Real Time Visualization of Agonist-mediated Redistribution and Internalization of a Green Fluorescent Protein-tagged Form of the Thyrotropin-releasing Hormone Receptor

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