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.     

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.     

Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins

Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements.     

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.     

Cell-free expression of visual arrestin. Truncation mutagenesis identifies multiple domains involved in rhodopsin interaction.

Visual arrestin plays an important role in regulating light responsiveness via its ability to specifically bind to the phosphorylated and light-activated form of rhodopsin. To further characterize rhodopsin/arrestin interactions we have utilized a rabbit reticulocyte lysate translation system to synthesize bovine visual arrestin. The translated arrestin (404 amino acids) was demonstrated to be fully functional in terms of its ability to specifically recognize and bind to phosphorylated light-activated rhodopsin (P-Rh*). Competitive binding studies revealed that the in vitro synthesized arrestin and purified bovine visual arrestin had comparable affinities for P-Rh*. In an effort to assess the functional role of different regions of the arrestin molecule, two truncated arrestin mutants were produced by cutting within the open reading frame of the bovine arrestin cDNA with selective restriction enzymes. In vitro translation of the transcribed truncated mRNAs resulted in the production of arrestins truncated from the carboxyl terminus. The ability of each of the mutant arrestins to bind to dark (Rh), light-activated (Rh*), dark phosphorylated (P-Rh), and light-activated phosphorylated rhodopsin were then compared. Arrestin lacking 39 carboxyl-terminal residues binds specifically not only to P-Rh* but also to Rh* and P-Rh. This suggests that the carboxyl-terminal domain of arrestin plays an important regulatory role in ensuring strict arrestin binding selectivity to P-Rh*. Arrestin that has only the first 191 amino-terminal residues predominately discriminates the phosphorylation state of the rhodopsin; however, it also retains some binding specificity for the activation state. These results suggest that the amino-terminal half of arrestin contains key rhodopsin recognition sites responsible for interaction with both the phosphorylated and light-activated forms of rhodopsin.     

Conformational differences between arrestin2 and pre-activated mutants as revealed by hydrogen exchange mass spectrometry

Arrestins are regulatory proteins that bind specifically to ligand-activated phosphorylated G protein-coupled receptors to terminate G protein-mediated signaling, cause the internalization of the receptor-arrestin complex, and initiate additional intracellular signaling cascades. Multiple lines of evidence suggest that arrestin normally exists in an inactive basal state and undergoes conformational activation in the process of receptor binding. "Pre-activated" phosphorylation-independent arrestin mutants display increased binding to ligand-activated but unphosphorylated receptors. The mutations are believed to expose key receptor-binding regions, allowing the mutants to mimic, to some extent, the transition of arrestin to its active state. In the present study, amide hydrogen exchange (HX) and mass spectrometry (MS) were used to examine the inactive conformation of wild-type arrestin2 and compare its solution conformation with two pre-activated mutants (R169E and 3A (I385A, V386A, F387A)). The results suggest an unexpected level of structural organization within arrestin elements containing clathrin and adaptin2-binding sites that were previously believed to be completely disordered. Increased deuterium incorporation was observed in both mutant forms compared with wild-type, indicating a change in the conformation of the mutants. Three regions demonstrated significant differences in deuterium incorporation: the first 33 residues of the N terminus and residues 243-255 (both previously implicated in receptor interaction), and residues 271-299. The results suggest that subtle differences in conformation are responsible for the significant difference in biological activity displayed by pre-activated arrestin mutants and that similar changes occur in the process of arrestin binding to the receptor.     

The Role of Arrestin α-Helix I in Receptor Binding

Arrestins rapidly bind phosphorylated activated forms of their cognate G protein-coupled receptors, thereby preventing G protein coupling and often switching signaling to other pathways. Amphipathic α-helix I (residues 100-111) has been implicated in receptor binding, but the mechanism of its action has not been determined yet. Here we show that several mutations in the helix itself and in adjacent hydrophobic residues in the body of the N-domain reduce arrestin1 binding to light-activated phosphorylated rhodopsin (P-Rh^?). On the background of phosphorylation-independent mutants that bind with high affinity to both P-Rh^? and light-activated unphosphorylated rhodopsin, these mutations reduce the stability of the arrestin complex with P-Rh^?, but not with light-activated unphosphorylated rhodopsin. Using site-directed spin labeling, we found that the local structure around α-helix I changes upon binding to rhodopsin. However, the intramolecular distances between α-helix I and adjacent β-strand I (or the rest of the N-domain), measured using double electron-electron resonance, do not change, ruling out relocation of the helix due to receptor binding. Collectively, these data demonstrate that α-helix I plays an indirect role in receptor binding, likely keeping β-strand I, which carries several phosphate-binding residues, in a position favorable for its interaction with receptor-attached phosphates.     

Elucidation of inositol hexaphosphate and heparin interaction sites and conformational changes in arrestin-1 by solution nuclear magnetic resonance

Arrestins specifically bind activated and phosphorylated G protein-coupled receptors and orchestrate both receptor trafficking and channel signaling through G protein-independent pathways via direct interactions with numerous nonreceptor partners. Here we report the first successful use of solution NMR in mapping the binding sites in arrestin-1 (visual arrestin) for two polyanionic compounds that mimic phosphorylated light-activated rhodopsin: inositol hexaphosphate (IP6) and heparin. This yielded an identification of residues involved in the binding with these ligands that was more complete than what has previously been feasible. IP6 and heparin appear to bind to the same site on arrestin-1, centered on a positively charged region in the N-domain. We present the first direct evidence that both IP6 and heparin induced a complete release of the arrestin C-tail. These observations provide novel insight into the nature of the transition of arrestin from the basal to active state and demonstrate the potential of NMR-based methods in the study of protein-protein interactions involving members of the arrestin family.     

The interaction with the cytoplasmic loops of rhodopsin plays a crucial role in arrestin activation and binding

The binding of arrestin to rhodopsin is initiated by the interaction of arrestin with the phosphorylated rhodopsin C-terminus and/or the cytoplasmic loops, followed by conformational changes that expose an additional high-affinity site on arrestin. Here we use an arrestin mutant (R175E) that binds similarly to phosphorylated and unphosphorylated, wild-type rhodopsin to identify rhodopsin elements other than C-terminus important for arrestin interaction. R175E-arrestin demonstrated greatly reduced binding to unphosphorylated cytoplasmic loop mutants L72A, N73A, P142A and M143A, suggesting that these residues are crucial for high-affinity binding. Interestingly, when these rhodopsin mutants are phosphorylated, R175E-arrestin binding is less severely affected. This effect of phosphorylation on R175E-arrestin binding highlights the co-operative nature of the multi-site interaction between arrestin and the cytoplasmic loops and C-terminus of rhodopsin. However, a combination of any two mutations disrupts the ability of phosphorylation to enhance binding of R175E-arrestin. N73A, P142A and M143A exhibited accelerated rates of dissociation from wild-type arrestin. Using sensitivity to calpain II as an assay, these cytoplasmic loop mutants also demonstrated reduced ability to induce conformational changes in arrestin that correlated with their reduced ability to bind arrestin. These results suggest that arrestin bound to rhodopsin is in a distinct conformation that is co-ordinately regulated by association with the cytoplasmic loops and the C-terminus of rhodopsin.     

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.     

Cone arrestin binding to JNK3 and Mdm2: conformational preference and localization of interaction sites

Arrestins are multi-functional regulators of G protein-coupled receptors. Receptor-bound arrestins interact with >30 remarkably diverse proteins and redirect the signaling to G protein-independent pathways. The functions of free arrestins are poorly understood, and the interaction sites of the non-receptor arrestin partners are largely unknown. In this study, we show that cone arrestin, the least studied member of the family, binds c-Jun N-terminal kinase (JNK3) and Mdm2 and regulates their subcellular distribution. Using arrestin mutants with increased or reduced structural flexibility, we demonstrate that arrestin in all conformations binds JNK3 comparably, whereas Mdm2 preferentially binds cone arrestin 'frozen' in the basal state. To localize the interaction sites, we expressed separate N- and C-domains of cone and rod arrestins and found that individual domains bind JNK3 and remove it from the nucleus as efficiently as full-length proteins. Thus, the arrestin binding site for JNK3 includes elements in both domains with the affinity of partial sites on individual domains sufficient for JNK3 relocalization. N-domain of rod arrestin binds Mdm2, which localizes its main interaction site to this region. Comparable binding of JNK3 and Mdm2 to four arrestin subtypes allowed us to identify conserved residues likely involved in these interactions.     

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.     

Formation and Decay of the Arrestin·Rhodopsin Complex in Native Disc Membranes

In the G protein-coupled receptor rhodopsin, light-induced cis/trans isomerization of the retinal ligand triggers a series of distinct receptor states culminating in the active Metarhodopsin II (Meta II) state, which binds and activates the G protein transducin (G_t). Long before Meta II decays into the aporeceptor opsin and free all-trans-retinal, its signaling is quenched by receptor phosphorylation and binding of the protein arrestin-1, which blocks further access of G_t to Meta II. Although recent crystal structures of arrestin indicate how it might look in a precomplex with the phosphorylated receptor, the transition into the high affinity complex is not understood. Here we applied Fourier transform infrared spectroscopy to monitor the interaction of arrestin-1 and phosphorylated rhodopsin in native disc membranes. By isolating the unique infrared signature of arrestin binding, we directly observed the structural alterations in both reaction partners. In the high affinity complex, rhodopsin adopts a structure similar to G_t-bound Meta II. In arrestin, a modest loss of β-sheet structure indicates an increase in flexibility but is inconsistent with a large scale structural change. During Meta II decay, the arrestin-rhodopsin stoichiometry shifts from 1:1 to 1:2. Arrestin stabilizes half of the receptor population in a specific Meta II protein conformation, whereas the other half decays to inactive opsin. Altogether these results illustrate the distinct binding modes used by arrestin to interact with different functional forms of the receptor.     

Arrestin-rhodopsin binding stoichiometry in isolated rod outer segment membranes depends on the percentage of activated receptors

In the rod cell of the retina, arrestin is responsible for blocking signaling of the G-protein-coupled receptor rhodopsin. The general visual signal transduction model implies that arrestin must be able to interact with a single light-activated, phosphorylated rhodopsin molecule (Rho*P), as would be generated at physiologically relevant low light levels. However, the elongated bi-lobed structure of arrestin suggests that it might be able to accommodate two rhodopsin molecules. In this study, we directly addressed the question of binding stoichiometry by quantifying arrestin binding to Rho*P in isolated rod outer segment membranes. We manipulated the "photoactivation density," i.e. the percentage of active receptors in the membrane, with the use of a light flash or by partially regenerating membranes containing phosphorylated opsin with 11-cis-retinal. Curiously, we found that the apparent arrestin-Rho*P binding stoichiometry was linearly dependent on the photoactivation density, with one-to-one binding at low photoactivation density and one-to-two binding at high photoactivation density. We also observed that, irrespective of the photoactivation density, a single arrestin molecule was able to stabilize the active metarhodopsin II conformation of only a single Rho*P. We hypothesize that, although arrestin requires at least a single Rho*P to bind the membrane, a single arrestin can actually interact with a pair of receptors. The ability of arrestin to interact with heterogeneous receptor pairs composed of two different photo-intermediate states would be well suited to the rod cell, which functions at low light intensity but is routinely exposed to several orders of magnitude more light.     

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.     

A Novel Polar Core and Weakly Fixed C-Tail in Squid Arrestin Provide New Insight into Interaction with Rhodopsin

Photoreceptors of the squid Loligo pealei contain a G-protein-coupled receptor (GPCR) signaling system that activates phospholipase C in response to light. Analogous to the mammalian visual system, signaling of the photoactivated GPCR rhodopsin is terminated by binding of squid arrestin (sArr). sArr forms a light-dependent, high-affinity complex with squid rhodopsin, which does not require prior receptor phosphorylation for interaction. This is at odds with classical mammalian GPCR desensitization where an agonist-bound phosphorylated receptor is needed to break stabilizing constraints within arrestins, the so-called “three-element interaction” and “polar core” network, before a stable receptor–arrestin complex can be established. Biophysical and mass spectrometric analysis of the squid rhodopsin–arrestin complex indicates that in contrast to mammalian arrestins, the sArr C-tail is not involved in a stable three-element interaction. We determined the crystal structure of C-terminally truncated sArr that adopts a basal conformation common to arrestins and is stabilized by a series of weak but novel polar core interactions. Unlike mammalian arrestin-1, deletion of the sArr C-tail does not influence kinetic properties of complex formation of sArr with the receptor. Hydrogen–deuterium exchange studies revealed the footprint of the light-activated rhodopsin on sArr. Furthermore, double electron–electron resonance spectroscopy experiments provide evidence that receptor-bound sArr adopts a conformation different from the one known for arrestin-1 and molecular dynamics simulations reveal the residues that account for the weak three-element interaction. Insights gleaned from studying this system add to our general understanding of GPCR–arrestin interaction.     

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.     

Aspartic acid 564 in the third cytoplasmic loop of the luteinizing hormone/choriogonadotropin receptor is crucial for phosphorylation-independent interaction with arrestin2

Arrestin2 binding to the active but unphosphorylated luteinizing hormone/choriogonadotropin receptor (LH/CG R) in ovarian follicles is triggered by activation of ADP-ribosylation factor 6 (ARF6) and leads to uncoupling of this receptor from cAMP signaling. We sought to determine how arrestin2 binds to LH/CG R, if binding is of high affinity, and if the receptor also binds arrestin3. Desensitization of intact LH/CG R was equally sensitive to ectopic constructs of arrestin2 that bind other G protein-coupled receptors (GPCRs) either in a phosphorylation-independent or -dependent manner. Intact LH/CG R was not desensitized by ectopic arrestin3 constructs. Surface plasmon resonance studies showed that arrestin2 bound a synthetic third intracellular (3i) LH/CG R loop peptide with picomolar affinity; arrestin3 bound with millimolar affinity. To determine whether Asp-564 in the 3i loop mimicked the phosphorylated residue of other GPCRs, human embryonic kidney (HEK) cells were transfected with wild-type (WT) and D564G LH/CG R. An agonist-stimulated ARF6-dependent arrestin2 undocking pathway to drive desensitization of WT receptor was recapitulated in HEK cell membranes, and ectopic arrestin2 promoted desensitization of WT LH/CG R. However, D564G LH/CG R in HEK cells was not desensitized, and synthetic 3i D564G peptide did not bind arrestin2. Synthetic 3i loop peptides containing D564E, D564V, or D564N also did not bind arrestin2. We conclude that the ARF6-mediated mechanism to release a pool of membrane-delimited arrestin to bind GPCRs may be a widespread mechanism to deliver arrestin to GPCRs for receptor desensitization. Unlike other GPCRs that additionally require receptor phosphorylation, LH/CG R activation is sufficient to expose a conformation in which Asp-564 in the 3i loop confers high affinity binding selectively to arrestin2.     

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.     

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.