Thyrotropin-releasing hormone-induced depletion of G qα/G 11α proteins from detergent-insensitive membrane domains

Abstract

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 11α 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 α 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 qα/G 11α-rich domains. Prolonged stimulation of E2M11 cells with TRH resulted in dramatic depletion of G qα/G 11α from all DIMs, which was paralleled by a concomitant G qα/G 11α 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.