Thyrotropin-releasing hormone-induced depletion of G(q)alpha/G(11)alpha proteins from detergent-insensitive membrane domains.
The role of detergent-insensitive membrane domains (DIMs) in desensitisation of the G protein-coupled receptor-mediated hormone response was studied in clone E2M11 of HEK293 cells which stably express high levels of both thyrotropin-releasing hormone (TRH) receptors and G(11)alpha G protein. DIMs were prepared by flotation in equilibrium sucrose density gradients and characterised by a panel of membrane markers representing peripheral, glycosylphosphatidylinositol-bound as well as integral membrane proteins (caveolin, CD29, CD55, CD59, CD147, the alpha subunit of Na, K-ATPase) and enzyme activities (alkaline phosphatase, adenylyl cyclase). Caveolin-containing DIMs represented only a small fraction of the overall pool of G(q)alpha/G(11)alpha-rich domains. Prolonged stimulation of E2M11 cells with TRH resulted in dramatic depletion of G(q)alpha/G(11)alpha from all DIMs, which was paralleled by a concomitant G(q)alpha/G(11)alpha increase in the high-density gradient fractions containing the bulk-phase membrane constituents soluble in 1% Triton X-100. Distribution of membrane markers was unchanged under these conditions. Membrane domains thus represent a substantial structural determinant of the G protein pool relevant to desensitisation of hormone action.
Pesanová
Z
Z
Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20, Prague, Czech Republic.
Novotný
J
J
Cerný
J
J
Milligan
G
G
Svoboda
P
P
D000262:Adenylyl Cyclases / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D018958:CD55 Antigens / Q000378:metabolism; D018957:CD59 Antigens / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003902:Detergents / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D019012:Integrin beta1 / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*
C495540:CAV1 protein, human; D018958:CD55 Antigens; D018957:CD59 Antigens; D051242:Caveolin 1; D022461:Caveolins; D003902:Detergents; D019012:Integrin beta1; D008565:Membrane Proteins; D013973:Thyrotropin-Releasing Hormone; D017830:Octoxynol; D000469:Alkaline Phosphatase; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000262:Adenylyl Cyclases; D000254:Sodium-Potassium-Exchanging ATPase