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.
Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone receptor-1 and Gqα /G11α induced by agonist stimulation
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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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