Agonist-induced Internalization of the G Protein G11α and Thyrotropin-releasing Hormone Receptors Proceed on Different Time Scales

Abstract

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 G11alpha and the rat thyrotropin-releasing hormone (TRH) receptor, the immunofluorescence signal of G11alpha was restricted almost exclusively to the plasma membrane. Exposure to TRH (10 microM) resulted first in partial relocation of G11alpha to discrete, segregated patches within the plasma membrane (10-60 min). Further exposure to TRH caused internalization of G11alpha to discrete, punctate, intracellular bodies (2-4 h) and subsequently to a virtually complete loss of G11alpha from plasma membranes and the cells (8-16 h). Short-term treatment with TRH followed by wash-out of the ligand allowed G11alpha immunofluorescence to be restored to the plasma membrane within 12 h. In subcellular membrane fractions, G11alpha 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 G11alpha to light-vesicular and cytosol fractions. At longer time intervals (4-16 h), an overall decrease in G11alpha content was observed.

Real Time Visualization of Agonist-mediated Redistribution and Internalization of a Green Fluorescent Protein-tagged Form of the Thyrotropin-releasing Hormone Receptor

“…Internalization was detectable with 3-5 min, virtually maximal within 10 -15 min, and was maintained up to at least 30 min. These results are consistent with related studies that have examined either the internalization of radiolabeled TRH (13,47) or have used epitope-tagged receptors that could be detected immunologically after cellular fixation (10,11,15). The TRHR has an extremely limited useful pharmacology, with the only widely available antagonist being chlordiazepoxide with K i in the region of 100 M. Despite this limitation we were able to demonstrate both concentration-dependent internalization of VSV-TRHR-GFP by TRH and that this was not mimicked by chlordiazepoxide.…”

“…Cellular shock with elevated osmolality is a well established means of interfering with this pathway (37,38) and has been used previously on studies of the TRH receptor (11,13). We demonstrated in initial studies that TRH bound to VSV-TRHR-GFP in intact cell binding assays with good affinity, which was at least as high as that to the isolated TRHR.…”

“…We demonstrated in initial studies that TRH bound to VSV-TRHR-GFP in intact cell binding assays with good affinity, which was at least as high as that to the isolated TRHR. The use of acid wash protocols to resolve plasma membrane-located and internalized TRHR is well established (11,13,36). We therefore used a combination of such a biochemical assay in concert with real time confocal visualization to explore this process in detail.…”

“…During studies of the TRHR we have noted that both the rat TRHR and the G proteins G q ␣ and G 11 ␣ are internalized in response to agonist (13,14), although internalization of the G proteins proceeds substantially more slowly than the receptor (13,14). The G proteins are subsequently down-regulated (7).…”

“…34 binding studies on intact cells, they were harvested using 0.5 mM EDTA in PBS, washed, and used directly for experiments. Membranes were prepared as described previously (6,13). Both types of experiment were performed with 10 nM [ 3 H]TRH and the addition of different concentrations of TRH at 4°C for 60 min.…”