Agonist-induced tyrosine phosphorylation of Gq/G11 alpha requires the intact structure of membrane domains.
Stimulation of receptors coupled to G(q)/G(11) protein may induce phosphorylation on a tyrosine residue of the alpha 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(11)alpha protein the maximal tyrosine phosphorylation of G(q)/G(11)alpha was reached within 10 min of TRH stimulation and then it faded away at longer time periods of agonist exposure. The G(q)/G(11)alpha protein levels did not change during this treatment. Incubation of intact cells with beta-cyclodextrin (beta CD) 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 beta CD. Isolation of caveolin-enriched, detergent-resistant membrane domains indicated destruction of these structures in beta CD-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(11)alpha.
Matousek
Petr
P
Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic.
Durchánková
Dana
D
Svandová
Ivana
I
Novotny
Jiri
J
Svoboda
Petr
P
D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D008560:Membrane Fluidity / Q000187:drug effects / Q000502:physiology; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation / Q000187:drug effects; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000378:metabolism; D013329:Structure-Activity Relationship; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism*
D018025:Receptors, Thyrotropin-Releasing Hormone; D014443:Tyrosine; D013973:Thyrotropin-Releasing Hormone; D043802:GTP-Binding Protein alpha Subunits, Gq-G11