Iloprost (IP) stimulation (1 microM, 2 h) of Flag-epitope-tagged human IP prostanoid receptor (FhIPR) expressed in HEK293 cells resulted in specific decrease of endogenous G(s)alpha protein in detergent-insensitive, caveolin-enriched, membrane domains (DIMs). Receptor protein FhIPR, caveolin, G(i)alpha and GPI-linked, domain markers CD55 and CD59 were unchanged. The same result was obtained in HEK293 cells expressing FhIPR-G(s)alpha fusion protein. The endogenous G(s)alpha decreased, but the level of Flag-hIPR-G(s)alpha protein did not change. The specific depletion of domain-bound pool of G(s)alpha 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.). 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 G(s)alpha 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-G(s)alpha was localised in high-density area of the gradient containing detergent-solubilised proteins. FhIPR and FhIPR-G(s)alpha distribution was similar to that of transmembrane plasma membrane (PM) markers (CD147, MHCI, CD29, Tapa1, the alpha 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 G(s)alpha 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.

• MeSH
• buněčné linie MeSH
• časové faktory MeSH
• down regulace účinky léků   MeSH
• iloprost farmakologie   MeSH
• kaveoliny MeSH
• lidé MeSH
• membránové mikrodomény chemie   MeSH
• membránové proteiny analýza   MeSH
• proteiny vázající GTP - alfa-podjednotky Gs analýza   účinky léků   MeSH
• receptory epoprostenolu MeSH
• receptory prostaglandinů agonisté   analýza   MeSH
• rekombinantní fúzní proteiny účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• iloprost MeSH
• kaveoliny MeSH
• membránové proteiny MeSH
• proteiny vázající GTP - alfa-podjednotky Gs MeSH
• PTGIR protein, human MeSH Prohlížeč
• receptory epoprostenolu MeSH
• receptory prostaglandinů MeSH
• rekombinantní fúzní proteiny MeSH