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 desensitisatio n of the G protein-coupled receptor-mediated hormone response was studied i n clone E2M11 of HEK293 cells which stably express high levels of both thyr otropin-releasing hormone (TRH) receptors and G(11 alpha) G protein. DIMs w ere prepared by flotation in equilibrium sucrose density gradients and char acterised by a panel of membrane markers representing peripheral, glycosylp hosphatidylinositol-bound as well as integral membrane proteins (caveolin, CD29, CD55, CD59, CD147, the alpha subunit of Na,K-ATPase) and enzyme activ ities (alkaline phosphatase, adenylyl cyclase), Caveolin-containing DIMs re presented 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 dr amatic depletion of G(q)alpha/G(11)alpha from all DIMs, which mas parallele d by a concomitant G(q)alpha/G(11)alpha increase in the high-density gradie nt fractions containing the bulk-phase membrane constituents soluble in 1% Triton X-100, Distribution of membrane markers mas unchanged under these co nditions. Membrane domains thus represent a substantial structural determin ant of the G protein pool relevant to desensitisation of hormone action, (C ) 1999 Federation of European Biochemical Societies.