An additional phosphate-binding element in arrestin molecule. Implications for the mechanism of arrestin activation

Arrestins quench the signaling of a wide variety of G protein-coupled receptors by virtue of high-affinity binding to phosphorylated activated receptors. The high selectivity of arrestins for this particular functional form of receptor ensures their timely binding and dissociation. In a continuing effort to elucidate the molecular mechanisms responsible for arrestin's selectivity, we used the visual arrestin model to probe the functions of its N-terminal beta-strand I comprising the highly conserved hydrophobic element Val-Ile-Phe (residues 11-13) and the adjacent positively charged Lys(14) and Lys(15). Charge elimination and reversal in positions 14 and 15 dramatically reduce arrestin binding to phosphorylated light-activated rhodopsin (P-Rh*). The same mutations in the context of various constitutively active arrestin mutants (which bind to P-Rh*, dark phosphorylated rhodopsin (P-Rh), and unphosphorylated light-activated rhodopsin (Rh*)) have minimum impact on P-Rh* and Rh* binding and virtually eliminate P-Rh binding. These results suggest that the two lysines "guide" receptor-attached phosphates toward the phosphorylation-sensitive trigger Arg(175) and participate in phosphate binding in the active state of arrestin. The elimination of the hydrophobic side chains of residues 11-13 (triple mutation V11A, I12A, and F13A) moderately enhances arrestin binding to P-Rh and Rh*. The effects of triple mutation V11A, I12A, and F13A in the context of phosphorylation-independent mutants suggest that residues 11-13 play a dual role. They stabilize arrestin's basal conformation via interaction with hydrophobic elements in arrestin's C-tail and alpha-helix I as well as its active state by interactions with alternative partners. In the context of the recently solved crystal structure of arrestin's basal state, these findings allow us to propose a model of initial phosphate-driven structural rearrangements in arrestin that ultimately result in its transition into the active receptor-binding state.     

Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge

Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms.     

Binding of GTP to transducin is not inhibited by arrestin and phosphorylated rhodopsin

In the presence of a photobleaching intermediate of unphosphorylated or phosphorylated rhodopsin (Rh*), the binding of GppNHp to transducin was measured with or without arrestin for elucidation of the shut-off mechanism of the visual transduction process in bovine rod outer segments. The ability of Rh* to catalyze the formation of the transducin-GppNHp complex in the absence of arrestin was independent of the degree of phosphorylation of Rh*. Furthermore, the catalyzing ability of the phosphorylated Rh* was not reduced by the addition of arrestin. These observations indicate that the interaction between phosphorylated Rh* and transducin was not inhibited by arrestin. Thus, the hypothesis was not supported that the PDE shut-off process is a simple competition between transducin and arrestin for binding to phosphorylated Rh*.     

Mapping the arrestin-receptor interface. Structural elements responsible for receptor specificity of arrestin proteins

Arrestins selectively bind to phosphorylated activated forms of their cognate G protein-coupled receptors. Arrestin binding prevents further G protein activation and often redirects signaling to other pathways. The comparison of the high-resolution crystal structures of arrestin2, visual arrestin, and rhodopsin as well as earlier mutagenesis and peptide inhibition data collectively suggest that the elements on the concave sides of both arrestin domains most likely participate in receptor binding directly, thereby dictating its receptor preference. Using comparative binding of visual arrestin/arrestin2 chimeras to the preferred target of visual arrestin, light-activated phosphorylated rhodopsin (PRh*), and to the arrestin2 target, phosphorylated activated m2 muscarinic receptor (P-m2 mAChR*), we identified the elements that determine the receptor specificity of arrestins. We found that residues 49-90 (beta-strands V and VI and adjacent loops in the N-domain) and 237-268 (beta-strands XV and XVI in the C-domain) in visual arrestin and homologous regions in arrestin2 are largely responsible for their receptor preference. Only 35 amino acids (22 of which are nonconservative substitutions) in the two elements are different. Simultaneous exchange of both elements between visual arrestin and arrestin2 fully reverses their receptor specificity, demonstrating that these two elements in the two domains of arrestin are necessary and sufficient to determine their preferred receptor targets.     

How does arrestin respond to the phosphorylated state of rhodopsin?

Visual arrestin quenches light-induced signaling by binding to light-activated, phosphorylated rhodopsin (P-Rh*). Here we present structure-function data, which in conjunction with the refined crystal structure of arrestin (Hirsch, J. A., Schubert, C., Gurevich, V. V., and Sigler, P. B. (1999) Cell, in press), support a model for the conversion of a basal or "inactive" conformation of free arrestin to one that can bind to and inhibit the light activated receptor. The trigger for this transition is an interaction of the phosphorylated COOH-terminal segment of the receptor with arrestin that disrupts intramolecular interactions, including a hydrogen-bonded network of buried, charged side chains, referred to as the "polar core." This disruption permits structural adjustments that allow arrestin to bind to the receptor. Our mutational survey identifies residues in arrestin (Arg175, Asp30, Asp296, Asp303, Arg382), which when altered bypass the need for the interaction with the receptor's phosphopeptide, enabling arrestin to bind to activated, nonphosphorylated rhodopsin (Rh*). These mutational changes disrupt interactions and substructures which the crystallographic model and previous biochemical studies have shown are responsible for maintaining the inactive state. The molecular basis for these disruptions was confirmed by successfully introducing structure-based second site substitutions that restored the critical interactions. The nearly absolute conservation of the mutagenically sensitive residues throughout the arrestin family suggests that this mechanism is likely to be applicable to arrestin-mediated desensitization of most G-protein-coupled receptors.     

Monomeric rhodopsin is sufficient for normal rhodopsin kinase (GRK1) phosphorylation and arrestin-1 binding

G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.     

Binding of inositol phosphates to arrestin.

Arrestin binds to phosphorylated rhodopsin in its light-activated form (metarhodopsin II), blocking thereby its interaction with the G-protein, transducin. In this study, we show that highly phosphorylated forms of inositol compete against the arrestin-rhodopsin interaction. Competition curves and direct binding assays with free arrestin consistently yield affinities in the micromolar range; for example, inositol 1,3,4,5-tetrakisphosphate (InP4) and inositol hexakisphosphate (InP6 bind to arrestin with dissociation constants of 12 microM and 5 microM, respectively. Only a small control amount of inositol phosphates is bound, when arrestin interacts with phosphorylated rhodopsin. This argues for a release of bound inositol phosphates by interaction with rhodopsin. Transducin, rhodopsin kinase, or cyclic GMP phosphodiesterase are not affected by inositol phosphates. These observations open a new way to purify arrestin and to inhibit its interaction with rhodopsin. Their physiological significance deserves further investigation.     

Sulfhydryl reactivity demonstrates different conformational states for arrestin, arrestin activated by a synthetic phosphopeptide, and constitutively active arrestin.

The sulfhydryl groups of the three cysteines in bovine arrestin react with DTNB very slowly (over a period of several hours). In the presence of the synthetic phosphopeptide comprising the fully phosphorylated carboxyl-terminal 19 amino acids of bovine rhodopsin, the reactivity of one of the sulfhydryls was enhanced while that of another was greatly reduced. Since this synthetic peptide was shown to activate arrestin with respect to its binding to unphosphorylated, light-activated rhodopsin, the reactivity of the sulfhydryl groups of a constitutively active R175Q arrestin mutant was examined. All three of the sulfhydryl groups of the mutant arrestin R175Q reacted rapidly with DTNB, but not as rapidly as with SDS-denatured arrestin. The arrestin mutant R175Q bound to light-activated, unphosphorylated rhodopsin in ROS disk membranes. The arrestin mutant R175Q also inhibited the light-activated PDE activity with an IC50 of 1.3 microM under the experimental conditions that were used. These data indicate that each of these forms of arrestin is a different conformation. The activated conformation of arrestin that binds to phosphorylated rhodopsin in vivo may be yet another conformation. We conclude that arrestin is a flexible molecule that is able to attain several different conformations, all of which are able to attain the activated functional state of arrestin.     

Insertional mutagenesis and immunochemical analysis of visual arrestin interaction with rhodopsin.

Visual arrestin inactivates the phototransduction cascade by specifically binding to light-activated phosphorylated rhodopsin. This study describes the combined use of insertional mutagenesis and immunochemical approaches to probe the structural determinants of arrestin function. Recombinant arrestins with insertions of a 10-amino acid c-Myc tag (EQKLISEEDL) were expressed in yeast and characterized. When the tag was placed on the C terminus after amino acid 399, between amino acids 99 and 100 or between residues 162 and 163, binding to rhodopsin was found to be very similar to that of wild-type arrestin. Two stable mutants with Myc insertions in the 68-78 loop were also generated. Binding to rhodopsin was markedly decreased for one (72myc73) and completely abolished for the other (77myc78). Limited proteolysis assays using trypsin in the absence or presence of heparin were performed on all mutants and confirmed their overall conformational integrity. Rhodopsin binding to either 162myc163 or 72myc73 arrestins in solution was completely inhibited in the presence of less than a 2-fold molar excess of anti-Myc antibody relative to arrestin. In contrast, the antibody did not block the interaction of the 399myc or 99myc100 arrestins with rhodopsin. These results indicate that an interactive surface for rhodopsin is located on or near the concave region of the N-domain of arrestin.     

Mechanism of rhodopsin kinase activation

The role of the cytoplasmic loops and C-terminal region of bovine rhodopsin (Rho) in binding and activating rhodopsin kinase was investigated. The ability of various enzymatically truncated forms of photolyzed rhodopsin (Rho*) to stimulate rhodopsin kinase activity was quantified. Following endopeptidase Asp-N cleavage of all phosphorylation sites on the C-terminal, the resulting truncated Rho* (329G-Rho*) was not phosphorylated by rhodopsin kinase. This suggests that rhodopsin kinase only phosphorylates C-terminal sites of Rho*. However 329G-Rho* could bind rhodopsin kinase and stimulate phosphorylation of exogenous peptide. Kinase stimulation was investigated for other truncated forms of Rho* in which the C-terminal region was either partially or completely eliminated, and the V-VI loop was either cleaved or left intact (339K-Rho*, 341E239E-Rho*, 329G239E-Rho*, 327P240S-Rho*). Results suggest that the V-VI loop is crucial for kinase binding (similar to the binding of GT). Mastoparan, a model peptide for G-protein-coupled receptors, was found to stimulate rhodopsin kinase in a mechanism similar to that of truncated Rho*. We conclude that rhodopsin kinase binds to the cytoplasmic loops of Rho* to cause a stimulation of its catalytic activity.     

Rhodopsin's carboxyl-terminal threonines are required for wild-type arrestin-mediated quench of transducin activation in vitro

Many recent reports have demonstrated that rhodopsin's carboxyl-terminal serine residues are the main targets for phosphorylation by rhodopsin kinase. Phosphorylation at the serines would therefore be expected to promote high-affinity arrestin binding. We have examined the roles of the carboxyl serine and threonine residues during arrestin-mediated deactivation of rhodopsin using an in vitro transducin activation assay. Mutations were introduced into a synthetic bovine rhodopsin gene and expressed in COS-7 cells. Individual serine and threonine residues were substituted with neutral amino acids. The ability of the mutants to act as substrates for rhodopsin kinase was analyzed. The effect of arrestin on the activities of the phosphorylated mutant rhodopsins was measured in a GTPgammaS binding assay involving purified bovine arrestin, rhodopsin kinase, and transducin. A rhodopsin mutant lacking the carboxyl serine and threonine residues was not phosphorylated by rhodopsin kinase, demonstrating that phosphorylation is restricted to the seven putative phosphorylation sites. A rhodopsin mutant possessing a single phosphorylatable serine at 338 demonstrated no phosphorylation-dependent quench by arrestin. These results suggest that singly phosphorylated rhodopsin is deactivated through a mechanism that does not involve arrestin. Analysis of additional mutants revealed that the presence of threonine in the carboxyl tail of rhodopsin provides for greater arrestin-mediated quench than does serine. These results suggest that phosphorylation site selection could serve as a mechanism to modulate the ability of arrestin to quench rhodopsin.     

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

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.     

Topographic study of arrestin using differential chemical modifications and hydrogen/deuterium exchange.

Arrestin is involved in the quenching of phototransduction by binding to photoactivated and phosphorylated rhodopsin (P-Rho*). To study its conformational changes and regions interacting with P-Rho*, arrestin was subjected to (1) differential acetylation at lysine residues in the presence and absence of P-Rho*, and (2) amide hydrogen/deuterium exchange. Labeled protein was proteolysed and analyzed by mass spectrometry. Three Lys residues, 28, 176, and 211, were protected from acetylation in native arrestin, although they were not located in regions exhibiting slow amide hydrogen exchange rates. The presence of P-Rho* protected lysine 201 from acetylation and partially protected 14 other lysyl residues, including (2, 5), (163, 166, 167), (232, 235, 236, 238), (267, 276), (298, 300), and 367, where parentheses indicate lysine residues found within the same peptide. In contrast, in the C-terminal region of arrestin, lysyl residues (386, 392, 395) were more exposed upon binding to P-Rho*. These data allowed us to identify functional regions in the arrestin molecule.     

Role of the carboxyl-terminal region of arrestin in binding to phosphorylated rhodopsin.

The structural and functional properties of arrestin were studied by subjecting the protein to limited proteolysis. Limited proteolysis by trypsin cleaves arrestin (48 kDa), producing 20-25-kDa fragments. Prior to this stage of proteolysis, trypsin produced 46.6-, 45.4-, and 42-kDa fragments. Structural analysis of the proteolytic fragments demonstrated major cleavage at the carboxyl terminus, indicating that the carboxyl terminus is highly exposed. We found that forms of arrestin truncated at their carboxyl terminus maintained their functional properties and bound to phosphorylated rhodopsin. Native arrestin binds only to photoexcited phosphorylated rhodopsin, whereas the truncated arrestin binds to phosphorylated rhodopsin independent of its exposure to light. The truncated forms of arrestin were separated from native arrestin by a chromatographic procedure and subsequently characterized in functional studies. The binding of the truncated forms of arrestin to phosphorylated photoexcited rhodopsin is more tight than the binding of native arrestin as determined by a direct binding assay and the phosphodiesterase assay. We suggest that the acidic carboxyl-terminal region of arrestin may act as a regulator for light-dependent binding to phosphorylated rhodopsin.     

Regulation of arrestin binding by rhodopsin phosphorylation level

Arrestins ensure the timely termination of receptor signaling. The role of rhodopsin phosphorylation in visual arrestin binding was established more than 20 years ago, but the effects of the number of receptor-attached phosphates on this interaction remain controversial. Here we use purified rhodopsin fractions with carefully quantified content of individual phosphorylated rhodopsin species to elucidate the impact of phosphorylation level on arrestin interaction with three biologically relevant functional forms of rhodopsin: light-activated and dark phosphorhodopsin and phospho-opsin. We found that a single receptor-attached phosphate does not facilitate arrestin binding, two are necessary to induce high affinity interaction, and three phosphates fully activate arrestin. Higher phosphorylation levels do not increase the stability of arrestin complex with light-activated rhodopsin but enhance its binding to the dark phosphorhodopsin and phospho-opsin. The complex of arrestin with hyperphosphorylated light-activated rhodopsin is less sensitive to high salt and appears to release retinal faster. These data suggest that arrestin likely quenches rhodopsin signaling after the third phosphate is added by rhodopsin kinase. The complex of arrestin with heavily phosphorylated rhodopsin, which appears to form in certain disease states, has distinct characteristics that may contribute to the phenotype of these visual disorders.     

Squid visual arrestin: cDNA cloning and calcium-dependent phosphorylation by rhodopsin kinase (SQRK)

Arrestin binding to rhodopsin is one of the major mechanisms of termination of photoresponses in both vertebrates and invertebrates. Here we report the cDNA cloning and characterization of a 48-kDa visual arrestin from squid (Loligo pealei). The cDNA encoded a protein that had 56-64% amino acid sequence similarity to reported arrestin sequences. This protein does not encode any distinct modular domains but contains five fingerprint regions that have been identified within arrestins. Antibodies raised to the recombinant arrestin protein detected arrestin expression only in the eye and recognized a doublet in photoreceptor membranes, representing unphosphorylated and phosphorylated arrestin. In squid eye membranes, arrestin was phosphorylated in a Ca2+-dependent manner and this phosphorylation was inhibited by antibodies raised against squid rhodopsin kinase, but not by inhibitors of protein kinase C or calmodulin kinase. Addition of purified squid rhodopsin kinase to washed rhabdomeric membranes resulted in phosphorylation of rhodopsin, and arrestin was also phosphorylated when calcium was present. This is the first report of a rhodopsin kinase phosphorylating an arrestin substrate, and suggests a dual role for this kinase in the inactivation of the squid visual system.     

The differential engagement of arrestin surface charges by the various functional forms of the receptor

G-protein-coupled receptor signaling is terminated by arrestin proteins that preferentially bind to the activated phosphorylated form of the receptor. Arrestins also bind active unphosphorylated and inactive phosphorylated receptors. Binding to the non-preferred forms of the receptor is important for visual arrestin translocation in rod photoreceptors and the regulation of receptor signaling and trafficking by non-visual arrestins. Given the importance of arrestin interactions with the various functional forms of the receptor, we performed an extensive analysis of the receptor-binding surface of arrestin using site-directed mutagenesis. The data indicated that a large number of surface charges are important for arrestin interaction with all forms of the receptor. Arrestin elements involved in receptor binding are differentially engaged by the various functional forms of the receptor, each requiring a unique subset of arrestin residues in a specific spatial configuration. We identified several additional phosphate-binding elements in the N-domain and demonstrated for the first time that the active receptor preferentially engages the arrestin C-domain. We also found that the interdomain contact surface is important for arrestin interaction with the non-preferred forms of the receptor and that residues in this region play a role in arrestin transition into its high affinity receptor binding state.     

Identification of regions of arrestin that bind to rhodopsin

Arrestin facilitates phototransduction inactivation through binding to photoactivated and phosphorylated rhodopsin (RP). However, the specific portions of arrestin that bind to RP are not known. In this study, two different approaches were used to determine the regions of arrestin that bind to rhodopsin: panning of phage-displayed arrestin fragments against RP and cGMP phosphodiesterase (PDE) activity inhibition using synthetic arrestin peptides spanning the entire arrestin protein. Phage display indicated the predominant region of binding was contained within amino acids 90-140. A portion of this region (residues 95-140) expressed as a fusion protein with glutathione S-transferase is capable of binding to rhodopsin regardless of the activation or phosphorylation state of the receptor. Within this region, the synthetic peptide of residues 109-130 was shown to completely inhibit the binding of arrestin to rhodopsin with an IC50 of 1.1 mM. The relatively high IC50 of this competition suggests that this portion of the molecule may be only one of several regions of binding between arrestin and RP. A survey of synthetic arrestin peptides in the PDE assay indicated that the two most effective inhibitors of PDE activity were peptides of residues 111-130 and 101-120. These results indicate that at least one of the principal regions of binding between arrestin and RP is contained within the region of residues 109-130.     

Rapid affinity purification of retinal arrestin (48 kDa protein) via its light-dependent binding to phosphorylated rhodopsin

Arrestin (also named '48 kDa protein' or 'S-antigen') is a soluble protein involved in controlling light-dependent cGMP phosphodiesterase activity in retinal rods, and is also known for its ability to induce autoimmune uveitis of the eye. We report a rapid and simple purification method based on the property of arrestin to bind specifically and reversibly to illuminated and phosphorylated rhodopsin [(1984) FEBS Lett. 176, 473-478]. This method does not require column chromatography and yields about 2-4 mg purified arrestin from 15 bovine retinas. Pure arrestin can be resolved by isoelectric focusing into at least 10 distinct bands, all of which stain with a monoclonal antibody specific for S-antigen.     

Conservation of the phosphate-sensitive elements in the arrestin family of proteins.

Arrestins play a key role in the homologous desensitization of G protein-coupled receptors (GPCRs). These cytosolic proteins selectively bind to the agonist-activated and GPCR kinase-phosphorylated forms of the GPCR, precluding its further interaction with the G protein. Certain mutations in visual arrestin yield "constitutively active" proteins that bind with high affinity to the light-activated form of rhodopsin without requiring phosphorylation. The crystal structure of visual arrestin shows that these activating mutations perturb two groups of intramolecular interactions that keep arrestin in its basal (inactive) state. Here we introduced homologous mutations into arrestin2 and arrestin3 and found that the resulting mutants bind to the beta(2)-adrenoreceptor in vitro in a phosphorylation-independent fashion. The same mutants effectively desensitize both the beta(2)-adrenergic and delta-opioid receptors in the absence of receptor phosphorylation in Xenopus oocytes. Moreover, the arrestin mutants also desensitize the truncated delta-opioid receptor from which the C terminus, containing critical phosphorylation sites, has been removed. Conservation of the phosphate-sensitive hot spots in non-visual arrestins suggests that the overall fold is similar to that of visual arrestin and that the mechanisms whereby receptor-attached phosphates drive arrestin transition into the active binding competent state are conserved throughout the arrestin family of proteins.