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J. Biol. Chem., Vol. 277, Issue 11, 8970-8978, March 15, 2002

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Arrestin Variants Display Differential Binding Characteristics for the Phosphorylated N-Formyl Peptide Receptor Carboxyl Terminus^*

Ross M. Potter, T. Alexander Key^§, Vsevolod V. Gurevich^¶, Larry A. Sklar^§, and Eric R. Prossnitz^**

From the  Department of Cell Biology and Physiology, ^§ Cancer Research and Treatment Center, and Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, the ^¶ Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, and the  National Flow Cytometry Resource, Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545

The phosphorylation-dependent binding of arrestins to cytoplasmic domains of G protein-coupled receptors (GPCRs) is thought to be a crucial step in receptor desensitization. In some GPCR systems, arrestins have also been demonstrated to be involved in receptor internalization, resensitization, and the activation of signaling cascades. The objective of the current study was to examine binding interactions of members of the arrestin family with the formyl peptide receptor (FPR), a member of the GPCR family of receptors. Peptides representing the unphosphorylated and phosphorylated carboxyl terminus of the FPR were synthesized and bound to polystyrene beads via a biotin/streptavidin interaction. Using fluorescein-conjugated arrestins, binding interactions between arrestins and the bead-bound FPR carboxyl terminus were analyzed by flow cytometry. Arrestin-2 and arrestin-3 bound to the FPR carboxyl-terminal peptide in a phosphorylation-dependent manner, with K_d values in the micromolar range. Binding of visual arrestin, which binds rhodopsin with high selectivity, was not observed. Arrestin-2-(1-382) and arrestin-3-(1-393), truncated mutant forms of arrestin that display phosphorylation-independent binding to intact receptors, were also observed to bind the bead-bound FPR terminus in a phosphorylation-dependent manner, but with much greater affinity than the full-length arrestins, yielding K_d values in the 5-50 nM range. Two additional arrestin mutants, which are full-length but display phosphorylation-independent binding to intact GPCRs, were evaluated for their binding affinity to the FPR carboxyl terminus. Whereas the single point mutant, arrestin-2 R169E, displayed an affinity similar to that of the full-length arrestins, the triple point mutant, arrestin-2 I386A/V387A/F388A, displayed an affinity more similar to that of the truncated forms of arrestin. The results suggest that the carboxyl terminus of arrestin is a critical determinant in regulating the binding affinity of arrestin for the phosphorylated domains of GPCRs.

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