Analysis of the C-Terminal Tail of the Rat Thyrotropin-Releasing Hormone Receptor-1 in Interactions and Cointernalization with β-Arrestin 1-Green Fluorescent Protein

2001 ◽  
Vol 59 (2) ◽  
pp. 375-385 ◽  
Author(s):  
D. Alex Groarke ◽  
Tomas Drmota ◽  
Daljit S. Bahia ◽  
Nicholas A. Evans ◽  
Shelagh Wilson ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Hormone Receptor ◽  
Fluorescent Protein ◽  
Thyrotropin Releasing Hormone ◽  
Releasing Hormone ◽  
Green Fluorescent

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

1999 ◽  
Vol 340 (2) ◽  
pp. 529-538 ◽  
Author(s):  
Tomas DRMOTA ◽  
Jiri NOVOTNY ◽  
Gwyn W. GOULD ◽  
Petr SVOBODA ◽  
Graeme MILLIGAN
Keyword(s):  
Plasma Membrane ◽  
G Proteins ◽  
Hormone Receptor ◽  
Fluorescent Protein ◽  
Thyrotropin Releasing Hormone ◽  
Hek293 Cells ◽  
Low Density ◽  
Rapid Separation ◽  
Releasing Hormone ◽  
Green Fluorescent

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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