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

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

Ca2+ binding capacity of cytoplasmic proteins from rod photoreceptors is mainly due to arrestin

Arrestin (also called S-antigen or 48-kDa protein) binds to photoexcited and phosphorylated rhodopsin and, thereby, blocks competitively the activation of transducin. Using Ca2+ titration in the presence of the indicator arsenazo III and 45Ca2+ autoradiography, we show that arrestin is a Ca2(+)-binding protein. The Ca2+ binding capacity of arresting-containing protein extracts from bovine rod outer segments is about twice as high as that of arrestin-depleted extracts. The difference in the Ca2+ binding of arrestin-containing and arrestin-depleted protein extracts was attributed to arrestin. Both, these difference-measurements of protein extracts and the measurements of purified arrestin yield dissociation constants for the Ca2+ binding of arrestin between 2 and 4 microM. The titration curves are consistent with a molar ratio of one Ca2+ binding site per arrestin. No Ca2+ binding in the micromolar range was found in extracts containing mainly transducin and cGMP-phosphodiesterase. Since arrestin is one of the most abundant proteins in rod photoreceptors occurring presumably up to millimolar concentrations in rod outer segments, we suggest that aside from its function to prevent the activation of transducin, arrestin acts probably as an intracellular Ca2+ buffer.     

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.     

Reduced rhodopsin phosphorylation during retinal dystrophy

During inherited retinal dystrophy in Irish Setter dogs, decreased activity of cGMP phosphodiesterase (PDE) results in high cGMP levels and retinal degeneration (1-3). This defect could be in PDE itself, or in its interactions with other proteins of the rod outer segment. We report herein that when retinas from 8-week-old dogs were phosphorylated with gamma-32P-ATP, and separated on SDS-PAGE, phosphorylation of rd dog rhodopsin was reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Since rd-mediated inhibition was prevented by 1 mM NaF, the results suggest that the cause of reduced rd phosphorylation is increased phosphatase activity. Together, these results demonstrate that decreased phosphorylation of rhodopsin due to increased phosphatase activity is a fundamental biochemical change which may partially account for the degenerative process and loss of visual acuity during inherited retinal dystrophy.     

Activation of phosphodiesterase by rhodopsin and its analogues

Activation of guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (EC 3.1.4.35.) in frog rod outer segment membrane by rhodopsin and its analogues was investigated. The Schiff-base linkage between opsin and retinal in rhodopsin was not always necessary for the phosphodiesterase activation. The binding of beta-ionone ring of retinal to a hydrophobic region of opsin was not enough to induce the enzyme activation. A striking photo-activation of the enzyme was induced by photo-isomerization of rhodopsin analogues from cis to trans form. It seems probable that an "expanded" conformation of opsin around the retinylidene chromophore induced by the cis to trans isomerization may be the trigger for the activation of phosphodiesterase. On the other hand, the phosphodiesterase in frog rod outer segment was activated by warming of bathorhodopsin to -12 degrees C and then incubating it at the same temperature. Thus, metarhodopsin II or an earlier intermediate than metarhodopsin II should be a direct intermediate for the enzyme activation.     

Regulation of rhodopsin dephosphorylation by arrestin

We have characterized the opsin phosphatase activities in extracts of rod outer segments and determined their relationship to known protein phosphatases. The opsin phosphatase activity in the extracts was not due to protein phosphatases 1, 2B, or 2C because it was neither stimulated by Mg2+ or Ca2+/calmodulin nor inhibited by protein phosphatase inhibitors-1 or -2. Opsin phosphatase activity in rod outer segment extracts was potently inhibited by okadaic acid (IC50 approximately 10 nM), a preferential inhibitor of protein phosphatase 2A. Moreover, during chromatography on DEAE-Sepharose, the opsin phosphatase activity co-eluted with three peaks of protein phosphatase 2A activity, termed protein phosphatases 2A0, 2A1, and 2A2. The opsin phosphatase activity of each peak was stimulated by polylysine, a known activator of protein phosphatase 2A. Finally, treatment of rod outer segment extracts with 80% ethanol at room temperature converted the activity from a high molecular weight form characteristic of the protein phosphatase 2A0, 2A1, and 2A2 species to a low molecular weight form characteristic of the protein phosphatase 2A catalytic subunit. We conclude that protein phosphatase 2A is likely to be the physiologically relevant rhodopsin phosphatase. The 48-kDa rod outer segment protein arrestin (S-antigen) was found to inhibit the dephosphorylation of freshly photolyzed rhodopsin by protein phosphatase 2A but did not inhibit the dephosphorylation of unbleached rhodopsin. Arrestin has no effect on the dephosphorylation of phorphorylase a, indicating that the effect was substrate-directed. It appears that dephosphorylation of the photoreceptor protein phosphorhodopsin occurs only after decay of the photoactivated protein and that this may be regulated in vivo by arrestin. The binding of arrestin to photolyzed phosphorylated rhodopsin, i.e. the binding of a regulatory protein to a protein phosphatase substrate to form a complex resistant to dephosphorylation represents a novel mechanism for the regulation of protein phosphatase 2A.     

Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments.

Frog (Rana catesbiana) rod outer segment disc membranes contain a cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which is activated by light in the presence of ATP. This enzyme is firmly bound to the disc membrane, but can be eluted from the membrane with 10 mM Tris-HCl buffer, pH 7.4 and 2 mM EDTA. The eluted phosphodiesterase has reduced activity, but can be activated approximately 10-fold by polycations such as protamine and polylysine. The eluted phosphodiesterase can no longer be activated by light in the presence of ATP, that is, activation by light apparently depends on the native orientation of phosphodiesterase in relationship to other disc membrane components. The eluted phosphodiesterase was purified to homogeneity as judged by analytical polyacrylamide gel electrophoresis and polyacrylamide gel isoelectric focusing. The over-all purification from intact retina was approximately 925-fold. The purification of phosphodiesterase from the isolated rod outer segment preparation was about 185-fold with a 28% yield. Phosphodiesterase accounts for approximately 0.5% of the disc membrane protein. The eluted phosphodiesterase (inactive form) has a sedimentation coefficient of 12.4 S corresponding to an approximate molecular weight of 240,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates the purified phosphodiesterase into two subunits of 120,000 and 110,000 daltons. With cyclic 3':5'-GMP (cGMP) as substrate the Km for the purified phosphodiesterase is 70 muM. Protamine increases the Vmax without changing the Km for cGMP. The isoelectric point (pI) of the native dimer is 5.7. Limited exposure of the eluted phosphodiesterase (inactive form) to trypsin produces a somewhat greater activation than is obtained with 0.5 mg/ml of protamine. The trypsin-activated phosphodiesterase has a sedimentation coefficient of 7.8 S corresponding to an approximate molecular weight of 170,000. The 110,000-dalton subunit is much less sensitive to trypsin hydrolysis and the 120,000-dalton subunit is rapidly replaced by smaller fragments. On the basis of the molecular weight of the purified phosphodiesterase (240,000) and the concentrations of phosphodiesterase and rhodopsin in the rod outer segment, it is estimated that the molar ratio ophosphodiesterase to rhodopsin in the rod outer segment is approximately 1:900. Since all of the disc phosphodiesterase molecules are activated when 0.1% of the rhodopsins are bleached, we conclude that in the presence of ATP 1 molecule of bleached rhodopsin can activate 1 molecule of phosphodiesterase.     

Binding of inositol phosphates to arrestin.

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

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.     

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.     

Inactivation of photoexcited rhodopsin in retinal rods: the roles of rhodopsin kinase and 48-kDa protein (arrestin)

The inactivation of excited rhodopsin in the presence of ATP, rhodopsin kinase, and/or arrestin has been studied from its effect on the two subsequent steps in the light-induced enzymatic cascade: metarhodopsin II catalyzed activation of G-protein and G-protein-dependent activation of cGMP phosphodiesterase. The inactivation of G-protein (from light-scattering measurements) and that of phosphodiesterase (from measurements of cGMP hydrolysis) have been studied and compared in reconstituted systems containing various combinations of the proteins involved (rhodopsin, G-protein, phosphodiesterase, kinase, and arrestin). Our results show that rhodopsin kinase alone can terminate the activation of G-protein and that arrestin speeds up the process at a relative concentration similar to that reported in the rod (half-maximal effect at 50 nM for 4.4 microM rhodopsin). Measurements of rhodopsin phosphorylation under identical conditions show that in the presence of arrestin total metarhodopsin II inactivation is achieved when only 0.5-1.4 phosphates are bound per bleached rhodopsin, whereas in the absence of arrestin it requires binding of 12-16 phosphates per bleached rhodopsin. Phosphodiesterase activity can similarly be turned off by kinase, and the process is similarly accelerated by arrestin.     

Light-stimulated protein movement in rod photoreceptor cells of the rat retina

We examined the intracellular distribution of three proteins involved in the cyclic GMP cascade of visual transduction; cGMP phosphodiesterase, the alpha-subunit of G-protein and arrestin. In adult rats, light-induced changes in the amounts of G and arrestin in the photoreceptor cell outer segments were observed both by polyacrylamide gel analysis of purified ROS and by immunocytochemical localization on retinal sections. In dark conditions, G was concentrated in the outer segments of photoreceptor cells while in the light G alpha was seen in the inner segments and the outer nuclear layer. Arrestin had the opposite distribution, appearing in the inner segments and outer nuclear layer under dark conditions and in the ROS under light conditions. In contrast, PDE, the enzyme which is activated by G and inhibited by arrestin showed no light-stimulated movement. In both light- and dark-adapted retinas, PDE was localized primarily in the outer segments of the photoreceptor cells.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. I.

Guanosine 3′,5′-cyclic monophosphate phosphodiesterase (EC 3.1.4.1) in frog rod outer segment prepared by a sucrose stepwise density gradient method was activated by light in the presence of GTP. Rhodopsin in rod outer segment was solubilized with sucrose laurylmonoester and then purified by concavanalin A-Sepharose column. Addition of photo-bleached preparation of the purified rhodopsin to the rod outer segment, which had been prepared by 43% (w/w) sucrose floatation, caused the activation of phosphodiesterase in the dark, while each component of the photo-product eluted from the column (all-trans retinal and opsin) did not. Regenerated rhodopsin prepared from 11-cis retinal and purified opsin activated phosphosdiesterase when it was bleached. From these facts it is suggested that an intermediate or a process of photolysis of rhodopsin causes activation of phosphodiesterase.     

Visual transduction in rod outer segments

The visual transduction cascade of the retinal rod outer segment responds to light by decreasing membrane current. This ion channel is controlled by cyclic GMP which is, in turn, controlled by its synthesis and degradation by guanylate cyclase and phosphodiesterase, respectively. When light bleaches rhodopsin there is an induced exchange of GTP for GDP bound to the alpha subunit of the retinal G-protein, transducin (T). The T alpha.GTP then removes the inhibitory constraint of a small inhibitory subunit (PDE gamma) on the retinal cGMP phosphodiesterase (PDE). This results in activation of the PDE and in hydrolysis of cGMP. Recently both low and high affinity binding sites have been identified for PDE gamma on the PDE alpha/beta catalytic subunits. The discovery of two PDE gamma subunits, each with different binding affinities, suggests that a tightly regulated shut-off mechanism may be present.     

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.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. II

Frog (Rana catesbeiana) rod outer segment membrane contains cyclic GMP phosphodiesterase (EC 3.1.4.1). Irradiation of dark-adapted rod outer segment membrane increased the enzyme activity by 5–20-fold in the presence of GTP. The phosphodiesterase in rod outer segment membrane is also activated by mixing a photo-product of 11-cis (regenerated), 9-cis or 7-cis rhodopsin which is stable at 0°C. However, neither opsin in the membrane nor all-trans retinal activates the enzyme. The phosphodiesterase in rod outer segment membrane is also activated by irradiation at ?4°C. Thus, we conclude that the phosphodiesterase is activated by a common photolysis intermediate of these rhodopsin isomers, perhaps before metarhodopsin II decays.     

Arrestin from nucleated red blood cells binds to bovine rhodopsin in a light-dependent manner

Using a panel of monoclonal antibodies, it has previously been demonstrated that the cytosol of nucleated red cells (trout and turkey) contains a protein similar to arrestin, a soluble protein found so far only in the photosensitive cells and which, by binding to photoexcited rhodopsin, inhibits the phototransduction process. The role of this arrestin-like protein in non-photosensitive cells is questionable. In this report we present evidence that partially purified red blood cell arrestin (RBC arrestin) behaves functionally like bovine retinal arrestin: it binds to phosphorylated bovine rhodopsin only when this receptor has been photoactivated. Thus RBC arrestin and bovine retinal arrestin are closely related both structurally and functionally. By analogy with the function of retinal arrestin, it is proposed that RBC arrestin is involved in desensitization of membrane transport proteins and/or adrenergic receptors.     

Chemical modification of transducin with dansyl chloride hinders its binding to light-activated rhodopsin

Transducin (T), the heterotrimeric guanine nucleotide binding protein in rod outer segments, serves as an intermediary between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase. Labeling of T with dansyl chloride (DnsCl) inhibited its light-dependent guanine nucleotide binding activity. Conversely, DnsCl had no effect on the functionality of rhodopsin. Approximately 2-3 mol of DnsCl were incorporated per mole of T. Since fluoroaluminate was capable of activating DnsCl-modified T, this lysine-specific labeling compound did not affect the guanine nucleotide-binding pocket of T. However, the labeling of T with DnsCl hindered its binding to photoexcited rhodopsin, as shown by sedimentation experiments. Additionally, rhodopsin completely protected against the DnsCl inactivation of T. These results demonstrated the existence of functional lysines on T that are located in the proximity of the interaction site with the photoreceptor protein.     

Sites of arrestin action during the quench phenomenon in retinal rods

The target proteins for arrestin (48 kDa protein) action during the quench of cGMP phosphodiesterase (PDE) activation in retinal rod disk membranes were identified by the use of a cross-linking reagent. A heterobifunctional, cleavable, photo-activatable cross-linker (sulfo-SADP) was coupled to purified arrestin. Under precise weak visible light bleach and nucleotide conditions of quench, the cross-linker was UV flash-activated at a time when quench was well established. The target proteins covalently linked to arrestin by cross-linker activation were identified by immunoblotting. In the presence of ATP arrestin cross-linked to both PDE and rhodopsin during the quench phenomenon. Removal of ATP from the reaction mixture essentially abolished the cross-link with PDE, just as ATP omission abolishes quench, but significantly increased the cross-link to rhodopsin. The absence of a cross-link to the plentiful beta-subunit of transductin, as well as the results of competition studies employing arrestin without attached cross-linker, suggest that the observed cross-links are specific and reflect true binding interactions of arrestin during quench. The data are consistent with a model of quench in which photolyzed rhodopsin (R*) catalyzes the formation of an activated form of arrestin, which dissociates from R* in the presence of ATP, and binds to PDEs, thereby deactivating them.     

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.