctivation of the chimeric G protein. Agonist-stimulated [S-35]guanosin e-5'-O-(3-thio)triphosphate binding and GTPase activity assays are the most commonly used strategies to examine interactions between G prote in-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 r ates typical of the G(i) family G proteins can overcome this and offer a novel means to examine agonist function at such receptors.