Thyrotropin‐releasing hormone‐induced depletion of Gqα/G11α 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 G11α 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 Gqα/G11α‐rich domains. Prolonged stimulation of E2M11 cells with TRH resulted in dramatic depletion of Gqα/G11α from all DIMs, which was paralleled by a concomitant Gqα/G11α 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.

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