Long-term agonist stimulation of IP prostanoid receptor depletes the cognate Gsα protein in membrane domains but does not change the receptor level

AbstractIloprost (IP) stimulation (1 μM, 2 h) of Flag-epitope-tagged human IP prostanoid receptor (FhIPR) expressed in HEK293 cells resulted in specific decrease of endogenous Gsα protein in detergent-insensitive, caveolin-enriched, membrane domains (DIMs). Receptor protein FhIPR, caveolin, Giα and GPI-linked, domain markers CD55 and CD59 were unchanged. The same result was obtained in HEK293 cells expressing FhIPR-Gsα fusion protein. The endogenous Gsα decreased, but the level of Flag-hIPR-Gsα protein did not change. The specific depletion of domain-bound pool of Gsα as consequence of iloprost stimulation was also demonstrated in membrane domains prepared according to alkaline treatment plus sonication protocol (detergent-free procedure of Song et al. [J. Biol. Chem. 271 (1996) 9690]). Our data further indicated that in control, quiescent cells only a very small amount of IP prostanoid receptor was present in DIMs together with large amount of its cognate Gsα protein. Expressed in quantitative terms, DIMs contained 30–40% of the total cellular amount of G proteins whereas the content of IP prostanoid receptors was 1–3%. The dominant portion (>95%) of FhIPR as well as FhIPR-Gsα was localised in high-density area of the gradient containing detergent-solubilised proteins. FhIPR and FhIPR-Gsα distribution was similar to that of transmembrane plasma membrane (PM) markers (CD147, MHCI, CD29, Tapa1, the α subunit of Na,K-ATPase, transmembrane form of CD58 and CD44). All these proteins are known to be fully solubilised by detergent and thus unable to float in density gradient.Our data indicate that (i) long-term agonist stimulation of IP prostanoid receptor is associated with preferential decrease of its cognate G protein Gsα from membrane domains; receptor level is unchanged. (ii) Very small fraction (1–3%) of total cellular amount of receptors is recovered in DIMs together with roughly 40% of G proteins. These data suggest a “supra-stoichiometric” arrangement of G proteins and corresponding receptors in DIMs