Iloprost (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-G sα 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.
Moravcová, Z., Rudajev, V., Stöhr, J., Novotný, J., Cerný, J., Parenti, M., et al. (2004). Long-term agonist stimulation of IP prostanoid receptor depletes the cognate Gsα protein in membrane domains but does not change the receptor level. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 1691(1), 51-65 [10.1016/j.bbamcr.2003.12.004].
Long-term agonist stimulation of IP prostanoid receptor depletes the cognate Gsα protein in membrane domains but does not change the receptor level
PARENTI, MARCO DOMENICO;
2004
Abstract
Iloprost (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-G sα 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.
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
