Oligomerization of the human thyrotropin receptor: fluorescent protein-tagged hTSHR reveals post-translational complexes.
To examine thyrotropin (TSH) receptor homophilic interactions we fused the human TSH receptor (hTSHR) carboxyl terminus to green fluorescent protein (GFP) and the corresponding chimeric cDNA was expressed in Chinese hamster ovary cells. Fluorescent TSH receptors on the plasma membrane were functional as assessed by TSH-induced cAMP synthesis. The binding of TSH, as well as TSHR autoantibodies, induced time- and dose-dependent receptor capping. Fluorescence resonance energy transfer between receptors differentially tagged with GFP variants (RFP and YFP) provided evidence for the close proximity of individual receptor molecules. This was consistent with previous studies demonstrating the presence of TSHR dimers and oligomers in thyroid tissue. Co-immunoprecipitation of GFP-tagged and Myc-tagged receptor complexes was performed using doubly transfected cells with Myc antibody. Western blotting of the immunoprecipitated complex revealed the absence of noncleaved TSH holoreceptors. This further suggested that cleavage of the holoreceptor into its two-subunit structure, comprising disulfide-linked TSHR-alpha and TSHR-beta subunits, was required for the formation of TSHR dimers and higher order complexes.
Latif
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Division of Endocrinology, Diabetes and Bone Diseases, Mount Sinai School of Medicine, New York, New York 10029-6574, USA. rauf.latif@mssm.edu
Graves
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Davies
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D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011233:Precipitin Tests; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D004220:Disulfides; D000939:Epitopes; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011989:Receptors, Thyrotropin; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP