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  • Title: Real time visualization of agonist-mediated redistribution and internalization of a green fluorescent protein-tagged form of the thyrotropin-releasing hormone receptor.
    Author: Drmota T, Gould GW, Milligan G.
    Journal: J Biol Chem; 1998 Sep 11; 273(37):24000-8. PubMed ID: 9727016.
    Abstract:
    The long isoform of the rat thyrotropin-releasing hormone receptor (TRHR) was modified by the addition of a vesicular stomatitis virus (VSV) epitope tag and green fluorescent protein (GFP). VSV-TRHR-GFP bound TRH with affinity similar to that of the unmodified receptor and stimulated [3H]inositol phosphate production. A clone stably expressing VSV-TRHR-GFP at some 120,000 copies/cell was selected to visualize this receptor during cellular exposure to TRH. Internalization was detected within 3-5 min after treatment with 1 x 10(-7) M TRH, with dramatic reductions in plasma membrane localization achieved within 10-15 min. The TRHR antagonist/inverse agonist chlordiazepoxide competitively inhibited internalization. Hyperosmotic sucrose inhibited internalization of VSV-TRHR-GFP, measured both by intact cell [3H]TRH binding studies and by confocal microscopy. Now TRH caused a redistribution of VSV-TRHR-GFP to highly punctate but plasma membrane-delineated foci. Pretreatment with the microtubule-disrupting agent nocodazole allowed internalization of the VSV-TRHR-GFP construct but only into vesicles that remained in close apposition to the plasma membrane. Covisualization of VSV-TRHR-GFP and Texas Red transferrin initially indicated entirely separate localizations. After exposure to TRH substantial amounts of VSV-TRHR-GFP were present in vesicles overlapping those containing Texas Red transferrin. Such results demonstrate the G protein-coupling capacity and provide real time visualization of the processes of internalization of a TRH-receptor-GFP construct in response to agonist.
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