Retinal Arrestin Research Articles

Retina-arrestin specific CD8+ T cells are not implicated in HLA-A29-positive birdshot chorioretinitis

BackgroundHLA-A29-positive birdshot chorioretinitis (BCR) is an inflammatory eye disorder that is generally assumed to be caused by an autoimmune response to HLA-A29-presented peptides from retinal arrestin (SAG), yet the epitopes recognized by CD8+ T cells from patients remain to be identified. ObjectivesThe identification of natural ligands of SAG presented by HLA-A29. To quantify CD8+ T cells reactive to antigenic SAG peptides presented by HLA-A29 in patients and controls. MethodsWe performed mass-spectrometry based immunopeptidomics of HLA-A29 of antigen-presenting cell lines from patients engineered to express SAG. MHC-I Dextramer technology was utilised to determine expansion of antigen-specific CD8+ T cells reactive to SAG peptides in complex with HLA-A29 in a cohort of BCR patients, HLA-A29-positive controls, and HLA-A29-negative controls. ResultsWe report on the naturally presented antigenic SAG peptides identified by sequencing the HLA-A29 immunopeptidome of antigen-presenting cells of patients. We show that the N-terminally extended SAG peptide precursors can be trimmed in vitro by the antigen-processing aminopeptidases ERAP1 and ERAP2. Unexpectedly, no enhanced antigen engagement by CD8+ T cells upon stimulation with SAG peptides was observed in patients or HLA-A29-positive controls. Multiplexed HLA-A29-peptide dextramer profiling of a case-control cohort revealed that CD8+ T cells specific for these SAG peptides were neither detectable in peripheral blood nor in eye biopsies of patients. ConclusionsCollectively, these findings demonstrate that SAG is not a CD8+ T cell autoantigen and sharply contrast the paradigm in the pathogenesis of BCR. Therefore, the mechanism by which HLA-A29 is associated with BCR does not involve SAG.

Gut microbiota linked to autoimmune uveitis.

Autoimmune uveitis is a group of intraocular inflammatory diseases that arise without known infectious etiology. Uveitis is one of the leading causes of blindness in the developed world and is responsible for up to 15% of severe visual handicap. The disease affects population of working age and has a significant impact on public health. Patients often show detectable immune responses to unique retinal proteins such as retinal arrestin and interphotoreceptor retinoid binding protein (IRBP), which are involved in visual function, and particular HLA haplotypes have been associated with disease, supporting the autoimmune nature (1). Although anecdotal evidence suggested a link between microbial infections and uveitis, our understanding of the etiology of disease, its driving mechanisms and treatment options are still limited. Animal models have been instrumental to advance our understanding on pathogenesis of uveitis. A ‘classic’ uveitis model in mice is referred as experimental autoimmune uveitis (EAU), involving active immunization with IRBP in complete Freund’s adjuvant (CFA) that contains heat-killed Mycobacterium tuberculosis . Co-administration of bacterial adjuvants is required to activate innate immune cells and create the proinflammatory milieu to induce adaptive immune responses and to trigger the autoimmune effector pathway (1). However, unlike experimental diseases in the animal models, most cases of human autoimmune uveitis cannot be connected to an exposure of the immune system to ocular antigens that are present behind the blood retinal barrier. These target antigens in uveitis are sequestered inside the eye and are not expressed in the periphery (2), but retina-specific T cells that would recognize them must be activated to enter the eye to drive pathology in uveitis. This presents a paradox and raises a fundamental question: where and how do autoreactive T cells, that can recognize retinal antigens and trigger uveitis, first become activated?

Uveitis-Associated Epitopes of Retinal Antigens Are Pathogenic in the Humanized Mouse Model of Uveitis and Identify Autoaggressive T Cells

Noninfectious uveitis is a leading cause of blindness and thought to involve autoimmune T cell responses to retinal proteins (e.g., retinal arrestin [soluble-Ag (S-Ag)]). There are no known biomarkers for the disease. Susceptibility is associated with HLA, but little is known about susceptible class II alleles or the potentially pathogenic epitopes that they present. Using a humanized HLA-transgenic mouse model of S-Ag-induced autoimmune uveitis, we identified several susceptible and resistant alleles of HLA-DR and -DQ genes and defined pathogenic epitopes of S-Ag presented by the susceptible alleles. The sequences of these epitopes overlap with some previously identified peptides of S-Ag ("M" and "N"), known to elicit memory responses in lymphocytes of uveitis patients. HLA-DR-restricted, S-Ag-specific CD4(+) T cells could be detected in blood and draining lymph nodes of uveitic mice with HLA class II tetramers and transferred the disease to healthy mice. Importantly, tetramer-positive cells were detected in peripheral blood of a uveitis patient. To our knowledge, these findings provide the first tangible evidence that an autoimmune response to retina is causally involved in pathogenesis of human uveitis, demonstrating the feasibility of identifying and isolating retinal Ag-specific T cells from uveitis patients and may facilitate their development as biomarkers for the disease.

Uveitis-associated epitopes of retinal antigens are pathogenic in the humanized mouse model of EAU and identify autoaggressive T cells (93.1)

Abstract Autoimmune uveitis affects 150,000 Americans annually and is a leading cause of blindness. Pathogenesis is thought to involve T cell mediated responses to retinal proteins, such as retinal arrestin (S-Ag). Susceptibility to autoimmune uveitis is strongly associated with HLA class I and class II, but relatively little is known about the nature of HLA class II alleles and about the epitopes that might be pathogenic in the context of different HLA class II molecules. Using a ‘humanized’ mouse model for experimental autoimmune uveitis (EAU) in HLA transgenic mice, we have identified permissive and non-permissive alleles of the HLA-DR and -DQ genes and characterized allele-specific uveitogenic epitopes. The sequences of these epitopes overlap with some of the previously identified peptides of S-Ag (M and N), which elicit memory responses in lymphocytes of uveitis patients. HLA-DR restricted S-Ag specific CD4+ T cells could be detected in blood and draining lymph nodes of immunized mice using HLA class II tetramers, demonstrating the potential use of this reagent to detect and isolate antigen specific T cells in uveitis patients. Our data provide new insights into the pathogenesis of human uveitis, and may help in the development of antigen specific therapies.

Cancer-associated retinopathy in patients with breast carcinoma

Introduction:Cancer-associated retinopathy (CAR) is a paraneoplastic neurological syndrome resulting in progressive loss of vision and clinical signs of retinal degeneration. It is associated with various types of cancer and is also considered to be an autoimmune disorder that involves cross-reaction between autoantibodies and retinal proteins. The aim of this study was to establish whether immunoreactivity to retinal antigens (RAs) observed in patients with breast cancer is accompanied by any visual impairments.Materials and Methods:Sera of 295 patients with diagnosed breast cancer were screened for the presence of anti-RAs antibodies using immunoblotting. Cellular immunoreactivity to RAs present in retinal extracts and to purified recoverin and arrestin was determined by means of a lymphocyte proliferation assay. Six patients with high-titer antibodies to RAs then underwent ophthalmic and neurological examinations.Results:Four serum samples contained high-titer antibodies to a 46-kDa protein, most probably retinal α-enolase, three had antibodies to a 48-kDa protein identified as retinal arrestin, while 56-, 43-, 41-, and 34-kDa antigens were recognized only by one serum sample each. Moreover, weak cellular response to all the RAs tested was observed in one patient and another patient responded only to retinal extract. Two of the examined patients displayed symptoms of CAR.Conclusions:Immunoreactivity to RAs in patients with breast cancer may also be present in cases without clinical signs of CAR.

Enolase and Arrestin are Novel Nonmyelin Autoantigens in Multiple Sclerosis

Although myelin autoimmunity is known to be a major factor in the pathogenesis of multiple sclerosis (MS), the role of nonmyelin antigens is less clear. Given the complexity of this disease, it is possible that autoimmunity against nonmyelin antigens also has a pathogenic role. Autoantibodies against enolase and arrestin have previously been reported in MS patients. The T-cell response to these antigens, however, has not been established. Thirty-five patients with MS were recruited, along with thirty-five healthy controls. T-cell proliferative responses against non-neuronal enolase, neuron-specific enolase (NSE), retinal arrestin, beta-arrestin, and myelin basic protein were determined. MS patients had a greater prevalence of positive T-cell proliferative responses to NSE, retinal arrestin, and beta-arrestin than healthy controls (p<0.0001). The proliferative response against NSE, retinal arrestin, and beta-arrestin correlated with the response against myelin basic protein (p < or = 0.004). Furthermore, the proliferative response against retinal arrestin was correlated to beta-arrestin (p<0.0001), whereas there was no such correlation between non-neuronal enolase and NSE (p = 0.23). There is accumulating evidence to suggest that the pathogenesis of MS involves more than just myelin autoimmunity/destruction. Autoimmunity against nonmyelin antigens may be a component of this myriad of immunopathological events. NSE, retinal arrestin, and beta-arrestin are novel nonmyelin autoantigens that deserve further investigation in this respect. Autoimmunity against these antigens may be linked to neurodegeneration, defective remyelination, and predisposition to uveitis in multiple sclerosis. Further investigation of the role of these antigens in MS is warranted.

Endogenous IRBP can be dispensable for generation of natural CD4+CD25+ regulatory T cells that protect from IRBP-induced retinal autoimmunity

Susceptibility to experimental autoimmune uveitis (EAU), a model for human uveitis induced in mice with the retinal antigen interphotoreceptor retinoid-binding protein (IRBP), is controlled by “natural” CD4+CD25+ regulatory T (T reg) cells. To examine whether endogenous expression of IRBP is necessary to generate these T reg cells, we studied responses of IRBP knockout (KO) versus wild-type (WT) mice. Unexpectedly, not only WT but also IRBP KO mice immunized with a uveitogenic regimen of IRBP in complete Freund's adjuvant (CFA) exhibited CD25+ regulatory cells that could be depleted by PC61 treatment, which suppressed development of uveitogenic effector T cells and decreased immunological responses to IRBP. These EAU-relevant T reg cells were not IRBP specific, as their activity was not present in IRBP KO mice immunized with IRBP in incomplete Freund's adjuvant (IFA), lacking mycobacteria (whereas the same mice exhibited normal T reg cell activity to retinal arrestin in IFA). We propose that mycobacterial components in CFA activate T reg cells of other specificities to inhibit generation of IRBP-specific effector T cells in a bystander fashion, indicating that effective T reg cells can be antigen nonspecific. Our data also provide the first evidence that generation of specific T reg cells to a native autoantigen in a mouse with a diverse T cell repertoire requires a cognate interaction.

Autoantigens act as tissue-specific chemoattractants

We have investigated the chemoattractant properties of self-antigens associated with autoimmune diseases and solid tumors. Many autoantigens induced leukocyte migration, especially by immature dendritic cells (iDC) by interacting with various chemoattractant Gi-protein-coupled receptors (GiPCR). Our initial observation that myositis-associated autoantigens, histidyl-tRNA synthetase and asparaginyl-tRNA synthetase, were chemotactic for CC chemokine receptor 5 (CCR5)- and CCR3-expressing leukocytes, while other nonautoantigenic aminoacyl-tRNA synthesases were not, suggested that only self-antigens capable of interacting with receptors on antigen-presenting cells were immunogenic. We next determined that self-antigens associated with autoimmune diseases, e.g., multiple sclerosis or experimental autoimmune encephalomyelitis, type I diabetes, scleroderma, systemic lupus erythematosus, autoimmune uveitis, or experimental autoimmune uveitis (EAU), were chemotactic for GiPCR expressed by iDC. The majority of autoantigens were DC chemoattractants at 10-100 ng/ml, but did not induce DC maturation until they reached 1000-fold higher concentrations. Interphotoreceptor retinoid-binding protein and retinal arrestin (S-antigen) are targets of autoantibodies in human uveitis and are chemotactic for CXC chemokine receptor 5 (CXCR5)- and/or CXCR3-expressing iDC. However, although S-antigen does not induce EAU in wild-type mice, it is nevertheless a chemoattractant for murine iDC. These unexpected observations suggested that the chemotactic activity of these tissue-specific self-antigens could be involved in promotion of tissue repair and restoration. Thus, the primary role of autoantigens may be to alert the immune system to danger signals from invaded and damaged tissues to facilitate repair, and autoimmune responses subsequently develop only in subjects with impaired immunoregulatory function.

Peripheral Expression of Rod Photoreceptor Arrestin Induces an Epitope-Specific, Protective Response Against Experimental Autoimmune Uveoretinitis

Purpose: To examine the immunological basis for reduced susceptibility to experimental autoimmune uveoretinitis (EAU) in rats expressing retinal photoreceptor cell arrestin in the periphery. Methods: Peripheral expression of arrestin in Lewis rats was achieved by engraftment of syngeneic bone marrow (BM) transduced with retroviruses encoding wild-type arrestin or a mutant arrestin lacking the immunodominant epitope Arr273 − 289 (Δ 273-Arr). EAU was induced by immunization with arrestin peptides Arr273−289 or Arr343−362. Cultured splenocytes and/or lymphocytes from immunized rats were assayed for antigen-induced proliferation, antibody production, and cytokines. Results: Rats expressing Δ 273-Arr were not protected from Arr273 − 289-induced EAU, showing that protection was epitope specific. Proliferation assays found little difference in the ability of draining lymph node cells from arrestin-transduced rats to proliferate in response to the antigen, indicating that antigen-responsive T cells were not deleted in BM recipients. Only rats immunized with Arr343 − 362 elicited antibodies, but no difference in titer was found between transduced and control animals. Higher levels of IFN-γ mRNA were made by Arr273 − 289-immunized rats than Arr343 − 366-immunized rats, but in either case, the levels did not correlate with chimeric status or EAU susceptibility. Arr273 − 289-immunized rats had higher levels of IL-10 mRNA than Arr343 − 362-immunized rats, and those levels were decreased in arrestin chimeric rats. Overall, immunization with the more potently uveitogenic Arr343 − 362 induced lower levels of IL-10 and IFN-γ than the less uveitogenic Arr273 − 289. A strong correlation was found between the ability of lymphocytes to make IL-4 in the arrestin-chimeric animals and inhibition of EAU. Conclusions: Peripheral expression of arrestin in a regenerating immune system induces an epitope-specific protective response to EAU induced by arrestin peptides. Although IL-4 and IL-10 levels were altered in arrestin-chimeric mice, the outcome was not consistently TH2-like. Only IL-4 production was clearly associated with reduced susceptibility to EAU.

Retinal Antigens Are Recognized by Antibodies Present in Sera of Patients with Multiple Sclerosis

Multiple sclerosis (MS) is frequently accompanied by visual symptoms including those related to retinal disorders. Since they may be a consequence of an autoimmune reaction, we examined whether sera of patients with diagnosed MS and changes in visual-evoked potentials contain antibodies against retinal antigens (retAgs). Immunoblot analysis revealed that MS sera recognized mainly a 46-kD antigen, a 41-kD antigen, retinal arrestin, to a smaller extent also 70-, 56-, 43-, and 36-kD proteins. Patients whose sera showed the highest reactivity with 41- and 46-kD antigens had deficiencies in visual acuity, visual fields, ophthalmoscopy, and electroretinograms. Our observation suggests that antibodies to these retAgs may play a role in the origin of ophthalmologic impairment in MS.

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.

Spectroscopic characterization of arrestin interactions with competitive ligands: study of heparin and phytic acid binding.

A combination of intrinsic fluorescence and circular dichroic (CD) spectroscopy has been used to characterize the complexes formed between bovine retinal arrestin and heparin or phytic acid, two ligands that are known to mimic the structural changes in arrestin attending receptor binding. No changes in the CD spectra were observed upon ligand binding, nor did the degree of tryptophan fluorescence quenching change significantly in the complexes. These data argue against any large-scale changes in protein secondary or tertiary structure accompanying ligand binding. The change in tyrosine fluorescence intensity was used to determine the dissociation constants for the heparin and phytic acid complexes of arrestin. The only change observed was a saturable diminution of tyrosine fluorescence signal from the protein. For both ligands, the data suggest two distinct binding interactions with the protein--a high--affinity interaction with Kd between 200 and 300 nM, and a lower affinity interaction with Kd between 2 and 8 microM. Study of collisional quenching of tyrosine fluorescence in free arrestin and the ligand-replete complexes indicates that 10 of the 14 tyrosine residues of the protein are solvent-exposed in the free protein; this value drops to between 5 and 6 solvent-exposed residues in the high-affinity complexes of the two ligands. These data suggest that ligand binding leads to direct occlusion of between 4 and 5 tyrosine residues on the solvent-exposed surface of the protein, but not to any large-scale changes in protein structure. The large activation energy previously reported to be associated with arrestin-receptor interactions may therefore reflect localized movements of the N- and C-termini of arrestin, which are proposed to interact in the free protein through electrostatic interactions. Binding of the anionic ligands heparin, phytic acid, or phosphorylated rhodopsin may compete with the C-terminus of arrestin for these electrostatic interactions, thus allowing the C-terminus to swing out of the binding region.

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 Å and 3.5 Å (space group P2 12 12, cell dimensions a=169.17 Å, b=185.53 Å, c=90.93 Å, T=100 K). The asymmetric unit contains four molecules with a solvent content of 68.5% by volume.

Immunodetection of a Protein Related to Mammalian Arrestin inEuglena gracilis

Six monoclonal antibodies directed against different epitopes of bovine retinal arrestin recognized a single polypeptide in extracts of achlorophyllousEuglena graciliscells. This polypeptide had an apparent molecular weight slightly lower: 45kDa vs. 48 kDa, and a more basic isoelectric point compared to that of bovine visual arrestin. It was located in an insoluble cytoplasmic fraction. Immunofluorescence assays show a cytoplasmic punctuated pattern suggesting a cluster distribution or a linkage to some cell structure. The presence of arrestine-like molecules in achlorophyllousEuglena graciliscells suggests that these proteins might be involved in a peculiar step of chemical signal transduction processes.

Isolation of canine retinal arrestin cDNA and exclusion of three candidate genes for Swedish Briard retinal dystrophy

Purpose. Mutations of genes encoding various retina-specific proteins are known to cause a wide spectrum of inherited retinal dystrophies in different species. In the canine, several types of genetic retinal dystrophies have been described affecting primarily the photoreceptors and/or the retinal pigment epithelium. We are performing a systematic analysis of canine candidate genes for such diseases to identify the one mutated in the retinal dystrophy in Swedish Briard dogs. Methods. We isolated and characterised the full length cDNA of canine retinal arrestin by the method of rapid amplification of cDNA ends (RACE). Results. The full length cDNA isolated by us is 1,575 base pairs (bp) long and contains a 1,218 bp-long open reading frame. Conclusions. The homology of the canine arrestin protein is highest with the human analogue (88.9%) and lowest with mouse arrestin (85.3%). The most obvious sequence differences among the different arrestins are in the extreme carboxyl terminus. PCR-SSCP (single strand conformation polymorphism) analysis and direct sequencing of retinal cDNA didn't provide any evidence that mutations in the canine arrestin gene are responsible for the retinal dystrophy seen in the Swedish strain of Briard dogs. Similar data were obtained for the genes encoding rhodopsin and the ß-subunit of photoreceptor-specific phosphodiesterase by segregation analysis.

A Splice Variant of Arrestin from Human Retina

Retinal arrestin is known to participate in the quenching of phototransduction through binding to light-activated and phosphorylated rhodopsin. Recently, a splice variant of retinal arrestin was identified in bovine photoreceptors which could bind unphosphorylated photoactive rhodopsin. In this report, a splice variant of retinal arrestin is identified in the human retina. The variant of human arrestin is produced by splicing out exon 12, unlike the bovine variant which is produced by splicing out exon 16. This 78bp deletion in the human splice variant produces a polypeptide with a calculated molecular weight of 42.2kDa that lacks 26 amino acids when compared to the full-length retinal arrestin. The use of quantitative competitive PCR indicates that the mRNA for the human splice variant of arrestin is present at approximately one-twentieth of the level of human arrestin mRNA.

Trout red blood cell arrestin (TRCarr), a novel member of the arrestin family: cloning, immunoprecipitation and expression of recombinant TRCarr.

Arrestins are cytosolic proteins involved in the desensitization of G-protein-coupled receptors. We report the cloning of trout red blood cell arrestin which shows 76, 82 and 52% identity with bovine beta-arrestin1, beta-arrestin2 and retinal arrestin respectively. Antibodies were generated against the C-terminus of trout red blood cell arrestin. These antibodies detected arrestin in erythrocyte cytosol and were able to precipitate the native protein. The Na+/H+ antiporter of trout red blood cell is activated by beta-adrenergic stimulation and is then desensitized whereas the transmembrane signalling pathway is not. To investigate the subcellular distribution of arrestin on beta-adrenergic activation and desensitization of the antiporter, precipitation experiments were carried out on trout erythrocytes. A desensitization-dependent shift in cytosolic arrestin to the membranes could not be detected using the immunoprecipitation technique but we cannot exclude the possibility that a small number of cytosolic arrestins might be involved in the regulation of membrane proteins in trout erythrocyte. Recombinant trout arrestin was produced in a protease-deficient Escherichia coli strain and its functionality was tested in a reconstituted rhodopsin assay. The recombinant protein provides a suitable tool for investigating the target for arrestin in trout red blood cell, which still remains to be identified.

Immunodetection of an arrestin-like protein in human retinal pigment epithelium.

The goal of this study was to determine if an arrestin/S-antigen-like protein is produced by human retinal pigment epithelial (HRPE) cells maintained in tissue culture. Arrestin immunoreactivity was examined in fixed, monolayer cultures of HRPE and on immunoblots of SDS-PAGE separations of whole cell lysates of HRPE using five monoclonal antibodies (mAbs A2G5, A9C6, 3C4.2, 3D1.2 and 5c6.47) that bind to different epitopes in bovine retinal S-antigen. Monolayers of HRPE cells showed immunoreactivity with four of the mAbs though the relative staining intensity varied among mAbs and donors. For example, mAb A2G5 which historically shows very limited crossreactivity among species, reacted strongly with cells from one donor, moderately with cells from a second donor and only weakly with other donor cultures examined. mAb 3D1.2 showed no reactivity with HRPE cells. Immunoblots of SDS-PAGE separations of whole cell lysates of HRPE established from ten different donors confirmed the presence of an arrestin-related polypeptide that comigrated with retinal arrestin. These results demonstrate the presence of an arrestin-like protein in HRPE cells which have been maintained in tissue culture. Though the function of this arrestin homologue in HRPE is not yet established, it could play a role in the downregulation of receptor and/or transport protein activity.

Synthetic phosphopeptide from rhodopsin sequence induces retinal arrestin binding to photoactivated unphosphorylated rhodopsin

A synthetic heptaphosphopeptide comprising the fully phosphorylated carboxyl terminal phosphorylation region of bovine rhodopsin, residues 330–348, was found to induce a conformational change in bovine arrestin. This caused an alteration of the pattern of limited proteolysis of arrestin similar to that induced by binding phosphorylated rhodopsin or heparin. Unlike heparin, the phosphopeptide also induced light-activated binding of arrestin to both unphosphorylated rhodopsin in disk membranes as well as to endoproteinase Asp-N-treated rhodopsin (des 330–348). These findings suggest that one function of phosphorylation of rhodopsin is to activate arrestin which can then bind to other regions of the surface of the photoactivated rhodopsin.

X-Arrestin: a new retinal arrestin mapping to the X chromosome

We have been using a differential cDNA cloning approach to isolate human retina-specific and retina-enriched genes [1]. A 1,314 bp cDNA was isolated by this approach, representing a highly retina-specific message encoding a 388 amino acid protein showing 58%, 50%, and 49% homology to bovine β-arrestin, and bovine and human retinal arrestin (S-antigen), respectively. Chromosomal mapping localized this new arrestin genc to the proximal long arm of the X chromosome, hence it was named X-arrestin. In situ hybridization demonstrated its expression in the inner and outer segments and the inner plexiform regions of the retina.