THYROTROPIN-RELEASING HORMONE-INDUCED SUBCELLULAR REDISTRIBUTION AND DOWN-REGULATION OF G(11-ALPHA) - ANALYSIS OF AGONIST REGULATION OF COEXPRESSED G(11-ALPHA) SPECIES VARIANTS

Human embryonic kidney 293 cells that had been transfected to express the long isoform of the rat thyrotropin-releasing hormone (TRH) recept or (clone E2) were further transfected with a cDNA encoding the murine version of G(11 alpha). A clone was isolated (clone E2M11) that stabl y expressed murine as well as the endogenous human G(11 alpha). Subcel lular fractionation demonstrated identical cellular distribution of th e two species variants of this G protein. Sustained exposure of clone E2M11 cells to TRH resulted in substantial cellular redistribution and reduction in total cellular levels of G(11 alpha) immunoreactivity. F ractions of both the exogenously introduced murine and endogenously ex pressed human isoforms of G(11 alpha) were transferred from plasma mem branes to low density membranes (detected as a shift from middle to lo w density regions on sucrose density gradients) and cytosol fractions. The plasma membrane redistribution to low density membrane was accomp anied by a parallel redistribution of G protein beta subunits; however , there was no increase in beta subunits in the cytosol. The total cel lular amount of G(11 alpha) subunits was decreased to 21% and 59% for human and murine isoforms, respectively, and beta subunits were decrea sed to 68% after sustained treatment with TRH compared with controls ( 100%). Such data are consistent with the notion that the agonist-occup ied long isoform of the rat TRH receptor may be able to partially diff erentiate between the endogenous (human) and exogenous (murine) G(11 a lpha). This was not a reflection that the murine G protein was express ed but incorrectly folded as both species variants of G(11 alpha) were solubilized equally from E2M11 membranes by sodium cholate, Using thi s system, we demonstrate both agonist-induced subcellular redistributi on and down-regulation of G(11 alpha) and beta subunit proteins in res ponse to activation of a phopholipase C coupled receptor.