X-ray crystal structure of arrestin from bovine rod outer segments.

Retinal arrestin is the essential protein for the termination of the light response in vertebrate rod outer segments. It plays an important role in quenching the light-induced enzyme cascade by its ability to bind to phosphorylated light-activated rhodopsin (P-Rh*). Arrestins are found in various G-protein-coupled amplification cascades. Here we report on the three-dimensional structure of bovine arrestin (relative molecular mass, 45,300) at 3.3 A resolution. The crystal structure comprises two domains of antiparallel beta-sheets connected through a hinge region and one short alpha-helix on the back of the amino-terminal fold. The binding region for phosphorylated light-activated rhodopsin is located at the N-terminal domain, as indicated by the docking of the photoreceptor to the three-dimensional structure of arrestin. This agrees with the interpretation of binding studies on partially digested and mutated arrestin.

Crystallization and preliminary X-ray analysis of arrestin from bovine rod outer segment.

We present the first X-ray study of a member of the arrestin family, the bovine retinal arrestin. Arrestin is essential for the fine regulation and termination of the light-induced enzyme cascade in vertebrate rod outer segments. It plays an important role in quenching phototransduction by its ability to preferentially bind to phosphorylated light-activated rhodopsin. The crystals diffract between 3 angstroms and 3.5 angstroms (space group P2(1)2(1)2, cell dimensions a = 169.17 angstroms, b = 185.53 angstroms, c = 90.93 angstroms, T = 100 K). The asymmetric unit contains four molecules with a solvent content of 68.5% by volume.

Duration and amplitude of the light-induced cGMP hydrolysis in vertebrate photoreceptors are regulated by multiple phosphorylation of rhodopsin and by arrestin binding.

The duration and amplitude of the light-induced cGMP hydrolysis in bovine rod outer segments were investigated using purified rhodopsin in nine different states of phosphorylation in a reconstituted system. Effects of varying amounts of arrestin at all states of rhodopsin phosphorylation were measured. The findings were the following: (1) At low bleaching levels, the activity of phosphodigesterase (PDE) depends strongly on the phosphorylation degree of the light-activated rhodopsin (R*), while at saturating light levels R* of all phosphorylation degrees activates PDE to the same extent. (2) The turnoff time for PDE is markedly shortened if R* is phosphorylated, independent of the number of phosphate groups incorporated into rhodopsin (P/R); i.e., the first phosphate which is bound to R* seems to be responsible for the shortened turnoff time. The lifetime of phosphorylated R* is shown to be dramatically reduced compared to that of unphosphorylated R*, as monitored by the ability of R* to activate PDE. (3) After activation with phosphorylated R*, addition of arrestin caused a further reduction of both the maximum activity and the turnoff time of PDE. Both effects were strongly dependent on (a) the phosphorylation degree of R*, (b) the concentration of arrestin, and (c) the concentration of R*. These results suggest that the light-induced phosphorylation of rhodopsin to different extents and the subsequent binding of arrestin are involved in the light adaptation and in the fine regulation of the light response in vertebrate photoreceptors.

Deactivation of photoactivated rhodopsin by rhodopsin-kinase and arrestin.

Photoactivated rhodopsin (R) catalyses, by repetitively interacting with many copies of a guanosine nucleotide binding protein (transducin), the amplified binding of GTP to transducin molecules which then activate cyclic GMP phosphodiesterase. Electrophysiologists recently have shown that cyclic GMP keeps ion channels in the plasma membrane of the rod outer segment open in darkness, and that light-induced hydrolysis of cyclic GMP leads to closure of the channels and therefore to hyperpolarization of the rod cell. Photoactivated rhodopsin interacts not only with transducin, but with two more proteins: a protein kinase that specifically phosphorylates R (in contrast to dark-adapted rhodopsin) at multiple sites; and an abundant soluble protein of 48 KDal (called 48 K-protein, S-antigen, or arrestin) that specifically binds to phosphorylated R. Phosphorylation partially suppresses the ability of R to catalyze transducin-mediated phosphodiesterase activation even in the absence of arrestin. Binding of arrestin to the phosphorylated R potentiates this inhibitory effect, most probably because arrestin competes with transducin for binding on the phosphorylated R. Phosphorylation, in conjunction with arrestin binding, therefore appears to be a mechanism that terminates the active state of the receptor, R.

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.

Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments.

Each photoexcited rhodopsin (R*) molecule catalyzes binding of GTP to many copies of the guanine nucleotide-binding protein transducin, which, in its GTP-binding form, then activates cGMP phosphodiesterase (PDEase). Subsequent deactivation of this light-activated enzyme cascade involves hydrolysis of the GTP bound to transducin, as well as decay of the activating capacity of R*. We report here that deactivation of PDEase in rod outer segment suspensions is highly enhanced by addition of ATP and purified 48-kDa protein, which is an intrinsic rod outer segment protein that is soluble in the dark but binds to photolyzed rhodopsin that has been phosphorylated by rhodopsin kinase and ATP [Kühn, H., Hall, S.W. & Wilden, U. (1984) FEBS Lett. 176, 473-478]. To analyze the mechanism by which ATP and 48-kDa protein deactivate PDEase, we used an ATP-free system consisting of thoroughly washed disk membranes, whose rhodopsin had been previously phosphorylated and chromophore-regenerated, and to which purified PDEase and transducin were reassociated. Such phosphorylated membranes exhibited a significantly lower (by a factor less than or equal to 5) light-induced PDEase-activating capacity than unphosphorylated controls. Addition of purified 48-kDa protein to phosphorylated membranes further suppressed their PDEase-activating capacity; suppression could be as high as 98% (as compared to unphosphorylated membranes), depending on the amount of 48-kDa protein and the flash intensity. Addition of ATP had little further effect. In contrast, PDEase activation or deactivation with unphosphorylated control membranes was not influenced by 48-kDa protein, even in the presence of ATP, provided rhodopsin kinase was absent. Our data suggest that 48-kDa protein binds to phosphorylated R* and thereby quenches its capacity to activate transducin and PDEase.

Light-induced binding of 48-kDa protein to photoreceptor membranes is highly enhanced by phosphorylation of rhodopsin.

The 48-kDa protein, a major protein of rod photoreceptor cells, is soluble in the dark but associates with the disk membranes when some (5-10%) of their rhodopsin has absorbed light and if this rhodopsin is additionally phosphorylated by ATP and rhodopsin kinase. If rhodopsin has been phosphorylated and regenerated prior to the protein binding experiment, the binding of 48-kDa protein depends on light but no longer on the presence of ATP. Another photoreceptor protein, GTP-binding protein, associates with both phosphorylated and unphosphorylated rhodopsin upon illumination. Excess GTP-binding protein thereby displaces 48-kDa protein from phosphorylated disks; this indicates competition between these two proteins for binding sites on illuminated phosphorylated rhodopsin molecules.