Agonist-induced internalization of the G protein G11α and thyrotropin-releasing hormone receptors proceed on different time scales

Using a combination of confocal immunofluorescence microscopy and subcellular fractionation, we demonstrate for the first time active internalization, trafficking, and down-regulation of a G protein alpha subunit subsequent to agonist occupation of a receptor, This proceeds on a much slower time scale than internalization of the corresponding receptor. In intact E2M11 HEK293 cells that express high levels of murine G(11)alpha and the rat thyrotropin-releasing hormone (TRH) receptor, the immunofluorescence signal of G(11)alpha was restricted almost exclusively to the plasma membrane. Exposure to TRH (10 mu M) resulted first in partial relocation of G(11)alpha to discrete, segregated patches within the plasma membrane (10-60 min). Further exposure to TRH caused internalization of G(11)alpha to discrete, punctate, intracellular bodies (2-4 h) and subsequently to a virtually complete loss of G(11)alpha from plasma membranes and the cells (8-16 h), Short-term treatment with TRH followed by wash-out of the ligand allowed G(11)alpha immunofluorescence to be restored to the plasma membrane within 12 h. In subcellular membrane fractions, G(11)alpha was centered on plasma membranes, and this was not altered by up to 1-2 h of incubation with TRH. Further exposure to TRH (2-4 h) resulted in transfer of a significant portion of G(11)alpha to light-vesicular and cytosol fractions. At longer time intervals (4-16 h), an overall decrease in G(11)alpha content was observed.