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Originally published In Press as doi:10.1074/jbc.M110894200 on April 4, 2002

J. Biol. Chem., Vol. 277, Issue 24, 21939-21946, June 14, 2002
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Identification of a Short Linear Sequence Present in the C-terminal Tail of the Rat Follitropin Receptor That Modulates Arrestin-3 Binding in a Phosphorylation-independent Fashion*

Hiroshi Kishi, Hanumanthappa Krishnamurthy, Colette Galet, Ravi Sankar Bhaskaran, and Mario AscoliDagger

From the Department of Pharmacology, The University of Iowa College of Medicine, Iowa City, Iowa 52242

The rat follitropin receptor (rFSHR) is an unusual G protein-coupled receptor in that agonist-induced activation leads to the phosphorylation of the first and third intracellular loops instead of the C-terminal tail. To determine regions of G protein-coupled receptors that affect internalization independently of phosphorylation we examined the effects of truncations of the C-terminal tail of the rFSHR on agonist-induced internalization. Our studies show that progressive truncations of a region flanked by residues 642 and 651 enhance the internalization of human follicle-stimulating hormone (hFSH). Further characterization of a mutant truncated at residue 649 (designated rFSHR-t649) and another mutant in which the 642-651 region was deleted in the context of the full-length rFSHR, designated rFSHR(Delta 642-651), showed that both of them internalized hFSH at rates that were 2-3 times faster than rFSHR-wild type (wt). Like rFSHR-wt, however, the internalization of hFSH mediated by rFSHR-t649 and rFSHR(Delta 642-651) can be inhibited with dominant-negative mutants of the non-visual arrestins or dynamin. Alanine-scanning mutagenesis of the 642-651 region suggests that the effects on internalization are not mediated by a single residue, however. In an attempt to understand the molecular basis of the enhanced internalization of hFSH mediated by these mutants we used an assay that can be readily used to assess the association of the rFSHR with the arrestin-3 in co-transfected cells. Using this assay we were able to show that, when compared with rFSHR-wt, rFSHR(Delta 642-651) displays an ~4-fold enhancement in binding affinity for arrestin-3 and an ~1.7-fold reduction in maximal arrestin-3 binding capacity. We conclude that a short linear sequence present in the C-terminal tail of the rFSHR (642SATHNFHARK651) that is not phosphorylated limits internalization by lowering the affinity of the rFSHR for the endogenous non-visual arrestins.

* This work was supported by National Institutes of Health Grant HD-28962 (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, 2-319A BSB, 51 Newton Rd., The 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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