Arrestin variants display differential binding characteristics for the phosphorylated N-formyl-peptide receptor carboxy terminus

doi:10.1074/jbc.M111086200

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Arrestin variants display differential binding characteristics for the phosphorylated N-formyl-peptide receptor carboxy terminus

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

Depatment of Cell Biology and Physiology, University of New Mexico, Albuquerque, NM 87131

Corresponding Author: eprossnitz{at}Salud.unm.edu

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 carboxy 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 carboxy terminus were analyzed by flow cytometry. Arrestin2 and arrestin3 bound to the FPR carboxy terminus peptide in a phosphorylation-dependent manner, with Kd values in the µM range. Binding of visual arrestin, which binds rhodopsin with high selectivity, was not observed. Arrestin2 (1-382) and arrestin3 (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 Kd 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 carboxy terminus. Whereas the single point mutant, arrestin2 R169E, displayed an affinity similar to that of the full length arrestins, the triple point mutant, arrestin2 I386A/V387A/F388A, displayed an affinity more similar to that of the truncated forms of arrestin. The results suggest that the carboxy terminus of arrestin is a critical determinant in regulating the binding affinity of arrestin for the phosphorylated domains of GPCRs.

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				C. M. Revankar, C. M. Vines, D. F. Cimino, and E. R. Prossnitz Arrestins Block G Protein-coupled Receptor-mediated Apoptosis J. Biol. Chem., June 4, 2004; 279(23): 24578 - 24584. [Abstract] [Full Text] [PDF]

				J. Bylund, A. Bjorstad, D. Granfeldt, A. Karlsson, C. Woschnagg, and C. Dahlgren Reactivation of Formyl Peptide Receptors Triggers the Neutrophil NADPH-oxidase but Not a Transient Rise in Intracellular Calcium J. Biol. Chem., August 15, 2003; 278(33): 30578 - 30586. [Abstract] [Full Text] [PDF]

				T. A. Key, T. D. Foutz, V. V. Gurevich, L. A. Sklar, and E. R. Prossnitz N-Formyl Peptide Receptor Phosphorylation Domains Differentially Regulate Arrestin and Agonist Affinity J. Biol. Chem., January 31, 2003; 278(6): 4041 - 4047. [Abstract] [Full Text] [PDF]

				M. G. H. Scott, E. Le Rouzic, A. Perianin, V. Pierotti, H. Enslen, S. Benichou, S. Marullo, and A. Benmerah Differential Nucleocytoplasmic Shuttling of beta -Arrestins. CHARACTERIZATION OF A LEUCINE-RICH NUCLEAR EXPORT SIGNAL IN beta -ARRESTIN2 J. Biol. Chem., September 27, 2002; 277(40): 37693 - 37701. [Abstract] [Full Text] [PDF]

				F. Huttenrauch, A. Nitzki, F.-T. Lin, S. Honing, and M. Oppermann beta -Arrestin Binding to CC Chemokine Receptor 5 Requires Multiple C-terminal Receptor Phosphorylation Sites and Involves a Conserved Asp-Arg-Tyr Sequence Motif J. Biol. Chem., August 16, 2002; 277(34): 30769 - 30777. [Abstract] [Full Text] [PDF]