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J. Biol. Chem., Vol. 277, Issue 47, 45059-45067, November 22, 2002
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From the Division of Endocrinology, Diabetes and Bone
Diseases, Mount Sinai School of Medicine, New York, New York
Recently, several studies have reported
oligomerization of G protein-coupled receptors, although the functional
implications of this phenomenon are still unclear. Using fluorescence
resonance energy transfer (FRET) and coimmunoprecipitation
(COIP), we previously reported that the human thyrotropin (TSH)
receptor tagged with green fluorescent protein
(TSHRGFP) and expressed in a heterologous system was
present as oligomeric complexes on the cell surface. Here, we have
extended this biophysical and biochemical approach to study the
regulation of such oligomeric complexes. Co-expression of
TSHRGFP and TSHRMyc constructs in Chinese
hamster ovary cells resulted in FRET-positive cells. The specificity of
the FRET signal was verified by the absence of energy transfer in
individually transfected TSHRGFP and
TSHRMyc:Cy3 cells cultured together and also by
acceptor photobleaching. Occupation of the receptor molecule by the
ligand (TSH) resulted in a dose-dependent decrease in the FRET index from 20% in the absence of TSH to <1% with
103 microunits/ml of TSH. Such reduction in
oligomeric forms was also confirmed by coimmunoprecipitation. Exposure
of TSHRGFP/Myc cells to forskolin or cytochalasin D caused
no change in the FRET index, confirming that the decrease in the
oligomeric complexes was a receptor-dependent phenomenon
and free of energy or microtuble requirements. The TSH-induced decrease
in TSHR oligomers was found to be secondary to dissociation of the TSHR
complexes as evidenced by an increase in fluorescent intensity of
photobleached spots of GFP fluorescence with 103
microunits/ml of TSH. These data indicated that the less active conformation of the TSHR was comprised of receptor complexes and that
such complexes were dissociated on the binding of ligand. Such
observations support the concept of a constitutively active TSHR dimer
or monomer that is naturally inhibited by the formation of higher order
complexes. Inhibition of these oligomeric forms by ligand binding
returns the TSHR to an activated state.
Ligand-dependent Inhibition of Oligomerization at the
Human Thyrotropin Receptor*
,
*
This work was supported in part by National Institutes of
Health Grants DK52464, DK35764, and DK45011 (to T. F. D.) and
by the David Owen Segal Endowment (to R. L.). Confocal laser
scanning microscopy was supported by National Institutes of Health
Grant 1S10RR9145-01 and National Science Foundation 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.: 212-241-4218; Fax:
212-241-4218; E-mail: rauf.latif@mssm.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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