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

Abstract

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 theFPRcarboxyl-terminalpeptideinaphosphorylationdependent 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 phosphorylationdependent 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.G protein-coupled receptors (GPCRs) 1 are the largest family of cellular surface receptors, the mediators of numerous physiological responses, and the targets of ϳ50% of therapeutic drugs (1). Their activation occurs through the binding of specific ligands to the extracellular face of the receptor, whereas cellular activation is mediated through interactions with cytoplasmic proteins, such as G proteins. Following ligand-mediated activation, GPCRs are the target of regulatory mechanisms aimed initially at terminating and then at reinitiating the signaling capacity of the cell. Furthermore, GPCR processing has also been shown to be important in the activation of Src and certain MAP kinase pathways (2). The protein arrestin was first characterized as a protein involved in GPCR desensitization following receptor phosphorylation, but has recently been demonstrated to play multiple roles in receptor function, including acting as an adapter for clathrin-mediated endocytosis and a scaffolding protein for...

N-Formyl Peptide Receptor Phosphorylation Domains Differentially Regulate Arrestin and Agonist Affinity

“…However, we have also shown that neither truncated arrestin-2 nor arrestin-3 exhibits specific binding to the nonphosphorylated FPR carboxyl terminus alone (30). Together, these results support a role for additional receptor domains in arrestin binding.…”

“…1 The abbreviations used are: GPCR, G protein-coupled receptor; FPR, N-formyl peptide receptor; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; GFP, green fluorescent protein; FITC, fluorescein 5-isothio-phosphorylation-and activation-independence with a number of GPCRs, including receptors from which the carboxyl terminus, containing critical phosphorylation sites, has been removed (25). We have recently demonstrated that the affinity of truncated arrestin-2 for a carboxyl-terminal peptide of the Nformyl peptide receptor (FPR) is significantly higher than that of wild type arrestin-2 (30). Thus, it is likely there is a complex interplay of multiple receptor and arrestin domains.…”

Reactivation of Formyl Peptide Receptors Triggers the Neutrophil NADPH-oxidase but Not a Transient Rise in Intracellular Calcium

“…The carboxyl terminus of FPR (as well as of other GPCRs) contains serine and threonine residues that may be phosphorylated by various kinases. Removal of eight possible phosphorylation sites in the cytoplasmic tail of FPR has been shown to block ligand-induced phosphorylation of the receptor, a loss that correlates to a lack of receptor desensitization and receptor internalization (39). These two processes are regulated separately, illustrated by the fact that an inability to phosphorylate some residues (but not others) completely abolishes receptor desensitization but is without effect on internalization (40).…”

The structural basis of arrestin-mediated regulation of G-protein-coupled receptors

“…The studies of arrestin interactions with receptor elements using surface plasmon resonance also yielded micromolar affinities (Cen et al, 2001a;Liu et al, 2004). Wild-type arrestins bound to the immobilized phosphorylated C-terminal peptide of the N-formyl peptide receptor with micromolar affinities, although several constitutively active mutants demonstrated submicromolar dissociation constants (Potter et al, 2002). Interestingly, in a few studies where the affinity of arrestin proteins for the native phosphoreceptor has been measured, it was found to be nanomolar for rhodopsin (Schleicher et al, 1989;Pulvermuller et al, 1997;Osawa et al, 2000) and sub-nanomolar for the b2AR or m2 muscarinic cholinergic receptor (Gurevich et al, 1993a(Gurevich et al, , 1995.…”