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J. Biol. Chem., Vol. 276, Issue 48, 45217-45224, November 30, 2001
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From the Division of Endocrinology, Diabetes and Bone Diseases,
Mount Sinai School of Medicine, New York, New York 10029-6574
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-
Oligomerization of the Human Thyrotropin Receptor
FLUORESCENT PROTEIN-TAGGED hTSHR REVEALS POST-TRANSLATIONAL
COMPLEXES*
,
and TSHR-
subunits, was
required for the formation of TSHR dimers and higher order complexes.
*
This work was supported in part by National Institutes of
Health Grants DK52464, DK35764, and DK45011 (to T. F. D.) and
the David Owen Segal Endowment (to R. L.). The confocal laser
scanning microscopy was performed at the MSSM-Microscopy Center, with
funding from National Institutes of Health shared instrumentation Grant 1S10RR9145-01 and National Science Foundation major research
instrumentation Grant DBI-9724504.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Medicine, Box
1055, Mount Sinai School of Medicine, One Gustave L. Levy Place,
New York, NY 10029-6574. Tel.: 212-241-8148; Fax: 212-241-4218; E-mail: rauf.latif@mssm.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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