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J Biol Chem, Vol. 273, Issue 34, 21699-21707, August 21, 1998

Agonist-induced Internalization of the G Protein G₁₁ and Thyrotropin-releasing Hormone Receptors Proceed on Different Time Scales

Tomas Drmota^§, Jiri Novotny, Gun-Do Kim^§, Karin A. Eidne^¶, Graeme Milligan^§, and Petr Svoboda^§

From the  Institute of Physiology, Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic, the ^§ Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, and the ^¶ Medical Research Council Reproductive Biology Unit, Edinburgh, EH3 9EW Scotland, United Kingdom

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  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 G₁₁ and the rat thyrotropin-releasing hormone (TRH) receptor, the immunofluorescence signal of G₁₁ was restricted almost exclusively to the plasma membrane. Exposure to TRH (10 µM) resulted first in partial relocation of G₁₁ to discrete, segregated patches within the plasma membrane (10-60 min). Further exposure to TRH caused internalization of G₁₁ to discrete, punctate, intracellular bodies (2-4 h) and subsequently to a virtually complete loss of G₁₁ from plasma membranes and the cells (8-16 h). Short-term treatment with TRH followed by wash-out of the ligand allowed G₁₁ immunofluorescence to be restored to the plasma membrane within 12 h. In subcellular membrane fractions, G₁₁ 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 G₁₁ to light-vesicular and cytosol fractions. At longer time intervals (4-16 h), an overall decrease in G₁₁ content was observed.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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