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J. Biol. Chem., Vol. 277, Issue 31, 28228-28237, August 2, 2002
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From the Department of Pharmacology and Physiology, University of
Rochester Medical Center, Rochester, New York 14642
Dimerization and phosphorylation of
thyrotropin-releasing hormone (TRH) receptors was characterized using
HEK293 and pituitary GHFT cells expressing epitope-tagged receptors.
TRH receptors tagged with FLAG and hemagglutinin epitopes were
co-precipitated only if they were co-expressed, and 10-30% of
receptors were isolated as hemagglutinin/FLAG-receptor dimers under
basal conditions. The abundance of receptor dimers was increased when
cells had been stimulated by TRH, indicating that TRH either stabilizes pre-existing dimers or increases dimer formation. TRH increased receptor dimerization and phosphorylation within 1 min in a
dose-dependent manner. TRH increased phosphorylation of
both receptor monomers and dimers, documented by incorporation of
32P and an upshift in receptor mobility reversed by
phosphatase treatment. The ability of TRH to increase receptor
phosphorylation and dimerization did not depend on signal transduction,
because it was not inhibited by the phospholipase C inhibitor U73122. Receptor phosphorylation required an agonist but was not blocked by the
casein kinase II inhibitor apigenin, the protein kinase C
inhibitor GF109203X, or expression of a dominant negative form of G
protein-coupled receptor kinase 2. TRH receptors lacking most of the
cytoplasmic carboxyl terminus formed dimers constitutively but failed
to undergo agonist-induced dimerization and phosphorylation. TRH also
increased phosphorylation and dimerization of TRH receptors expressed
in GHFT pre-lactotroph cells.
Dimerization and Phosphorylation of Thyrotropin-releasing Hormone
Receptors Are Modulated by Agonist Stimulation*
,
*
This work was supported by National Institutes of Health
Grant DK19974, a Wilmot Cancer Research Fellowship (to C.-C. Z.), and
a Pharmaceutical Manufacturers' Association Advanced Predoctoral Fellowship (to L. B. C.).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.
Present address: Dept. of Pathology, New York University School of
Medicine, New York, NY 10016.
§
To whom correspondence should be addressed: Dept. of Pharmacology
and Physiology, University of Rochester Medical Center, 601 Elmwood
Ave., Rochester, NY 14642. Tel.: 585-275-4933; Fax: 585-461-0397;
E-mail: patricia_hinkle@urmc.rochester.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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