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PubMed1995

Rhodopsin kinase inhibition by recoverin. Function of recoverin myristoylation.

Recoverin is a Ca(2+)-binding protein that may play a role in vertebrate photoreceptor light adaptation by imparting Ca2+ sensitivity to rhodopsin kinase. It is heterogeneously acylated (mostly myristoylated) at its amino-terminal glycine. Recent studies have shown that recoverin myristolyation is necessary for its Ca(2+)-dependent membrane association and cooperative Ca2+ binding. We have addressed several issues concerning the role of recoverin myristoylation with respect to inhibition of rhodopsin kinase. We find that 1) myristoylation of recoverin is not necessary for inhibition of rhodopsin kinase, 2) myristoylation of recoverin induces a cooperative Ca(2+)-dependence for rhodopsin kinase inhibition, and 3) each Ca(2+)-binding site on the nonmyristoylated recoverin partially inhibits rhodopsin kinase. The available data suggest that the functions of recoverin myristoylation in the living rod are to induce a sharp Ca2+ dependence of rhodopsin kinase inhibition and to bring this dependence into the rod's physiological Ca2+ concentration range.

Animals↗

PubMedDate not supplied

Recoverin immunoreactivity in mammalian cone bipolar cells.

Human, macaque monkey, and rat retinas were immunostained with a polyclonal antibody preparation against purified recoverin, a 23-kD calcium-binding protein isolated from bovine retina that localizes to rods and cones (Dizhoor et al., 1991). In addition to immunoreactive photoreceptors, we have identified subpopulations of recoverin-positive bipolar cells in all three species. Results from immunostaining with progressive dilutions of anti-recoverin and preadsorption of the antibody with a dilution series of purified recoverin showed that photoreceptors and bipolar cells had similar affinities for the antibody and suggested that the molecule recognized by the antibody in both cell types is recoverin. Immunoreactivity for recoverin and protein kinase C, a selective marker for all rod bipolar cells, was found in separate bipolar cell populations. Recoverin immunoreactivity is therefore a characteristic of certain cone bipolar cell types. In rat retina, anti-recoverin labeled two morphologically distinct subpopulations of cone bipolar cells whose axonal arbors stratified at different depths in the inner plexiform layer (IPL). The bipolar cells labeled with anti-recoverin did not correspond to those that were reactive for calbindin, another cone bipolar cell marker. Human and monkey retinas also had two populations of cone bipolar cells that were recoverin-positive. One population showed a distinct pattern of narrow bistratification at the outer border of the IPL and a regular mosaic arrangement of its axonal arbors, suggesting that the entire population of a single cone bipolar type was labeled. Cell density, dendritic morphology, and axonal-field size and stratification indicate that anti-recoverin selectively strains the flat midget (presumed OFF-center) cone bipolar cell type observed previously in Golgi preparations. By contrast the second bipolar cell population had axonal stratification in the inner half of the IPL and showed an unusual but consistent morphology and spatial distribution. Individual cells were intensely stained but were present at an extremely low density (approximately 2-5 cells/mm2). These cells had multibranched dendritic trees characteristic of the diffuse bipolar cell class, but very small axonal fields in the size range of the midget bipolar class. Neither of the two recoverin-positive bipolar cell types in monkey was labeled with anti-calbindin or anti-cholecystokinin. An antibody preparation against bovine pineal hydroxyindole-O-methyltransferase (HIOMT) labeled photoreceptors and bipolar cells that closely resembled the recoverin-positive bipolar cells in human and rat retinas.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Nuclear magnetic resonance evidence for Ca(2+)-induced extrusion of the myristoyl group of recoverin.

Recoverin, a recently discovered member of the EF-hand protein superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group covalently attached to the amino terminus of recoverin enables it to translocate to retinal disc membranes when the Ca2+ level is elevated. Two-dimensional 1H-13C shift correlation NMR spectra of recoverin containing a 13C-labeled myristoyl group were obtained to selectively probe the effect of Ca2+ on the environment of the attached myristoyl group. In the Ca(2+)-free state, each pair of methylene protons bonded to carbon atoms 2, 3, 11, and 12 of the myristoyl group gives rise to two peaks. The splittings, caused by nonequivalent methylene proton chemical shifts, indicate that the myristoyl group interacts intimately with the protein in the Ca(2+)-free state. By contrast, only one peak is seen for each pair of methylene protons in the Ca(2+)-bound state, indicating that the myristoyl group is located in an isotropic environment in this form. Furthermore, the 1H-13C shift correlation NMR spectrum of Ca(2+)-bound recoverin is very similar to that of myristic acid in solution. 1H-(13)C shift correlation NMR experiments were also performed with 13C-labeled recoverin to selectively probe the resonances of methyl groups in the hydrophobic core of the protein. The spectrum of Ca(2+)-bound myristoylated recoverin is different from that of Ca(2+)-free myristoylated recoverin but similar to that of Ca(2+)-bound unmyristoylated recoverin. Hence, the myristoyl group interacts little with the hydrophobic core of myristoylated recoverin in the Ca(2+)-bound state. Three-dimensional (13C/F1)-edited (13C/F3)-filtered heteronuclear multiple quantum correlation-nuclear Overhauser effect spectroscopy spectra of recoverin containing a 13C-labeled myristoyl group were obtained to selectively probe protein residues located within 5 A of the myristoyl group. The myristoyl group makes close contact with a number of aromatic residues in Ca(2+)-free recoverin, whereas the myristoyl group makes no observable contacts with the protein in the Ca(2+)-bound state. These NMR data demonstrate that the binding of Ca2+ to recoverin induces the extrusion of its myristoyl group into the solvent, which would enable it to interact with a lipid bilayer or a hydrophobic site of a target protein.

Calcium↗

PubMed1999

Unique retina cell phenotypes revealed by immunological analysis of recoverin expression in rat retina cells.

Among retina-specific proteins, recoverin is unique with respect to its cellular regulation in that it is found in rods, cones, some bipolar cells, and a rare population of cells in the ganglion cell layer. Recoverin is a calcium-binding protein which inhibits rhodopsin kinase from phosphorylating rhodopsin. Because cells in the inner layers of the retina do not contain rhodopsin kinase, arrestin, or other phototransduction proteins, it seems likely that recoverin has a different function in those cell types. To study this protein more fully, antibodies were generated against the entire mouse recoverin protein, as well as against peptides from the amino and from the carboxyl termini. These antibodies confirmed the localization of recoverin in vivo and clearly demonstrated, in culture, cells which were recoverin positive and rhodopsin negative. Surprisingly, two unique cell phenotypes were seen in cell culture which are not found in vivo. These cells are [rhodopsin(+), recoverin(-)] and [arrestin(+), recoverin(-)]. These phenotypes appear to represent the same population of cells and suggest that the recoverin gene can be regulated independent of genes for other phototransduction proteins. This cell culture system will be useful for investigating environments and factors which participate in the expression of the recoverin gene, and may identify regulatory features of the recoverin gene which cause it to be illicitly expressed in small-cell lung carcinomas in cancer-associated retinopathy (CAR).

Amino Acid Sequence↗

PubMed2000

Recoverin negative photoreceptor cells.

Recoverin, a calcium-binding protein, is unique with respect to its cellular regulation. It is present in retinal rods, cones, cone bipolar cells, and in a rare population of cells in the ganglion cell layer. Inappropriate turn-on or turn-off of recoverin expression has been reported both in small cell lung carcinoma cells from patients with cancer-associated retinopathy (Matsusara et al. [1996] Br. J. Cancer 74:1419-1422; Adamus et al. [1998] J. Autoimmun. 11: 523-533; Ohguro et al. [1999] Invest. Ophthalmol. Vis. Sci. 40:82-89) and in cultured retinal neurons (McGinnis et al. [1999] J. Neursci. Res. 55:252-260). In a recent report using double labeling immunofluorescence microscopy methods with antibodies against either rhodopsin and recoverin or arrestin and recoverin, two unique cell phenotypes, rhodopsin-positive and recoverin-negative, and arrestin-positive, and recoverin-negative were observed in vitro. These two unique cell types could be nonphotoreceptor cells in which rhodopsin and arrestin are inappropriately turned on or they are photoreceptor cells in which the recoverin gene is inappropriately turned off. In this study, multiple antibodies were used to study, on a single-cell basis, whether the photoreceptor cell-specific marker, rhodopsin, is inappropriately expressed in nonphotoreceptor cells in our retinal neuronal culture system. We also examined the hypothesis that the two unique cell phenotypes represent the same population of cells. A triple labeling method has been established to visualize recoverin, rhodopsin, and arrestin protein expression simultaneously in cultured retinal neurons. Our data clearly and directly demonstrate that the previously described unique cell phenotypes are the same population of cells, rod photoreceptors. The existence of recoverin-negative photoreceptors demonstrates that the recoverin gene can be regulated independently of other photoreceptor cell-specific proteins and suggests that this primary cell culture may be useful as a model system for investigating the illicit expression of the recoverin gene in cancer associated retinopathy.

Animals↗

PubMed1992

Recoverin in pineal organs and retinae of various vertebrate species including man.

Recoverin is a recently discovered 26 kDa calcium-binding protein, which activates guanylate cyclase in retinal photoreceptors when the intracellular concentration of free calcium drops upon photoexcitation. In this study we examined the distribution of recoverin in retinae and pineal organs of Xenopus laevis larvae, 1-day-old chicken, adult pigeon, albino rat, sheep and man by means of immunocytochemistry. Recoverin immunoreaction was found in all species investigated except for the chicken. In the retina, recoverin immunoreaction was restricted to photoreceptors; all other cell types were immunonegative. In the pineal organ, the recoverin immunoreaction labeled 'pinealocytes of the sensory line', i.e. classical pineal photoreceptors of Xenopus laevis larvae, modified pineal photoreceptors of pigeon, and pinealocytes of mammals. The number of recoverin immunoreactive pinealocytes varied considerably among species of mammals: very few cells were stained in the rat pineal organ, whereas in rabbit, sheep and man, numerous pinealocytes were found to be recoverin-immunoreactive. No immunocytochemical staining was observed after preabsorption of the recoverin antibody with the recombinant protein. Immunoblotting experiments showed that the immunoreaction is due to a protein of 26 kDa in both retina and pineal tissue. Thus, recoverin appears to belong to the family of proteins which are expressed in both retina and pineal organ and are highly conserved in the course of phylogeny. Recoverin may be involved in phototransduction in the directly light-sensitive pineal organs of poikilothermic vertebrates and birds. However, the functional role of recoverin in the mammalian pineal organ, which is not photosensitive, remains unknown.

Animals↗

PubMed1995

Recoverin, a photoreceptor-specific calcium-binding protein, is expressed by the tumor of a patient with cancer-associated retinopathy.

Recoverin is a member of the EF-hand family of calcium-binding proteins involved in the transduction of light by vertebrate photoreceptors. Recoverin also was identified as an autoantigen in the degenerative disease of the retina known as cancer-associated retinopathy (CAR), a paraneoplastic syndrome whereby immunological events lead to the degeneration of photoreceptors in some individuals with cancer. In this study, we demonstrate that recoverin is expressed in the lung tumor of a CAR patient but not in similar tumors obtained from individuals without the associated retinopathy. Recoverin was identified intially by Western blot analysis of the CAR patient's biopsy tissue by using anti-recoverin antibodies generated against different regions of the recoverin molecule. In addition, cultured cells from the biopsy tissue expressed recoverin, as demonstrated by reverse transcription-PCR using RNA extracted from the cells. The immunodominant region of recoverin also was determined in this study by a solid-phase immunoassay employing overlapping heptapeptides encompassing the entire recoverin sequence. Two linear stretches of amino acids (residues 64-70, Lys-Ala-Tyr-Ala-Gln-His-Val; and 48-52, Gln-Phe-Gln-Ser-Ile) made up the major determinants. One of the same regions of the recoverin molecule (residues 64-70) also was uniquely immunopathogenic, causing photoreceptor degeneration upon immunization of Lewis rats with the corresponding peptide. These data demonstrate that the neural antigen recoverin more than likely is responsible for the immunological events associated with vision loss in some patients with cancer. These data also establish CAR as one of the few autoimmune-mediated diseases for which the specific self-antigen is known.

Amino Acid Sequence↗

PubMed1997

Functional significance of recoverin localization in multiple retina cell types.

Knowledge of the cellular localization of recoverin in photoreceptor cells has enabled its interaction with other proteins to be postulated, tested and verified. Recoverin, a calcium sensing protein, is now thought to act by prolongation of the light state through interference with the interaction of arrestin and rhodopsin. Because of the detection of recoverin in multiple cell populations, the specificity of the cellular localization of recoverin was investigated in the retina of the mouse, rat, rabbit, chicken, frog, and chameleon and compared to that for opsin, phosducin, and arrestin. In addition to photoreceptor cell staining, the application of affinity-purified antibodies against recoverin demonstrated immunoreactive cells in the inner nuclear layer and a rare immunopositive cell in the ganglion cell layer of the mouse, rat and rabbit retina. Only photoreceptor cells were stained with recoverin antibodies in the chameleon and frog retina, whereas no cells were recoverin-positive in the chicken retina. In all six species studied, only photoreceptor cells were labelled with antibodies against opsin, phosducin or arrestin. Based on intensity of staining, two distinct populations of anti-recoverin-immunoreactive cells were distinguished in the photoreceptor cell layer of the retinas of the rat and rabbit, with the more darkly stained cells (probably cones) representing about 3% of the photoreceptor cells. The presence of recoverin in cells other than photoreceptors suggests it has an alternative or additional function and indicates the presence of multiple cell type-specific expression signals in the regulatory region of the recoverin gene.

Amino Acid Sequence↗

PubMed1996

Recoverin in cultured human retinoblastoma cells: enhanced expression during morphological differentiation.

Recoverin is a calcium-binding protein expressed in retinal photoreceptors. It appears to delay the termination of the phototransduction cascade by blocking the phosphorylation of photoexcited rhodopsin. The goal of this study was to determine if recoverin mRNA and protein are expressed in cultured human Y79 retinoblastoma cells, so that this cell line could be used as a model to study the mechanism of recoverin gene expression in the retina. A cDNA encoding human recoverin was PCR cloned and used for prokaryotic expression of recoverin protein. Polyclonal antibodies raised against pure recombinant recoverin were used for western blotting and immunocytochemistry of Y79 cells grown as attachment cultures in the presence of the differentiating agents dibutyryl cyclic AMP (dbcAMP) or butyrate. Northern blot analysis was performed on mRNA extracted from Y79 cells that were also treated with the differentiating agents. In Y79 cell monolayer cultures, recoverin was immunolocalized to the cell cytoplasm, and immunoreactivity was increased dramatically by the addition of 2 mM butyrate to the culture medium. Butyrate treatment also caused an increase in the development of neurite-like cellular processes. Addition of 4 mM dbcAMP resulted in a moderate increase in both recoverin immunoreactivity and number of cellular processes. Western and northern blots of butyrate and dbcAMP-treated Y79 cell cultures demonstrated an increase in recoverin protein and RNA expression, respectively, comparable with that observed with immunocytochemistry. These data suggest that, under the influence of the differentiating agent butyrate, Y79 cells exhibit an increase in expression of the photoreceptor protein recoverin and a concomitant morphological differentiation toward a neuronal phenotype.

Antibody Specificity↗

PubMed2000

Aberrant expression of photoreceptor-specific calcium-binding protein (recoverin) in cancer cell lines.

Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions to retinal antigens aberrantly expressed in tumor cells lead to the degeneration of retinal photoreceptor cells. In our previous study (H. Ohguro et al., Invest. Ophthalmol. Vis. Sci., 40: 82-89, 1999), recoverin, a retina-specific calcium-binding protein, and heat shock cognate protein 70 (hsc 70) were identified as autoantigens recognized by sera from patients with CAR. Therefore, we suggested that autoimmune reactions against both recoverin and hsc 70 might be involved in the pathogenesis of CAR. To elucidate the initial step of the molecular pathology of CAR, we examined the expression of recoverin and hsc 70 by reverse transcription-PCR and Western blot using cell lines of several kinds of cancers, including lung small cell carcinoma, lung adenocarcinoma, gastric cancer, pancreatic cancer, breast cancer, uterine cervical cancer, endometrial cancer, and leukemia. Recoverin was expressed in 21 of the 31 cancer cell lines. The expression levels of hsc 70 were significantly higher in cancer cell lines than in noncancerous cell lines. However, no difference in the expression levels of hsc 70 was observed between recoverin-positive and -negative cell lines. Immunofluorescence labeling by the affinity-purified recoverin antibody revealed the immunoreactivity to recoverin as a granular pattern within the cancer cells. Lung adenocarcinoma A549 cells, which did not express recoverin, exhibited a significant reduction in cell proliferation upon transfection with human recoverin cDNA. Taken together, our present data suggest that the retina-specific calcium-binding protein recoverin is expressed in more than 50% of a variety of cancer cells and may play a significant role in the cell proliferation of these tumor cells.

Antigens, Neoplasm↗

PubMed1994

Recoverin: a potent uveitogen for the induction of photoreceptor degeneration in Lewis rats.

Recoverin is a calcium-binding protein identified as an autoantigen in a paraneoplastic degenerative disease of the human retina known as cancer-associated retinopathy (CAR). In this study we investigated whether recoverin could elicit an immune response leading to the degeneration of photoreceptor cells in a rodent retina, and whether an animal model of CAR could be developed. Injection of Lewis rats with recoverin caused degeneration of the photoreceptor cells. Several features of uveoretinitis were observed, including vitreous cells, perivasculitis, retinal lesions and complete loss of the photoreceptor cell layer. The first clinical signs of retinal inflammation were observed 10-14 days after immunization. The earliest histological changes in the retina also were observed 14 days after immunization. Infiltration of the photoreceptor cell layer and inner layers of the retina with lymphocytic and some polymorphonuclear cells was frequently observed. Photoreceptors were damaged and later fully degenerated. This sequence of events was associated with high antibody titers against recoverin in all animals tested. Cellular responses to recoverin assayed between days 7 and 28 after immunization showed strong in vitro proliferative activities to recoverin. In addition, all aspects of the degenerative events could be reproduced in naive animals by the adoptive transfer of stimulated lymphocytes obtained from animals previously immunized with recoverin. This study demonstrates the successful induction of photoreceptor degeneration using recoverin as an immunogen. We demonstrate that recoverin is both a potent antigen and uveitogen. These observations may be relevant to our understanding of CARs in humans.

Animals↗

PubMed1997

Calcium binding to recoverin: implications for secondary structure and membrane association.

Recoverin is an EF-hand calcium-binding protein reportedly involved in the transduction of light by vertebrate photoreceptor cells. It also is an autoantigen in a cancer-associated degenerative disease of the retina. Measurements by circular dichroism presented here demonstrate that the binding of calcium to recoverin causes large structural changes. increasing the alpha-helical content of the protein and decreasing its beta-turn, beta-sheet and 'other' structures. The maximum helical content (67%) was observed at 100 microM free calcium and, unlike calmodulin, decreased as the calcium concentration was modulated in either direction from this value. Fluorescence measurements indicated that recoverin may aggregate or undergo structural changes independent of calcium binding as the calcium concentration is increased above 100 microM. EGTA also appeared to affect the structure of recoverin independent of its chelation of calcium. While calcium-induced conformational changes have been proposed to alter the membrane binding of recoverin through association of its myristoylated amino terminus, in the experiments presented here the partitioning of recoverin between the cytoplasmic and membrane compartments of the rod photoreceptor outer segment was unaffected by the concentration of calcium, therefore it appears unlikely that a calcium-myristoyl switch acts alone to anchor recoverin directly to the membrane. These experiments were conducted with native recoverin which is heterogeneously acylated, but mass spectrometry confirmed that simple chromatographic methods could be devised to isolate the different forms of recoverin for further studies.

Animals↗

PubMed1995

Role of heterogeneous N-terminal acylation of recoverin in rhodopsin phosphorylation.

Recoverin, a new member of the EF-hand superfamily, plays a critical role in the light/dark adaptation of retinal rods by regulating rhodopsin phosphorylation in a Ca(2+)-dependent manner. Recoverin is composed of four isoforms, each of which is modified at its N terminus by myristate (C14:0) or its structurally related fatty acid (C12:0, C14:2, or C14:1). Although the N-fatty acylation is implicated in protein-membrane and protein-protein interactions, the functional difference among the recoverin isoforms and the significance of the heterogeneous acylation have not been defined. Here we separated the heterogeneous recoverin into three fractions, C14:0-recoverin, C14:1-recoverin, and a mixture of C14:2- and C12:0- (C14:2/C12:0-) recoverin to evaluate the individual properties. Recoverin in every fraction bound Ca2+ as assessed by fluorescence spectroscopy and inhibited the light-dependent rhodopsin phosphorylation in the same range of free Ca2+ concentration (0.3-0.8 microM). However, the magnitude of the inhibition at higher Ca2+ concentration was different among the isoforms and ranked in the same order of the hydrophobicity of the N-fatty acyl groups: C14:0 > C14:1 > C14:2/C12:0. These results indicate that the diverged hydrophobicity of the recoverin N terminus plays an important role in the interaction with the membranes and/or its target protein but not with Ca2+.

Acylation↗

PubMed1995

Amino-terminal myristoylation induces cooperative calcium binding to recoverin.

Recoverin, a new member of the EF-hand protein superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group covalently attached to the amino terminus of recoverin enables it to bind to disc membranes when the Ca2+ level is elevated. Ca(2+)-bound recoverin prolongs the lifetime of photoexcited rhodopsin, most likely by blocking its phosphorylation. We report here Ca2+ binding studies of myristoylated and unmyristoylated recombinant recoverin using flow dialysis, fluorescence, and NMR spectroscopy. Unmyristoylated recoverin exhibits heterogeneous and uncooperative binding of two Ca2+ with dissociation constants of 0.11 and 6.9 microM. In contrast, two Ca2+ bind cooperatively to myristoylated recoverin with a Hill coefficient of 1.75 and an apparent dissociation constant of 17 microM. Thus, the attached myristoyl group lowers the calcium affinity of the protein and induces cooperativity in Ca2+ binding. One-dimensional 1H and two-dimensional 15N-1H shift correlation NMR spectra of myristoylated recoverin measured as a function of Ca2+ concentration show that a concerted conformational change occurs when two Ca2+ are bound. The Ca2+ binding and NMR data can be fit to a concerted allosteric model in which the two Ca2+ binding sites have different affinities in both the T and R states. The T and R conformational states are defined in terms of the Ca(2+)-myristoyl switch; in the T state, the myristoyl group is sequestered inside the protein, whereas in the R state, the myristoyl group is extruded. Ca2+ binds to the R state at least 10,000-fold more tightly than to T. In this model, the dissociation constants of the two sites in the R state of the myristoylated protein are 0.11 and 6.9 microM, as in unmyristoylated recoverin. The ratio of the unliganded form of T to that of R is estimated to be 400 for myristoylated and < 0.05 for unmyristoylated recoverin. Thus, the attached myristoyl group has two related roles: it shifts the T/R ratio of the unliganded protein more than 8000-fold, and serves as a membrane anchor for the fully liganded protein.

Allosteric Site↗

PubMed1993

Role of the acylated amino terminus of recoverin in Ca(2+)-dependent membrane interaction.

Recoverin, a calcium ion (Ca2+)-binding protein of vertebrate photoreceptors, binds to photoreceptor membranes when the Ca2+ concentration is greater than 1 micromolar. This interaction requires a fatty acyl residue covalently linked to the recoverin amino (NH2)-terminus. Removal of the acyl residue, either by proteolytic cleavage of the NH2-terminus or by production of nonacylated recoverin, prevented recoverin from binding to membranes. The acylated recoverin NH2-terminus could be cleaved by trypsin only when Ca2+ was bound to recoverin. These results suggest that the hydrophobic NH2-terminus is constrained in Ca(2+)-free recoverin and liberated by Ca2+ binding. The hydrophobic acyl moiety of recoverin may interact with the membrane only when recoverin binds Ca2+.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1998

Antibodies to recoverin induce apoptosis of photoreceptor and bipolar cells in vivo.

Autoantibodies against recoverin are found in the sera of patients with cancer-associated retinopathy (CAR) syndrome. In these studies we examined the effect of anti-recoverin antibodies from the sera of patients with CAR and rat monoclonal antibody on the retinas of Lewis rats. Anti-recoverin autoanti-bodies penetrated into the photoreceptor and bipolar cell layers following intravitreal injection. Their presence in the retina could be detected by immunofluorescence 24 h after injection. At the same time, individual cells undergoing apoptosis were identified throughout photoreceptor and bipolar cell layers using terminal transferase-mediated dUTP nick-end labeling (TUNEL) and electron microscopy. Normal antibodies used in control experiments did not produce TUNEL labeling. At 24 h, DNA fragmentation was confirmed by DNA ladder electrophoresis. At the electron microscopic level, there was clear evidence of cells undergoing apoptotic cell death in the retinas treated with anti-recoverin antibodies. At 24 and 96 h, nuclear chromatin condensation and increased vacuolization of photoreceptor outer segments were observed. An examination of retinas from animals receiving anti-retinal antibodies revealed a loss of 1-2 rows of nuclei in the outer and inner nuclear layers whereas all controls (sham, normal IgG, phosphate buffered saline) showed an unchanged number of nuclei rows. In addition, there was an increase in spacing between the rows of nuclei of the outer nuclear layer in retinas treated with anti- recoverin antibodies, indicating additional cell loss. These studies provide clear evidence that anti-recoverin antibodies are capable of penetrating photoreceptor and bipolar cells, the normal site of recoverin expression in the retina, and that anti-recoverin antibodies produce apoptotic cell death. A similar mechanism may occur in patients with CAR, which may lead to visual loss and blindness.

Animals↗

PubMed1997

Calcium-dependent binding of recoverin to membranes monitored by surface plasmon resonance spectroscopy in real time.

Recoverin is an N-myristoylated Ca2+-binding protein that serves as a Ca2+-sensor in visual transduction. We studied the dynamics of its Ca2+-dependent membrane association which depends on the myristoyl modification (Ca2+-myristoyl switch) by surface plasmon resonance spectroscopy. Either recoverin or phospholipid vesicles were immobilized on a sensor chip surface, and the respective binding partner was supplied in the mobile phase. Binding of recoverin to artificial liposomes or rod outer segment membranes was strictly dependent on Ca2+ and the myristoyl group. The Ca2+-myristoyl switch was half-maximal between 4.0 and 7.7 microM Ca2+, depending on whether recoverin or liposomes were in the mobile phase. At saturating [Ca2+], the dissociation constant (KD) of recoverin for phospholipid liposomes was approximately 150 microM. The association and dissociation of recoverin to membranes was fast and biphasic (fast and slow components) with time constants on the order of 0.1 s-1 and 0.01 s-1, respectively. Dissociation of the recoverin-membrane complex was 3-fold faster at low than at high free [Ca2+]. We discuss the analogy between the liposome-sensor chip and the disk surface and conclude that a minor fraction of the total recoverin in a rod outer segment is associated with membranes at resting dark levels of free [Ca2+].

Animals↗

PubMed1996

Expression of a photoreceptor protein, recoverin, as a cancer-associated retinopathy autoantigen in human lung cancer cell lines.

Recently, a photoreceptor protein, recoverin, has been recognised as an autoantigen of cancer-associated retinopathy (CAR), a rare paraneoplastic neurological syndrome often associated with patients with small-cell lung cancer (SCLC). Although until quite recently the specific expression of recoverin in cancer cells had not been indicated, Polans et al. (Polans AS, Witkowska D, Haley TL, Amundson D, Baizer L, Adamus G 1995, Proc. Natl. Acad. Sci. USA, 92, 9176-9180) demonstrated the specific expression of recoverin in lung tumour and primary cultured tumour cells from a CAR patient. We examined the expression of recoverin in human lung cancer cell lines by reverse transcription polymerase chain reaction (PCR), Northern blotting and Western immunoblotting. Recoverin was expressed in only one SCLC cell line from a patient with CAR. The sequence of recoverin cDNA from the cells was identical to the human recoverin sequence. These findings strongly support the hypothesis that the ectopic expression of wild-type recoverin in SCLC induces the cancer-retina immunological cross-reaction, leading to visual loss in CAR.

Biomarkers, Tumor↗