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J. Biol. Chem., Vol. 279, Issue 53, 55744-55753, December 31, 2004

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Activation-dependent Conformational Changes in -Arrestin 2^*

Kunhong Xiao, Sudha K. Shenoy, Kelly Nobles, and Robert J. Lefkowitz¶||

From the Departments of Medicine and Biochemistry and the ^¶Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

-Arrestins are multifunctional adaptor proteins, which mediate desensitization, endocytosis, and alternate signaling pathways of seven membrane-spanning receptors (7MSRs). Crystal structures of the basal inactive state of visual arrestin (arrestin 1) and -arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in -arrestins upon binding to the activated phosphorylated receptor. Here we characterize the conformational changes in -arrestin 2 (arrestin 3) by comparing the limited tryptic proteolysis patterns and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles of -arrestin 2 in the presence of a phosphopeptide (V₂R-pp) derived from the C terminus of the vasopressin type II receptor (V₂R) or the corresponding nonphosphopeptide (V₂R-np). V₂R-pp binds to -arrestin 2 specifically, whereas V₂R-np does not. Activation of -arrestin 2 upon V₂R-pp binding involves the release of its C terminus, as indicated by exposure of a previously inaccessible cleavage site, one of the polar core residues Arg³⁹⁴, and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg⁸. Interestingly, binding of the polyanion heparin also leads to release of the C terminus of -arrestin 2; however, heparin and V₂R-pp have different binding site(s) and/or induce different conformational changes in -arrestin 2. Release of the C terminus from the rest of -arrestin 2 has functional consequences in that it increases the accessibility of a clathrin binding site (previously demonstrated to lie between residues 371 and 379) thereby enhancing clathrin binding to -arrestin 2 by 10-fold. Thus, the V₂R-pp can activate -arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of -arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor--arrestin interactions can be modeled in vitro.

Received for publication, August 25, 2004 , and in revised form, October 19, 2004.

^* This work was supported in part by Grants R01HL16037 and HL 70631 (to R. J. L.) from the National Institutes of Health and by an American Heart Association postdoctoral fellowship (to K. X.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| An Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Depts. of Medicine and Biochemistry, Box 3821, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875; E-mail: lefko001{at}receptor-biol.duke.edu.

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