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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.

Arrestins are regulators of the active state of G-protein-coupled receptors. Towards elucidating the function of different arrestin subfamilies in sensory cells, we have isolated a novel arrestin 1, Am Arr1, from the UV photoreceptors of the neuropteran Ascalaphus macaronius. Am Arr1 forms a phylogenetic clade with antennal and visual Arr1 isoforms of invertebrates. Am Arr1 undergoes a light-dependent binding cycle to photoreceptor membranes, as reported earlier only for members of the arrestin 2 subfamily. This suggests a common control mechanism for the active state of invertebrate rhodopsins and G-protein-coupled receptors of antennal sensory cells. Furthermore, it implies that a strict correlation of distinct arrestin isoforms to distinct functions is not a general principle for invertebrate sensory cells.

In the presence of a photobleaching intermediate of unphosphorylated or phosphorylated rhodopsin (Rh ∗ ), the binding of GppNHp to transducin was measured with or without arrestin for elucidation of the shut-off mechanism of the visual transduction process in bovine rod outer segments. The ability of Rh ∗ to catalyze the formation of the transducin-GppNHp complex in the absence of arrestin was independent of the degree of phosphorylation of Rh ∗ . Furthermore, the catalyzing ability of the phosphorylated Rh ∗ was not reduced by the addition of arrestin. These observations indicate that the interaction between phosphorylated Rh ∗ and transducin was not inhibited by arrestin. Thus, the hypothesis was not supported that the PDE shut-off process is a simple competition between transducin and arrestin for binding to phosphorylated Rh ∗ .

The luteinizing hormone/choriogonadotropin hormone receptor (LH/CG R) signals to regulate ovulation, corpus luteum formation, and fetal survival during pregnancy. Agonist binding to the LH/CG R is poorly reversible, emphasizing the importance of a cellular mechanism to temper signaling by a potentially persistently active receptor. Like other G protein-coupled receptors (GPCRs), signaling by this receptor is modulated by its binding of an arrestin. We have identified ADP ribosylation factor 6 (ARF6) as a protein whose activation state is regulated by the LH/CG R and which functions to regulate the availability of plasma membrane-docked arrestin 2 to this receptor. We hypothesize that ARF6 might also serve GPCRs other than the LH/CG R to regulate the availability of arrestin 2 for receptor desensitization.

Arrestins constitute a superfamily of regulatory proteins that down-regulate phosphorylated G-protein membrane receptors, including rod and cone photoreceptors and adrenergic receptors. The potential role of arrestin in color visual processes led us to identify a cDNA encoding a cone-like arrestin in Xenopus laevis, the principle amphibian biological model system. Alignment of 18 deduced amino acid sequences of all known arrestins from both invertebrate and vertebrate species reveals five arrestin families. Further analysis identifies 7 variable and 4 conservative arrestin structural motifs that may identify potential functional domains. The adaptive evolutionary relationship of Xenopus cone arrestin to the arrestin gene tree suggests high intrafamily homology and early gene duplication events.

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.

The soluble protein arrestin (also named 48K-protein or retinal-S-antigen) is involved in controlling light-dependent transducin and cGMP phosphodiesterase activity in retinal rods. It is also known for its ability to induce autoimmune reactions in the eye causing the eye disease uveitis. We report here a rapid binding of ATP to arrestin with K A = 2 × 10 21 (1/mol) 3 and a coordination number n = 3. This ATP binding to arrestin supports the notion of a nucleotide exchange which initiates the rapid inhibitory action of this enzyme during the primary step of vertebrate phototransduction. Rhodopsin; Arrestin; Transducin; ATP binding; ADP/ATP exchange

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.

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.

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.

Cytosolic extracts of trout and turkey erythrocytes were tested for their immunoreactivity with polyclonal and monoclonal antibodies to retinal arrestin (S-antigen), a cytosolic protein of photoreceptor cells involved in the desensitization of rhodopsin. After adsorption or immunoaffinity chromatography of the extracts, these antibodies specifically recognized a protein having a molecular weight similar to that of retinal arrestin. Because the G-protein-mediated transduction systems, such as visual and β-adrenergic systems, display a high degree of structural and functional homology, the presence of arrestin-like proteins in non-photosensitive cells suggests that these proteins are involved in the transduction of chemical signals, with a possible role in receptor desensitization.