Agonist-induced tyrosine phosphorylation of G q/G 11α requires the intact structure of membrane domains

Abstract

Stimulation of receptors coupled to G q/G 11 protein may induce phosphorylation on a tyrosine residue of the α subunit of this G protein, which is an essential event for G q/G 11 activation. Here we observed that in HEK293 cells stably expressing high levels of thyrotropin-releasing hormone (TRH) receptors and G 11α protein the maximal tyrosine phosphorylation of G q/G 11α was reached within 10 min of TRH stimulation and then it faded away at longer time periods of agonist exposure. The G q/G 11α protein levels did not change during this treatment. Incubation of intact cells with β-cyclodextrin (βCD) for 40 min prior to hormone exposure significantly decreased the rapid transient tyrosine phosphorylation. Subsequent replenishment of cholesterol levels reversed the former negative effect of βCD. Isolation of caveolin-enriched, detergent-resistant membrane domains indicated destruction of these structures in βCD-treated cells. These data indicate that the preserved integrity of plasma membrane domains/caveolae is required for complete agonist-induced phosphorylation of G q/G 11α.