Long-term agonist stimulation of IP prostanoid receptor depletes the cognate G(s)alpha protein in membrane domains but does not change the receptor level
Language English Country Netherlands Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
15053924
DOI
10.1016/j.bbamcr.2003.12.004
PII: S0167488903002088
Knihovny.cz E-resources
- MeSH
- Cell Line MeSH
- Time Factors MeSH
- Down-Regulation drug effects MeSH
- Iloprost pharmacology MeSH
- Caveolins MeSH
- Humans MeSH
- Membrane Microdomains chemistry MeSH
- Membrane Proteins analysis MeSH
- GTP-Binding Protein alpha Subunits, Gs analysis drug effects MeSH
- Receptors, Epoprostenol MeSH
- Receptors, Prostaglandin agonists analysis MeSH
- Recombinant Fusion Proteins drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Iloprost MeSH
- Caveolins MeSH
- Membrane Proteins MeSH
- GTP-Binding Protein alpha Subunits, Gs MeSH
- PTGIR protein, human MeSH Browser
- Receptors, Epoprostenol MeSH
- Receptors, Prostaglandin MeSH
- Recombinant Fusion Proteins MeSH
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.
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