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J. Biol. Chem., Vol. 282, Issue 29, 21370-21381, July 20, 2007

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The Active Conformation of -Arrestin1

DIRECT EVIDENCE FOR THE PHOSPHATE SENSOR IN THE N-DOMAIN AND CONFORMATIONAL DIFFERENCES IN THE ACTIVE STATES OF -ARRESTINS1 AND -2^*

Kelly N. Nobles, Ziqiang Guan¹, Kunhong Xiao, Terrence G. Oas, and Robert J. Lefkowitz^¶2

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

-Arrestins are multifunctional adaptor proteins that regulate seven transmembrane-spanning receptor (7TMR) desensitization and internalization and also initiate alternative signaling pathways. Studies have shown that -arrestins undergo a conformational change upon interaction with agonist-occupied, phosphorylated 7TMRs. Although conformational changes have been reported for visual arrestin and -arrestin2, these studies are not representative of conformational changes in -arrestin1. Accordingly, in this study, we determine conformational changes in -arrestin1 using limited tryptic proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis in the presence of a phosphopeptide derived from the C terminus of the V₂ vasopressin receptor (V₂Rpp) or the corresponding unphosphorylated peptide (V₂Rnp). V₂Rpp binds specifically to -arrestin1 causing significant conformational changes, whereas V₂Rnp does not alter the conformation of -arrestin1. Upon V₂Rpp binding, we show that the previously shielded Arg³⁹³ becomes accessible, which indicates release of the C terminus. Moreover, we show that Arg²⁸⁵ becomes more accessible, and this residue is located in a region of -arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of -arrestin1, and we also show a functional consequence of the release of the C terminus of -arrestin1 by enhanced clathrin binding. In addition, we show marked protection of the N-domain of -arrestin1 in the presence of V₂Rpp, which is consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. A striking difference in conformational changes is observed in -arrestin1 when compared with -arrestin2, namely the flexibility of the interdomain hinge region. This study represents the first direct evidence that the "receptor-bound" conformations of -arrestins1 and 2 are different.

Received for publication, December 14, 2006 , and in revised form, April 25, 2007.

^* This work was supported in part by Grants HL16037 and HL70631 (to R. J. L.) from the National Institutes of Health. 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.

¹ Supported by LIPID MAPS Large Scale Collaborative Grant GM069338 from the National Institutes of Health.

² To whom correspondence should be addressed: Howard Hughes Medical Institute, Duke University Medical Center, Box 3821, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875; E-mail: lefko001{at}receptor-biol.duke.edu.

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