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.

Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free G␤␥ subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with G␤␥ and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by G␤␥. Alternatively, mutation of residues distal to the C-terminal amphipathic ␣-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to G␤␥. Interestingly, mutation of Arg-587 in ␤-sheet 3, a region not previously thought to interact with G␤␥, resulted in a specific and profound loss of G␤␥ responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound G␤␥ similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind G␤␥. In order to examine the role of phospholipid and G␤␥ interaction in cells, wild-type and mutant GRK2s were expressed with a ␤ 2 -adrenergic receptor (␤ 2 AR) mutant that is responsive to GRK2 phosphorylation (␤ 2 AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of ␤ 2 AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the -opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for G␤␥ and phospholipids, and (ii) demonstrated that G␤␥ binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.Diverse extracellular stimuli are perceived at the plasma membrane by G protein-coupled receptors (GPCRs).1 Agonistoccupied receptors promote the activation and dissociation of the heterotrimeric G protein ␣ and ␤␥ subunits, each of which goes on to regulate various effector molecules thereby producing a physiological response. This process is regulated in an agonist-dependent fashion by a family of G protein-coupled receptor kinases (GRKs), which phosphorylate activated GPCRs promoting binding of a second family of proteins, termed arrestins, which serve to uncouple the GPCR from further G protein activation (1, 2). Arrestin binding also promotes receptor internalization, which facilitates the processes of receptor resensitization and down-regulation (1).In general, all G...

The ␤-adrenergic receptor kinase (␤ARK) is a member of growing family of G protein coupled receptor kinases (GRKs). ␤ARK and other members of the GRK family play a role in the mechanism of agonist-specific desensitization by virtue of their ability to phosphorylate G protein-coupled receptors in an agonist-dependent manner. ␤ARK activation is known to occur following the interaction of the kinase with the agonist-occupied form of the receptor substrate and heterotrimeric G protein ␤␥ subunits. Recently, lipid regulation of GRK2, GRK3, and GRK5 have also been described. Using a mixed micelle assay, GRK2 (␤ARK1) was found to require phospholipid in order to phosphorylate the ␤ 2 -adrenergic receptor. As determined with a nonreceptor peptide substrate of ␤ARK, catalytic activity of the kinase increased in the presence of phospholipid without a change in the K m for the peptide. The molecular mechanisms involved in signal transduction of G protein-coupled receptors are best understood in the visual system where rhodopsin serves as the "receptor" for light (1) and the ␤-adrenergic pathway in which the ␤-adrenergic receptor (␤AR) 1 binds catecholamines (2, 3). A feature common to both model systems as well as many other G protein receptors is the diminished responsiveness with time to a signal of equal intensity. This phenomenon is known as desensitization (4) and exhibits both an agonist-specific and nonspecific pattern. Rapid, agonist-specific desensitization of rhodopsin and the ␤ 2 -adrenergic receptor (␤ 2 AR) occurs in response to the phosphorylation of the receptor by the enzymes rhodopsin kinase and the ␤-adrenergic receptor kinase (␤ARK) (5). Rhodopsin kinase and ␤ARK are members of a family known as G proteincoupled receptor kinases (GRKs). A common feature to the GRK family of kinases is multi-site phosphorylation of receptor substrates in response to agonist occupancy (6). The relationship between agonist occupancy and receptor phosphorylation by GRKs is key to the specificity of the desensitization process, while other kinases such as protein kinase A and C play a role in nonspecific or heterologous desensitization. Two possible mechanisms could explain the enhanced phosphorylation of the activated form of the receptor by kinases of the GRK family. First, receptor occupancy may induce a conformational change exposing potential phosphorylation sites previously sequestered from the kinase. Alternatively, interaction of the kinase with the agonist-bound form of the receptor could result in enhanced catalytic activity of the kinase. The bulk of the experimental evidence supports the latter hypothesis (7-9). In addition to the enhanced catalytic activity of GRKs in the presence of agonist-occupied receptor, GRK2 and GRK3 activity is also increased by heterotrimeric G protein ␤␥ subunits (10 -13). The potential for finely controlled desensitization by the interplay of receptors and ␤␥ subunits is an exciting possibility given the evidence for dual regulation of GRK2 and GRK3 by these proteins (14). While G prot...

Previously, Lefkowitz and coworkers (8) cloned and sequenced cDNAs for the first member of this proposed receptor kinase family, the f3-adrenergic receptor kinase (j3ARK) (8). This kinase phosphorylates and regulates the function of 8-adrenergic (and possibly other G-proteincoupled) receptors by catalyzing phosphorylation of the receptor on a serine/threonine-rich cluster found at its carboxyl terminus (9). Functionally, a very analogous enzyme is rhodopsin kinase (RK). Discovered almost two decades ago (4-6), this enzyme is known to phosphorylate rhodopsin, thereby initiating its deactivation. The biochemical and functional properties of RK are such as to suggest that it might be closely related to DARK (10). Accordingly we set out to clone cDNAs for RK so as to elucidate its primary structure and thereby clarify structural, evolutionary, and functional relationships with PARK, as well as to understand the nature and diversity of the proposed family of receptor kinase molecules.tt MATERIALS AND METHODSProtein Purification and Peptide Sequencing. RK was purified from bovine retina as described (11). The enzyme was extracted from photobleached rod outer segment membranes with 60 mM KC1. The kinase was further purified with a DEAE-cellulose step, and pooled activity was then applied to a hydroxyapatite column. RK was eluted in a buffer of 20 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane, 1 mM Mg(OAc)2, and 95 mM KC1. Fractions (1 ml) were concentrated to 300 A.l under reduced vacuum (Speed-Vac, Savant) in individual Eppendorf tubes. To each of the 10 tubes was added 700 p.l of CNBr in formic acid (99%) to give a final CNBr concentration of 10 mM; the digestion was allowed to proceed for 24 hr in the dark at room temperature. Each sample was dried under reduced pressure and rehydrated with 500 ,ul of 90%o water/10%o acetonitrile/0.1% trifluoroacetic acid. The 10 samples were individually applied to an Aquapore C4 reverse-phase column (Applied Biosystems; 2.1 x 30 mm) at a flow rate of 200 p.l/min; the column was washed with 1.5 ml of 0.1% trifluoroacetic acid in water between each sample application. Once the last sample was loaded, a gradient of 0-70% acetonitrile was developed. Fractions were collected at 1-min intervals. A control digest, consisting of 1 ml of buffer handled exactly as the kinasecontaining samples and subjected to CNBr treatment, was chromatographed under identical conditions. Peaks unique to the RK-containing digest were submitted for gas-phase peptide sequencing (R. Randall, Howard Hughes Medical Institute Biopolymer Lab, Durham, NC).cDNA Library Screening and Polymerase Chain Reaction (PCR). Screening of a bovine Okayama-Berg cDNA library (12) with the peptide-derived oligodeoxynucleotide 5'-Abbreviations: RK, rhodopsin kinase; fARK, fi-adrenergic receptor kinase; RACE, rapid amplification of cDNA ends.

1 The P-adrenoceptor is one of a number of G protein-coupled receptors which have been proposed to contain seven transmembrane a-helices. The function of this receptor appears to be regulated by phosphorylation by a specific enzyme, the ,-adrenoceptor kinase. Synthetic peptides which comprise each of the proposed intra-and extracellular domains of the 32-adrenoceptor have been tested as potential substrates and inhibitors of the ,-adrenoceptor kinase. 2 Two peptides which encompass the middle and terminal portions of the carboxyl tail of the receptor served as substrates by ,B-adrenoceptor kinase. The kinetics of the phosphorylation reaction, however, suggest that these peptides are 106-fold poorer substrates than the agonist occupied receptor. potential sites of interaction with P-adrenoceptor kinase. Moreover, these regions may serve as potential targets for the development of specific inhibitors of ,-adrenoceptor kinase which could be used to block homologous desensitization.

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