Agonist-induced internalization of the G protein G11alpha 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 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.
Drmota
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Institute of Physiology, Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic.
Novotny
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J
Kim
G D
GD
Eidne
K A
KA
Milligan
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G
Svoboda
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P
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000819:agonists* / Q000378:metabolism; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism; D013997:Time Factors; D014746:Vimentin / Q000378:metabolism
D018025:Receptors, Thyrotropin-Releasing Hormone; D014746:Vimentin; D013973:Thyrotropin-Releasing Hormone; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D019204:GTP-Binding Proteins