Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells.
Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors. At least some types of G-protein α subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists. However, the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol. Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of G(q/11)α protein and to determine their localization in isolated fractions and stability in naïve and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G(11)α protein. We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the G(q/11) protein, which were found to be membrane-bound. Both of these complexes dissociated after prolonged treatment with TRH. Still other G(q/11)α protein complexes of lower molecular weight were determined in the cytosol. These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h) hormone treatment. These results support the notion that a portion of G(q/11)α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor. At the same time, these findings indicate that the redistribution of G(q/11)α is brought about by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the G(q/11)α protein.
Department of Physiology, Faculty of Science, Charles University, Vinicna 7, 128 44 Prague 2, Czech Republic.
Zdenka
Z
Drastichova
Jiri
J
Novotny
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000302:isolation & purification* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000378:metabolism; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism
D018025:Receptors, Thyrotropin-Releasing Hormone; D013973:Thyrotropin-Releasing Hormone; D043802:GTP-Binding Protein alpha Subunits, Gq-G11