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Originally published In Press as doi:10.1074/jbc.M106082200 on November 5, 2001

J. Biol. Chem., Vol. 277, Issue 1, 702-710, January 4, 2002
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The Association of Arrestin-3 with the Human Lutropin/Choriogonadotropin Receptor Depends Mostly on Receptor Activation Rather than on Receptor Phosphorylation*

Le Min, Colette Galet, and Mario AscoliDagger

From the Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242-1109

Although the involvement of the nonvisual arrestins in the agonist-induced internalization of the human lutropin receptor (hLHR) has been documented previously with the use of dominant-negative mutants, a physical association of the nonvisual arrestins with the hLHR in intact cells has not been established. In the studies presented herein, we used a cross-linking/coimmunoprecipitation/immunoblotting approach as well as confocal microscopy to document the association of the hLHR with the nonvisual arrestins in co-transfected 293 cells. We also used this approach to examine the relative importance of receptor activation and receptor phosphorylation in the formation of this complex. Using hLHR mutants that impair phosphorylation, activation, or both, we show that the formation of the hLHR-nonvisual arrestin complex depends mostly on the agonist-induced activation of the hLHR rather than on the phosphorylation of the hLHR. These results stand in contrast to those obtained with several other G protein-coupled receptors (i.e. the beta 2-adrenergic receptor, the m2 muscarinic receptor, rhodopsin, and the type 1A angiotensin receptor) where arrestin binding depends mostly on receptor phosphorylation rather than on receptor activation. We have also examined the association of the nonvisual arrestins with naturally occurring gain-of-function mutations of the hLHR found in boys with Leydig cell hyperplasia or Leydig cell adenomas. Our results show that these mutants associate with the nonvisual arrestins in an agonist-independent fashion.

* This work was supported by National Institutes of Health Grant CA-40629 (to M. A).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.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, 2-319B BSB, 51 Newton Rd., University of Iowa, Iowa City, IA 52242-1109. Tel.: 319-335-9907; Fax: 319-335-8930; E-mail: mario-ascoli@uiowa.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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