Arrestins play an important role in quenching signal transduction initiated by G protein-coupled receptors. To explore the specificity of arrestin-receptor interaction, we have characterized the ability of various wild-type arrestins to bind to rhodopsin, the beta 2-adrenergic receptor (beta 2AR), and the m2 muscarinic cholinergic receptor (m2 mAChR). Visual arrestin was found to be the most selective arrestin since it discriminated best between the three different receptors tested (highest binding to rhodopsin) as well as between the phosphorylation and activation state of the receptor (> 10-fold higher binding to the phosphorylated light-activated form of rhodopsin compared to any other form of rhodopsin). While beta-arrestin and arrestin 3 were also found to preferentially bind to the phosphorylated activated form of a given receptor, they only modestly discriminated among the three receptors tested. To explore the structural characteristics important in arrestin function, we constructed a series of truncated and chimeric arrestins. Analysis of the binding characteristics of the various mutant arrestins suggests a common molecular mechanism involved in determining receptor binding selectivity. Structural elements that contribute to arrestin binding include: 1) a C-terminal acidic region that serves a regulatory role in controlling arrestin binding selectivity toward the phosphorylated and activated form of a receptor, without directly participating in receptor interaction; 2) a basic N-terminal domain that directly participates in receptor interaction and appears to serve a regulatory role via intramolecular interaction with the C-terminal acidic region; and 3) two centrally localized domains that are directly involved in determining receptor binding specificity and selectivity. A comparative structure-function model of all arrestins and a kinetic model of beta-arrestin and arrestin 3 interaction with receptors are proposed.

The rapid decrease of a response to a persistent stimulus, often termed desensitization, is a widespread biological phenomenon. Signal transduction by numerous G protein-coupled receptors appears to be terminated by a strikingly uniform two-step mechanism, most extensively characterized for the ␤ 2 -adrenergic receptor (␤ 2 AR), m2 muscarinic cholinergic receptor (m2 mAChR), and rhodopsin. The model predicts that activated receptor is initially phosphorylated and then tightly binds an arrestin protein that effectively blocks further G protein interaction. Here we report that complexes of ␤ 2 AR-arrestin and m2 mAChR-arrestin have a higher affinity for agonists (but not antagonists) than do receptors not complexed with arrestin. The percentage of phosphorylated ␤ 2 AR in this high affinity state in the presence of full agonists varied with different arrestins and was enhanced by selective mutations in arrestins. The percentage of high affinity sites also was proportional to the intrinsic activity of an agonist, and the coefficient of proportionality varies for different arrestin proteins. Certain mutant arrestins can form these high affinity complexes with unphosphorylated receptors. Mutations that enhance formation of the agonistreceptor-arrestin complexes should provide useful tools for manipulating both the efficiency of signaling and rate and specificity of receptor internalization.Agonist binding activates G protein 1 -coupled receptors and initiates two intimately intertwined cascades of events, resulting in signal transduction and signal termination (desensitization). The receptor-agonist complex initially interacts with G protein(s) to form a transient agonist-receptor-G protein ternary complex that is the first intermediate in transmembrane signaling (1, 2). This ternary complex has a higher affinity for agonists than receptor alone (1, 2). Formation of this complex promotes GDP release from the G protein, which is followed by rapid GTP binding and dissociation of the active G␣⅐GTP and G␤␥ subunits. The agonist-occupied receptors are then phosphorylated by G protein-coupled receptor kinases, resulting in arrestin binding and consequent disruption of receptor-G protein interaction (3). Recent studies suggest that arrestin binding also targets the receptors for internalization (4, 5), apparently by virtue of the ability of non-visual arrestins to interact with clathrin (6), a process that appears to be a prerequisite for resensitization (3). Thus, the formation of the arrestin-receptor complex is not only the final step of signal termination but also an initial step of subsequent resensitization, representing a critical juncture in the signaling process. Because of this the arrestin-receptor complex appears to be a tempting target for a more detailed characterization. EXPERIMENTAL PROCEDURESArrestin Expression in Escherichia coli and Purification-Bovine arrestin cDNAs were subcloned using the NcoI and HindIII sites of pTrcB (Invitrogen). BL-21 cells transformed with the pTrcB-arrestin constructs were grown...

The beta-adrenergic receptor kinase (beta-ARK) phosphorylates G protein coupled receptors in an agonist-dependent manner. Since the exact sites of receptor phosphorylation by beta-ARK are poorly defined, the identification of substrate amino acids that are critical to phosphorylation by the kinase are also unknown. In this study, a peptide whose sequence is present in a portion of the third intracellular loop region of the human platelet alpha 2-adrenergic receptor is shown to serve as a substrate for beta-ARK. Removal of the negatively charged amino acids surrounding a cluster of serines in this alpha 2-peptide resulted in a complete loss of phosphorylation by the kinase. A family of peptides was synthesized to further study the role of acidic amino acids in peptide substrates of beta-ARK. By kinetic analyses of the phosphorylation reactions, beta-ARK exhibited a marked preference for negatively charged amino acids localized to the NH2-terminal side of a serine or threonine residue. While there were no significant differences between glutamic and aspartic acid residues, serine-containing peptides were 4-fold better substrates than threonine. Comparing a variety of kinases, only rhodopsin kinase and casein kinase II exhibited significant phosphorylation of the acidic peptides. Unlike beta-ARK, RK preferred acid residues localized to the carboxyl-terminal side of the serine. A feature common to beta-ARK and RK was a much greater Km for peptide substrates as compared to that for intact receptor substrates.