The rat thyrotropin-releasing hormone receptor-1 (TRHR-1) was modified by the addition of green fluorescent protein (GFP) and expressed stably in HEK293 cells. Extensive overlap of plasma membrane distribution of autofluorescent TRHR-1-GFP with that of the phosphoinositidase C-linked G-proteins Gqα/G11α, identified by indirect immunofluorescence, was monitored concurrently. Addition of thyrotropin-releasing hormone resulted in rapid separation of TRHR-1-GFP and Gqα/G11α signals as the receptor was internalized. This situation persisted for more than an hour. At longer time periods a fraction of the cellular Gqα/G11α was also internalized, although much of the Gqα/G11α immunoreactivity remained associated with the plasma membrane. Parallel experiments, in which the cellular distribution of TRHR-1-GFP and Gqα/G11α immunoreactivity were monitored in sucrose-gradient fractions following cell disruption, also demonstrated a rapid, agonist-induced movement of TRHR-1-GFP away from the plasma membrane to low-density vesicular fractions. At later time points, a fraction of the cellular Gqα/G11α immunoreactivity was also redistributed to overlapping, but non-identical, low-density-vesicle-containing fractions. Pretreatment of the cells with cytochalasin D or nocodazole prevented agonist-induced redistribution of G-protein but not TRHR-1-GFP, further indicating resolution of the mechanics of these two processes. The combination of a GFP-modified receptor and immunostaining of the G-proteins activated by that receptor allows, for the first time, concurrent analysis of the varying dynamics and bases of internalization and redistribution of two elements of the same signal-transduction cascade.
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Research Article|
May 25 1999
Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone receptor-1 and Gqα /G11α induced by agonist stimulation
Tomas DRMOTA;
Tomas DRMOTA
*Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
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Jiri NOVOTNY;
Jiri NOVOTNY
*Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
†Institute of Physiology, Czech Academy of Sciences, Videnska 1083, Prague, Czech Republic
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Gwyn W. GOULD;
Gwyn W. GOULD
*Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
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Petr SVOBODA;
Petr SVOBODA
*Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
†Institute of Physiology, Czech Academy of Sciences, Videnska 1083, Prague, Czech Republic
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Graeme MILLIGAN
Graeme MILLIGAN
1
*Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
1To whom correspondence should be addressed (e-mail g.milligan@bio.gla.ac.uk).
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Publisher: Portland Press Ltd
Received:
January 28 1999
Revision Received:
March 08 1999
Accepted:
March 24 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 340 (2): 529–538.
Article history
Received:
January 28 1999
Revision Received:
March 08 1999
Accepted:
March 24 1999
Citation
Tomas DRMOTA, Jiri NOVOTNY, Gwyn W. GOULD, Petr SVOBODA, Graeme MILLIGAN; Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone receptor-1 and Gqα /G11α induced by agonist stimulation. Biochem J 1 June 1999; 340 (2): 529–538. doi: https://doi.org/10.1042/bj3400529
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