Real time visualization of agonist-mediated redistribution and internalization of a green fluorescent protein-tagged form of the thyrotropin-releasing hormone receptor.
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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Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ Scotland, United Kingdom.
Gould
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Milligan
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002707:Chlordiazepoxide / Q000494:pharmacology*; D017931:DNA Primers; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013395:Sucrose; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D006982:Hypertonic Solutions; D007295:Inositol Phosphates; D008164:Luminescent Proteins; D018025:Receptors, Thyrotropin-Releasing Hormone; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013395:Sucrose; D013973:Thyrotropin-Releasing Hormone; D002707:Chlordiazepoxide; D015739:Nocodazole