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.     

Phosphorylation modulates the affinity of light-activated rhodopsin for G protein and arrestin

Reduced effector activity and binding of arrestin are widely accepted consequences of GPCR phosphorylation. However, the effect of receptor multiphosphorylation on G protein activation and arrestin binding parameters has not previously been quantitatively examined. We have found receptor phosphorylation to alter both G protein and arrestin binding constants for light-activated rhodopsin in proportion to phosphorylation stoichiometry. Rod disk membranes containing different average receptor phosphorylation stoichiometries were combined with G protein or arrestin, and titrated with a series of brief light flashes. Binding of G(t) or arrestin to activated rhodopsin augmented the 390 nm MII optical absorption signal by stabilizing MII as MII.G or MII.Arr. The concentration of active arrestin or G(t) and the binding constant of each to MII were determined using a nonlinear least-squares (Simplex) reaction model analysis of the titration data. The binding affinity of phosphorylated MII for G(t) decreased while that for arrestin increased with each added phosphate. G(t) binds more tightly to MII at phosphorylation levels less than or equal to two phosphates per rhodopsin; at higher phosphorylation levels, arrestin binding is favored. However, arrestin was found to bind much more slowly than G(t) at all phosphorylation levels, perhaps allowing time for phosphorylation to gradually reduce receptor-G protein interaction before arrestin capping of rhodopsin. Sensitivity of the binding constants to ionic strength suggests that a strong membrane electrostatic component is involved in both the reduction of G(t) binding and the increase of arrestin binding with increasing rhodopsin phosphorylation.     

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.     

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.     

The arrestin-bound conformation and dynamics of the phosphorylated carboxy-terminal region of rhodopsin

Visual arrestin binds to the phosphorylated carboxy-terminal region of rhodopsin to block interactions with transducin and terminate signaling in the rod photoreceptor cells. A synthetic seven-phospho-peptide from the C-terminal region of rhodopsin, Rh(330-348), has been shown to bind arrestin and mimic inhibition of signal transduction. In this study, we examine conformational changes in this synthetic peptide upon binding to arrestin by high-resolution proton nuclear magnetic resonance (NMR). We show that the peptide is completely disordered in solution, but becomes structured upon binding to arrestin. A control, unphosphorylated peptide that fails to bind to arrestin remains highly disordered. Specific NMR distance constraints are used to model the arrestin-bound conformation. The models suggest that the phosphorylated carboxy-terminal region of rhodopsin, Rh(330-348), undergoes significant conformational changes and becomes structured upon binding to arrestin.     

Interaction of a fragment of the cannabinoid CB1 receptor C-terminus with arrestin-2

Desensitization of the cannabinoid CB1 receptor is mediated by the interaction with arrestin. In this study, we report the structural changes of a synthetic diphosphorylated peptide corresponding to residues 419-439 of the CB1 C-terminus upon binding to arrestin-2. This segment is pivotal to the desensitization of CB1. Using high-resolution proton NMR, we observe two helical segments in the bound peptide that are separated by the presence a glycine residue. The binding we observe is with a diphoshorylated peptide, whereas a previous study reported binding of a highly phosphorylated rhodopsin fragment to visual arrestin. The arrestin bound conformations of the peptides are compared.     

Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits

The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subunit of G(t), are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the gamma-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G(t) (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the alpha subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the alpha subunit of G(t) and plays a role in the regulation of betagamma subunit binding.     

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.     

Characterization of a truncated form of arrestin isolated from bovine rod outer segments.

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48-kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44-kDa form of arrestin, called p44, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C-terminal 35 residues (positions 370-404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, p44 blocks Gt activation. The binding characteristics of p44 set it apart from tryptic forms of arrestin (truncated at the N- and C-termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of Gt activation.     

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.     

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.     

Experimental and computational studies of the desensitization process in the bovine rhodopsin-arrestin complex

The deactivation of the bovine G-protein-coupled receptor, rhodopsin, is a two-step process consisting of the phosphorylation of specific serine and threonine residues in the cytoplasmic tail of rhodopsin by rhodopsin kinase. Subsequent binding of the regulatory protein arrestin follows this phosphorylation. Previous results find that at least three phosphorylatable sites on the rhodopsin tail (T340) and at least two of the S338, S334, or S343 sites are needed for complete arrestin-mediated deactivation. Thus, to elucidate the details of the interaction between rhodopsin with arrestin, we have employed both a computational and an in vitro experimental approach. In this work, we first simulated the interaction of the carboxy tail of rhodopsin with arrestin using a Monte Carlo simulated annealing method. Since at this time phosphorylation of specific serines and threonines is not possible in our simulations, we substitute either aspartic or glutamic acid residues for the negatively charged phosphorylated residues required for binding. A total of 17 simulations were performed and analysis of this shows specific charge-charge interactions of the carboxy tail of rhodopsin with arrestin. We then confirmed these computational results with assays of comparable constructed rhodopsin mutations using our in vitro assay. This dual computational/experimental approach indicates that sites S334, S338, and T340 in rhodopsin and K14 and K15 on arrestin are indeed important in the interaction of rhodopsin with arrestin, with a possible weaker S343 (rhodopsin)/K15 (arrestin) interaction.     

Mechanistic studies on rhodopsin kinase. Light-dependent phosphorylation of C-terminal peptides of rhodopsin.

The phosphorylation of a synthetic peptide, corresponding to the C-terminal 11 amino acids of bovine rhodopsin (VII, residues 338-348), was studied under different conditions. The peptide was only phosphorylated in the presence of photoactivated rhodopsin. Using the same protocol, 12 other peptides, mapping in the rhodopsin C-terminal, were screened for their effectiveness as substrates for rhodopsin kinase. It was found that the peptides became poorer substrates with increasing length, and the best substrates comprised the most C-terminal 9-12 amino acids as opposed to other parts of the C-terminus. It was noted that the absence of the two-terminal residues Pro347 and Ala348 impaired peptide phosphorylation. The effect of the decay of metarhodopsin II on the phosphorylation of rhodopsin and the peptides was determined, and it was found that the rhodopsin and peptide phosphorylations decayed with half times of approximately 33 min and 28 min, respectively. The sites of phosphorylation on the peptides were determined and in all cases the phosphorylation was found to be predominantly on serine residues. Only the 11-residue peptide (VII, residues 338-348) contained significant threonine phosphorylation, which was about 25% that on serine residues. Cumulatively, the results suggest that Ser343 is the preferred site of phosphorylation in vitro. The reason for the poor substrate effectiveness of the larger peptides was examined by competitive experiments in which it was shown that a poorly phosphorylated larger peptide successfully inhibited the phosphorylation of a 'good' peptide substrate. The studies above support a mechanism for rhodopsin kinase that we have termed the 'kinase-activation hypothesis'. This requires that the kinase exists in an inactive form and is activated only after binding to photoactivated rhodopsin.     

Substrate recognition determinants for rhodopsin kinase: studies with synthetic peptides, polyanions, and polycations

Rhodopsin kinase phosphorylates serine- and threonine-containing peptides from bovine rhodopsin's carboxyl-terminal sequence. Km's for the peptides decrease as the length of the peptide is increased over the range 12-31 amino acids, reaching 1.7 mM for peptide 318-348 from the rhodopsin sequence. The Km for phosphorylation of rhodopsin is about 10(3) lower than that for the peptides, which suggests that binding of rhodopsin kinase to its substrate, photolyzed rhodopsin, involves more than just binding to the carboxyl-terminal peptide region that is to be phosphorylated. A synthetic peptide from the rhodopsin sequence that contains both serines and threonines is improved as a substrate by substitution of serines for the threonines, suggesting that serine residues are preferred as substrates. Analogous 25 amino acid peptides from the human red or green cone visual pigment, a beta-adrenergic receptor, or M1 muscarinic acetylcholine receptors are better substrates for bovine rhodopsin kinase than is the peptide from bovine rhodopsin. An acidic serine-containing peptide from a non-receptor protein, alpha s1B-casein, is also a good substrate for rhodopsin kinase. However, many basic peptides that are substrates for other protein kinases--histone IIA, histone IIS, clupeine, salmine, and a neurofilament peptide--are not phosphorylated by rhodopsin kinase. Polycations such as spermine or spermidine are nonessential activators of phosphorylation of rhodopsin or its synthetic peptide 324-348. Polyanions such as poly(aspartic acid), dextran sulfate, or poly(adenylic acid) inhibit the kinase. Poly(L-aspartic acid) is a competitive inhibitor with respect to rhodopsin (KI = 300 microM) and shows mixed type inhibition with respect to ATP.     

Arrestin and its splice variant Arr1-370A (p44). Mechanism and biological role of their interaction with rhodopsin

Deactivation of G-protein-coupled receptors relies on a timely blockade by arrestin. However, under dim light conditions, virtually all arrestin is in the rod inner segment, and the splice variant p(44) (Arr(1-370A)) is the stop protein responsible for receptor deactivation. Using size exclusion chromatography and biophysical assays for membrane-bound protein-protein interaction, membrane binding, and G-protein activation, we have investigated the interactions of Arr(1-370A) and proteolytically truncated Arr(3-367) with rhodopsin. We find that these short arrestins do not only interact with the phosphorylated active receptor but also with inactive phosphorylated rhodopsin or opsin in membranes or solution. Because of the latter interaction they are not soluble (like arrestin) but membrane-bound in the dark. Upon photoexcitation, Arr(3-367) and Arr(1-370A) interact with prephosphorylated rhodopsin faster than arrestin and start to quench G(t) activation on a subsecond time scale. The data indicate that in the course of rhodopsin deactivation, Arr(1-370A) is handed over from inactive to active phosphorylated rhodopsin. This mechanism could provide a new aspect of receptor shutoff in the single photon operating range of the rod cell.     

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.     

N-formyl peptide receptor phosphorylation domains differentially regulate arrestin and agonist affinity

Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide receptor (FPR). In contrast to results with wild type arrestins, the truncated arrestin-2 protein bound to the unphosphorylated wild type FPR, although with lower affinity and a low affinity for the agonist as revealed by competition studies with heterotrimeric G proteins. Using FPR mutants, we further demonstrated that the phosphorylation status of serines and threonines between residues 328-332 is a key determinant that regulates the affinity of the FPR for arrestins. Furthermore, we found that the phosphorylation status of serine and threonine residues between amino acids 334 and 339 regulates the affinity of the receptor for agonist when arrestin is bound. These results suggest that the agonist affinity state of the receptor is principally regulated by phosphorylation at specific sites and is not simply a consequence of arrestin binding as has previously been proposed. Furthermore, this is the first demonstration that agonist affinity of a GPCR and the affinity of arrestin binding to the phosphorylated receptor are regulated by distinct receptor phosphodomains.     

Conformational changes in the phosphorylated C-terminal domain of rhodopsin during rhodopsin arrestin interactions

Phosphorylation of activated G-protein-coupled receptors and the subsequent binding of arrestin mark major molecular events of homologous desensitization. In the visual system, interactions between arrestin and the phosphorylated rhodopsin are pivotal for proper termination of visual signals. By using high resolution proton nuclear magnetic resonance spectroscopy of the phosphorylated C terminus of rhodopsin, represented by a synthetic 7-phosphopolypeptide, we show that the arrestin-bound conformation is a well ordered helix-loop structure connected to rhodopsin via a flexible linker. In a model of the rhodopsin-arrestin complex, the phosphates point in the direction of arrestin and form a continuous negatively charged surface, which is stabilized by a number of positively charged lysine and arginine residues of arrestin. Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin. In conjunction with other binding sites, the helix-loop structure provides a mechanism of shielding phosphates in the center of the rhodopsin-arrestin complex and appears critical in guiding arrestin for high affinity binding with rhodopsin.     

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.     

Binding of arrestin to cytoplasmic loop mutants of bovine rhodopsin

The binding of arrestin to rhodopsin is a multistep process that begins when arrestin interacts with the phosphorylated C terminus of rhodopsin. This interaction appears to induce a conformational change in arrestin that exposes a high-affinity binding site for rhodopsin. Several studies in which synthetic peptides were used have suggested that sites on the rhodopsin cytoplasmic loops are involved in this interaction. However, the precise amino acids on rhodopsin that participate in this interaction are unknown. This study addresses the role of specific amino acids in the cytoplasmic loops of rhodopsin in binding arrestin through the use of site-directed mutagenesis and direct binding assays. A series of alanine mutants within the three cytoplasmic loops of rhodopsin were expressed in HEK-293 cells, reconstituted with 11-cis-retinal, prephosphorylated with rhodopsin kinase, and examined for their ability to bind in vitro-translated, 35S-labeled arrestin. Mutations at Asn-73 in loop I as well as at Pro-142 and Met-143 in loop II resulted in dramatic decreases in the level of arrestin binding, whereas the level of phosphorylation by rhodopsin kinase was similar to that of wild-type rhodopsin. The results indicate that these amino acids play a significant role in arrestin binding.