Activation of bovine rod outer segment phospholipase C by arrestin.

Phospholipase C (PLC) enzyme activity in rod outer segment (ROS) membranes bleached in the presence of ATP and GTP was assayed using exogenously added [3H]phosphatidylinositol 4,5-bisphosphate vesicles as substrate. The addition of the soluble ROS protein arrestin (also known as S-antigen or 48K protein) to ROS membranes activated PLC 2-3.4-fold. This activation was dose-dependent, and maximal activation was observed at an arrestin concentration of congruent to 110-220 nM. PLC activation by arrestin was dependent on ROS protein concentration and free Ca2+. Soluble PLC (s-PLC) enzyme activity present in hypotonic extracts of bleached ROS was also activated 2-4-fold by arrestin. Maximum activation of s-PLC by arrestin was observed at free Ca2+ of 80 nM. Arrestin activation of s-PLC was not affected by urea-treated and extensively washed ROS membranes, suggesting that rhodopsin was not required for the observed effect of arrestin on s-PLC. The results are indicative of a direct interaction of arrestin with s-PLC, resulting in the activation of the latter. Based on these results and the documented binding of arrestin to bleached and phosphorylated rhodopsin, a model for the light activation of PLC in ROS is proposed.     

Dopamine D1 receptor interaction with arrestin3 in neostriatal neurons

Dopamine D1 receptor interactions with arrestins have been characterized using heterologously expressed D1 receptor and arrestins. The purpose of this study was to investigate the interaction of the endogenous D1 receptor with endogenous arrestin2 and 3 in neostriatal neurons. Endogenous arrestin2 and 3 in striatal homogenates bound to the C-terminus of the D1 receptor in a glutathione-S-transferase (GST) pulldown assay, with arrestin3 binding more strongly. The D1 C-terminus and, to a lesser extent, the third cytoplasmic loop also bound purified arrestin2 and 3. In neostriatal neurons, 2, 5, and 20 min agonist treatment increased the colocalization of the D1 receptor and arrestin3 immunoreactivity without altering the colocalization of the D1 receptor and arrestin2. Further, agonist treatment for 5 and 20 min caused translocation of arrestin3, but not arrestin2, to the membrane. The binding of arrestin3, but not arrestin2, to the D1 receptor was increased as assessed by coimmunoprecipitation after agonist treatment for 5 and 20 min. Agonist treatment of neurons induced D1 receptor internalization (35-45%) that was maximal within 2-5 min, a time-course similar to that of the increase in colocalization of the D1 receptor with arrestin3. These data indicate that the D1 receptor preferentially interacts with arrestin3 in neostriatal neurons.     

Arrestin 3 mediates endocytosis of CCR7 following ligation of CCL19 but not CCL21

Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 +/- 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 +/- 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2(-/-)/arrestin 3(-/-) murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.     

Interactions of metarhodopsin II. Arrestin peptides compete with arrestin and transducin

Arrestin blocks the interaction of rhodopsin with the G protein transducin (G(t)). To characterize the sites of arrestin that interact with rhodopsin, we have utilized a spectrophotometric peptide competition assay. It is based on the stabilization of the active intermediates metarhodopsin II (MII) and phosphorylated MII by G(t) and arrestin, respectively (extra MII monitor). The protocol involves native disc membranes and three sets of peptides 10-30 amino acids in length spanning the arrestin sequence. In the absence of arrestin, not one of the peptides by itself had an effect on the amount of MII formed. However, inhibition of arrestin-dependent extra MII was found for the peptides at residues 11-30 and 51-70 (IC(50) < 100 microm) and residues 231-260 (IC(50) < 200 microm). A similar pattern of inhibition by arrestin peptides was seen when arrestin was replaced by G(t) or the farnesylated G(t)gamma C-terminal peptide. Only arrestin-(11-30) inhibited MII.G(t) less (IC(50) = 300 microm) than phosphorylated MII.arrestin. We interpreted the data by competition of the arrestin peptides for interaction sites at rhodopsin, exposed in the MII conformation and specific for both arrestin and G(t). The arrestin sites are located in both the C- and N-terminal domains of the arrestin structure.     

Studies of ligand binding to arrestin

A striking homology is observed between the regions 70-83 and 361-374 of the sequence of bovine arrestin and the calcium-binding loops of calmodulin and troponin C. However, the predicted alpha-helices flanking the calcium-binding site in calmodulin and troponin C are not present in arrestin. Direct measurements therefore were made in order to assess whether arrestin can bind calcium. We found that arrestin does not bind Ca2+ at physiological ionic strength, as determined by equilibrium dialysis, gel filtration, and fluorescence spectroscopy. Rapid and quantitative precipitation of arrestin occurs with Tb3+. The precipitation is reversed by EDTA and blocked by Mg2+ but not by Ca2+. Prompted by several reports, we also investigated whether nucleotides bind to arrestin. Neither ATP nor GTP binds under the conditions tested. Binding of arrestin to photolyzed, phosphorylated rhodopsin also does not influence the binding of calcium or nucleotides.     

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.     

Evidence for ATP-ase activity of arrestin from bovine photoreceptors.

In vertebrate photoreceptors the soluble protein arrestin (45 kDa) is involved in controlling the light dependent activity of receptor proteins such as transducin or the cGMP-phosphodiesterase. Arrestin has further been identified as the retinal-S-antigen which is assumed to cause the autoimmune disease uveitis. In a first communication a binding of the nucleotide ATP to arrestin was described. In this subsequent study it is shown that arrestin is also able to hydrolyse ATP at a rate of (5.1 +/- 0.3) x 10(-3) U/mg.min with C1/2 = 93 +/- 5 nM and a Hill coefficient n = 1.8 +/- 0.1 at pH 7.2 and 20 degrees C. These findings suggest a new insight into the process of regulating photoreceptor activity.     

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.     

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.     

Role of ßarrestins in bradykinin B2 receptor-mediated signalling

G protein-coupled receptors (GPCRs) can engage multiple pathways to activate ERK1/2 via both G proteins and/or ßarrestin. Receptor recruitment of ßarrestin is also important for GPCR desensitization, internalization and resensitization. Modulation of the receptor/ßarrestin interaction through modification of either component would presumably alter the output generated by receptor activation. Here we examined how ßarrestins regulate bradykinin (BK) B2 receptor (B2R) signalling and desensitization by either truncating ßarrestin1 or ßarrestin2 or by alanine substitution of a serine/threonine cluster in the C-terminal tail of B2R (B2R-4A), conditions which all affect the avidity of the B2R/ßarrestin complex. We first demonstrate that BK-mediated ERK1/2 activation is biphasic containing an early peak (between 2-5 min) followed by sustained activation for at least 60 min. The early but not the sustained phase was predictably affected by inhibition of either Gαq/11 or Gαi/o, whereas loss of ßarrestin2 but not ßarrestin1 resulted in diminished prolonged ERK1/2 activation. ßarrestin2's role was further examined using a truncation mutant with augmented avidity for the agonist-occupied receptor, revealing an increase in both immediate and extended ERK1/2 signalling. We also show that ERK1/2 is recruited to the B2R/ßarrestin complex on endosomes as well as the plasma membrane. Moreover, we investigated ßarrestin's role using the B2R-4A, which is deficient in ßarrestin binding and does not internalize. We show that ERK1/2 signalling downstream of the receptor is entirely G protein-dependent and receptor-mediated intracellular calcium mobilization studies revealed a lack of desensitization. Functionally, the lack of desensitization resulted in increased cell growth and migration compared to the wild-type receptor, which was sensitive to MEK inhibition. These results highlight ßarrestin's crucial role in the maintenance of proper B2R signalling.     

Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes

AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (gamma-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappa-mediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism.     

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.     

The solution structure and activation of visual arrestin studied by small-angle X-ray scattering.

Visual arrestin is converted from a 'basal' state to an 'activated' state by interaction with the phosphorylated C-terminus of photoactivated rhodopsin (R*), but the conformational changes in arrestin that lead to activation are unknown. Small-angle X-ray scattering (SAXS) was used to investigate the solution structure of arrestin and characterize changes attendant upon activation. Wild-type arrestin forms dimers with a dissociation constant of 60 micro m. Small conformational changes, consistent with local movements of loops or the mobile N- or C-termini of arrestin, were observed in the presence of a phosphopeptide corresponding to the C-terminus of rhodopsin, and with an R175Q mutant. Because both the phosphopeptide and the R175Q mutation promote binding to unphosphorylated R*, we conclude that arrestin is activated by subtle conformational changes. Most of the arrestin will be in a dimeric state in vivo. Using the arrestin structure as a guide [Hirsch, J.A., Schubert, C., Gurevich, V.V. & Sigler, P.B. (1999) Cell 97, 257-269], we have identified a model for the arrestin dimer that is consistent with our SAXS data. In this model, dimerization is mediated by the C-terminal domain of arrestin, leaving the N-terminal domains free for interaction with phosphorylated R*.     

Structural properties of arrestin studied by chemical modification and circular dichroism.

A unique conformation of arrestin is crucial for its interaction with phosphorylated photolyzed rhodopsin. Conformational changes in arrestin were investigated using chemical modification and circular dichroism. We studied the kinetics of sulfhydryl modification of bovine arrestin in order to determine whether its conformation is altered by the presence of ligands or salts at different ionic strengths. We found that all three cysteines (stoichiometry was 2.7 +/- 0.06 3-carboxy-4-nitrophenyl sulfide (NbS)/arrestin) are accessible for modification by NbS2. Under pseudo-first-order conditions (30-100-fold excess of NbS2 over arrestin), the modifications of the 3 cysteines are indistinguishable. At higher concentrations of NbS2 (150-300-fold excess), the pseudo-first-order plot is not linear, and the reaction can be resolved into two processes that involve two classes of sulfhydryl groups. Addition of CaCl2, MgCl2, inorganic phosphate, MgATP, or MgGTP had little effect on the rate of modification of the cysteine residues; however, heparin and inositol hexakisphosphate, which have been shown to induce conformational changes in arrestin, block modification of one sulfhydryl group of arrestin and accelerate the modification of the remaining two. Analysis of CD spectra revealed that arrestin has virtually no alpha-helical structure, about 40% beta-structure, about 18% beta-turns, and about 40% other structure. The CD spectrum for arrestin did not change in the presence of heparin. These studies suggest that arrestin exists in equilibrium between two or more conformational states. However, it is proposed that conversion between these conformations occur without altering significantly the secondary structure of arrestin.     

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.     

Arrestin2 expression selectively increases during neural differentiation

Arrestins and G protein-coupled receptor kinases (GRKs) are key players in homologous desensitization of G protein-coupled receptors. Two non-visual arrestins, arrestin2 and 3, and five GRKs (GRK2, 3, 4, 5 and 6) are involved in desensitization of many receptors. Here, we demonstrate a steady increase in arrestin2 expression during prenatal development. The density of arrestin2 mRNA is higher in differentiated areas as compared with proliferative zones, whereas arrestin3 mRNA shows the opposite distribution. At embryonic day 14, concentrations of arrestin proteins are similar (32-34 nM). Later in development, arrestin2 expression rises, leading to a fourfold excess of arrestin2 over arrestin3 at birth (48 vs. 11 ng/mg protein or 102 vs. 25 nM). Among GRKs, only GRK5 increased with embryonic age from 124 nm at E14 to 359 nM at birth. Similarly, in vitro differentiation of cultured precursor cells, neurospheres, leads to a significant up-regulation of arrestin2 resulting in > 20-fold excess of arrestin2 (160 vs. 7 nM). GRK5 is the only subtype increased with neurosphere differentiation, although the change is only about twofold. The data demonstrate selective increases in the expression of arrestin2 associated with neural development and suggest specific yet unappreciated roles for arrestin2 in neural differentiation.     

Subspecies of arrestin from bovine retina. Equal functional binding to photoexcited rhodopsin but various isoelectric focusing phenotypes in individuals

Arrestin (also named 48-kDa protein or S-antigen) binds to photoexcited and phosphorylated rhodopsin and thereby prevents activation of cGMP phosphodiesterase (EC 3.1.4.35) by transducin in retinal rods. We report here that retinal arrestin consists of several subspecies (isoelectric points between pH 5.5-6.2), which can be separated by FPLC anion-exchange chromatography and by FPLC chromatofocusing resulting in highly enriched individual subspecies. The entire heterogeneity pattern of arrestin is present in rod outer segments, independently of whether arrestin orginated from the outer or mostly from the inner segment of rod cells. The different subspecies show a similar binding behavior to photoexcited rhodopsin phosphorylated to various degrees and they quench the cGMP phosphodiesterase activity equally well. In the presence of rod outer segment membranes, arrestin is phosphorylated light-dependently by protein kinase C (0.2 mol phosphate/mol arrestin). This implies that the heterogeneity of arrestin is not primarily due to phosphorylation. Arrestin from different individuals exists as four isoelectric focusing patterns which occur with remarkably different frequencies in calf and cattle. The complexity of the IEF pattern does not increase with aging. Distinct subspecies of arrestin may reflect differences in their primary structure, or may result from differentially regulated post-translational modifications in individuals.     

Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein-protein interactions

In rod photoreceptors, arrestin localizes to the outer segment (OS) in the light and to the inner segment (IS) in the dark. Here, we demonstrate that redistribution of arrestin between these compartments can proceed in ATP-depleted photoreceptors. Translocation of transducin from the IS to the OS also does not require energy, but depletion of ATP or GTP inhibits its reverse movement. A sustained presence of activated rhodopsin is required for sequestering arrestin in the OS, and the rate of arrestin relocalization to the OS is determined by the amount and the phosphorylation status of photolyzed rhodopsin. Interaction of arrestin with microtubules is increased in the dark. Mutations that enhance arrestin-microtubule binding attenuate arrestin translocation to the OS. These results indicate that the distribution of arrestin in rods is controlled by its dynamic interactions with rhodopsin in the OS and microtubules in the IS and that its movement occurs by simple diffusion.     

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.