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PubMed2001

Retinal degeneration under the effect of antibodies to recoverin.

Serious changes in the retina, diagnosed as retinal degeneration and uveitis, are observed only in the presence of high titers of antibodies to recoverin (Ca2+-binding protein, a paraneoplastic antigen) in the blood of rabbits. Negligible changes in the retina of rabbits with low antibody titers are detected only by cytohistochemical analysis of the retina. No changes in the retina develop in rabbits intravenously injected with antibodies to recoverin.

Animals↗
PubMedDate not supplied

The neurons of the ground squirrel retina as revealed by immunostains for calcium binding proteins and neurotransmitters.

Ground squirrel retinas were immunostained with antibodies against calcium binding proteins (CBPs) and classical neurotransmitters in order to describe neuronal phenotypes in a diurnal mammalian retina and to then compare these neurons with those of more commonly studied nocturnal retinas like cats' and rabbits'. Double immunostained tissue was examined by confocal microscopy using antibodies against the following: rhodopsin and the CBPs, calbindin, calretinin, parvalbumin, calmodulin and recoverin (CB, CR, PV, CM, RV), glycine, GABA, choline acetyltransferase (CHAT) and tyrosine hydroxylase (TOH). In ground squirrel retina, the traditional cholinergic mirror symmetric amacrine cells colocalize CHAT with PV and GABA and faintly with glycine. A second cholinergic amacrine cell type colocalizes glycine alone. CR is found in at least 3 different amacrine cell types. The CR-immunoreactive (IR) cell population is a mixture of glycinergic and GABAergic types. The dopamine cell type IR to tyrosine hydroxylase has the typical morphology of a wide field cell with dendrites in S1 but the "rings" seen in cat or rabbit retina are not as numerous. TOH-IR amacrine cells send large club-shaped processes to the outer plexiform layer. CB and CR are in bipolar cells, A- and B-type horizontal cells and several amacrine cell types. Anti-rhodopsin labels the low density rod photoreceptor population in this species. Anti-recoverin labels cones and some bipolar cells while PKC is found in several different bipolar cell types. One ganglion cell with dendritic branching in S3 is strongly CR-IR. We find no evidence for an AII amacrine cell in the ground squirrel, with either anti-CR or anti-PV. An amacrine cell with similarity to the DAP1-3 cell of rabbit is CR-IR and glycine-IR. We discuss this labeling pattern in relationship to other mammalian species. The differences in staining patterns and phenotypes revealed suggest a functional diversity in the populations of amacrine cells according to whether the retinas are rod or cone dominated.

Amacrine Cells↗
PubMed2000

Study of conformational rearrangement and refinement of structural homology models by the use of heteronuclear dipolar couplings.

For an increasing fraction of proteins whose structures are being studied, sequence homology to known structures permits building of low resolution structural models. It is demonstrated that dipolar couplings, measured in a liquid crystalline medium, not only can validate such structural models, but also refine them. Here, experimental 1H-15N, 1Halpha-13Calpha, and 13C'-13Calpha dipolar couplings are shown to decrease the backbone rmsd between various homology models of calmodulin (CaM) and its crystal structure. Starting from a model of the Ca2+-saturated C-terminal domain of CaM, built from the structure of Ca2+-free recoverin on the basis of remote sequence homology, dipolar couplings are used to decrease the rmsd between the model and the crystal structure from 5.0 to 1.25 A. A better starting model, built from the crystal structure of Ca2+-saturated parvalbumin, decreases in rmsd from 1.25 to 0.93 A. Similarly, starting from the structure of the Ca2+-ligated CaM N-terminal domain, experimental dipolar couplings measured for the Ca2+-free form decrease the backbone rmsd relative to the refined solution structure of apo-CaM from 4.2 to 1.0 A.

Amino Acid Sequence↗
PubMed1998

Calsenilin: a calcium-binding protein that interacts with the presenilins and regulates the levels of a presenilin fragment.

Most early-onset familial Alzheimer disease (AD) cases are caused by mutations in the highly related genes presenilin 1 (PS1) and presenilin 2 (PS2). Presenilin mutations produce increases in beta-amyloid (Abeta) formation and apoptosis in many experimental systems. A cDNA (ALG-3) encoding the last 103 amino acids of PS2 has been identified as a potent inhibitor of apoptosis. Using this PS2 domain in the yeast two-hybrid system, we have identified a neuronal protein that binds calcium and presenilin, which we call calsenilin. Calsenilin interacts with both PS1 and PS2 in cultured cells, and can regulate the levels of a proteolytic product of PS2. Thus, calsenilin may mediate the effects of wild-type and mutant presenilins on apoptosis and on Abeta formation. Further characterization of calsenilin may lead to an understanding of the normal role of the presenilins and of the role of the presenilins in Alzheimer disease.

Age of Onset↗
PubMed1993

Rhodopsin phosphorylation as a mechanism of cyclic GMP phosphodiesterase regulation by S-modulin.

During light-adaptation by the vertebrate eye, the rods are desensitized and the light response is accelerated. When light is absorbed by the rods, a phosphodiesterase is activated that hydrolyses cyclic GMP. A light-induced decrease in cytoplasmic Ca2+ concentration is part of this light-adaptation process. The protein S-modulin (M(r) 26,000) is known to increase the fraction of light-activated cyclic GMP-phosphodiesterase (PDE) at high Ca2+ concentrations in frog rod photoreceptors. Here I present evidence that S-modulin lengthens the lifetime of active PDE (PDE*) at high Ca2+ concentrations. These S-modulin effects are observed in the physiological range of Ca2+ concentration (30 nM to 1 microM; half-maximum effects at 200-400 nM). At the high Ca2+ concentrations at which S-modulin prolongs the lifetime of PDE*, S-modulin inhibits rhodopsin phosphorylation (half-maximum effect at approximately 100 nM Ca2+). ATP is necessary for the S-modulin effects on PDE activation. I therefore conclude that the Ca(2+)-dependent regulation of PDE by S-modulin is mediated by rhodopsin phosphorylation. This regulation seems to be the principal mechanism of light adaptation in vertebrate photoreceptors.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

Sequestration of the membrane-targeting myristoyl group of recoverin in the calcium-free state.

Recoverin, a retinal calcium-binding protein of relative molecular mass (M(r)) 23K, participates in the recovery phase of visual excitation and in adaptation to background light. The Ca(2+)-bound form of recoverin prolongs the photoresponse, probably by blocking phosphorylation of photoexcited rhodopsin. Retinal recoverin contains a covalently attached myristoyl group or related acyl group at its amino terminus and two Ca(2+)-binding sites. Ca2+ binding to myristoylated, but not unmyristoylated, recoverin induces its translocation to bilayer membranes, indicating that the myristoyl group is essential to the read-out of calcium signals (calcium-myristoyl switch). Here we present the solution structure of Ca(2+)-free, myristoylated recombinant recoverin obtained by heteronuclear multidimensional NMR spectroscopy. The myristoyl group is sequestered in a deep hydrophobic pocket formed by many aromatic and other hydrophobic residues from five flanking helices.

Amino Acid Sequence↗
PubMed1997

Molecular mechanics of calcium-myristoyl switches.

Many eukaryotic cellular and viral proteins have a covalently attached myristoyl group at the amino terminus. One such protein is recoverin, a calcium sensor in retinal rod cells, which controls the lifetime of photoexcited rhodopsin by inhibiting rhodopsin kinase. Recoverin has a relative molecular mass of 23,000 (M[r] 23K), and contains an amino-terminal myristoyl group (or related acyl group) and four EF hands. The binding of two Ca2+ ions to recoverin leads to its translocation from the cytosol to the disc membrane. In the Ca2+-free state, the myristoyl group is sequestered in a deep hydrophobic box, where it is clamped by multiple residues contributed by three of the EF hands. We have used nuclear magnetic resonance to show that Ca2+ induces the unclamping and extrusion of the myristoyl group, enabling it to interact with a lipid bilayer membrane. The transition is also accompanied by a 45-degree rotation of the amino-terminal domain relative to the carboxy-terminal domain, and many hydrophobic residues are exposed. The conservation of the myristoyl binding site and two swivels in recoverin homologues from yeast to humans indicates that calcium-myristoyl switches are ancient devices for controlling calcium-sensitive processes.

Calcium↗
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↗
PubMed2003

Genetic manipulations of adenovirus type 5 fiber resulting in liver tropism attenuation.

The development of genetically modified adenoviral vectors capable of specifically transducing a given cell population requires the addition and functional presentation of particular tropism determinants within the virus capsid, together with the abrogation of the molecular determinants that dictate their natural tropism in vivo. The human adenovirus serotype 5 (Ad5) first attaches to the cell surface following high-affinity binding of the C-terminal knob of the fiber capsid protein to the coxsackie and adenovirus receptor (CAR). Here we have assessed whether genetic shortening of the fiber shaft (virus BS1), or replacing the Ad5 fiber shaft and knob with their Ad3 counterparts (virus DB6), could cripple this interaction in vitro and in vivo. A 10-fold decrease in the binding of the modified capsids to soluble CAR was evidenced, which correlated with a similar reduction of their ability to transduce CAR-positive cells in vitro. The ability of BS1 to interact with cellular integrins was also impaired, suggesting that the penton base and the short-shafted fiber when embedded in the capsid preclude each other from efficiently interacting with their cognate cell surface receptors (CAR and integrins respectively). BS1 and DB6 intravenous injections in mice further supported a profound impairment of the ability of the capsid-modified viruses to transduce the liver as demonstrated by a 10-fold reduction of intracellular viral DNA and transgene expression. Interestingly enough, the host humoral response was also specifically weakened in BS1- and DB6-inoculated animals. Taken together, these observations indicate that (i) fiber shortening and (ii) pseudo-typing of Ad5-based vectors with the shaft and knob from non-CAR-binding serotypes constitute two promising strategies to successfully attenuate their native tropism in vitro and most importantly in vivo.

Animals↗
PubMed2003

CAR is a cell-cell adhesion protein in human cancer cells and is expressionally modulated by dexamethasone, TNFalpha, and TGFbeta.

The coxsackie adenovirus receptor (CAR) has become of interest for gene therapy due to its crucial function in adenoviral cell entry. In clinical trials with adenoviral vectors, dexamethasone is applied to reduce side effects such as inflammatory reactions or emesis. By using a beta-galactosidase-expressing adenovirus (AdGal), we observed that dexamethasone treatment resulted in decreased adenoviral gene transfer into human cancer cells. Expression of CAR and integrin alpha5beta1 was transcriptionally downregulated by dexamethasone as shown for HeLa cervical cancer cells and U87MG glioblastoma cells. TNFalpha increased CAR expression in HeLa and ovarian cancer cells but decreased CAR expression in U87MG cells. In all tested cancer cell lines, TNFalpha induced a significant increase in the expression of adenovirus-binding integrins alpha5beta1, alphavbeta3 and alphavbeta5. Pretreatment with TNFalpha increased AdGal gene transfer into cancer cells and enhanced the cytotoxic effect of a p53-expressing adenovirus. In contrast, TGFbeta reduced CAR expression level and adenoviral gene transfer into OV-UL-2 ovarian cancer cells. Confocal immunofluorescence analysis revealed localization of CAR at cell-cell adhesions in several human cancer cell lines and disruption of cell-cell contacts increased adenoviral gene transfer into human cancer cells. In clinical cancer gene therapy, efficiency of adenoviral gene delivery could be altered by cell adhesion, TNFalpha, TGFbeta, and dexamethasone.

Adenoviridae↗
PubMed2003

Neuronal calcium sensor proteins are direct targets of the insulinotropic agent repaglinide.

The NCS (neuronal calcium sensor) proteins, including neurocalcins, recoverins and visinin-like proteins are members of a family of Ca2+-sensitive regulators, each with three Ca2+-binding EF-hand motifs. In plants, lily CCaMK [chimaeric Ca2+/CaM (calmodulin)-dependent protein kinase] and its PpCaMK ( Physcomitrella patens CCaMK) homologue are characterized by a visinin-like domain with three EF-hands. In the present study, in an effort to discover NCS antagonists, we screened a total of 43 compounds using Ca2+-dependent drug affinity chromatography and found that the insulinotropic agent repaglinide targets the NCS protein family. Repaglinide was found to bind to NCS proteins, but not to CaM or S100 proteins, in a Ca2+-dependent manner. Furthermore, the drug antagonized the inhibitory action of recoverin in a rhodopsin kinase assay with IC50 values of 400 microM. Moreover, repaglinide tightly bound to the visinin-like domain of CCaMK and PpCaMK in a Ca2+-dependent manner and antagonized the regulatory function of the domain with IC50 values of 55 and 4 microM for CCaMK and PpCaMK respectively. Although both repaglinide and a potent insulin secretagogue, namely glibenclamide, blocked K(ATP) channels with similar potency, glibenclamide had no antagonizing effect on the Ca2+-stimulated CCaMK and PpCaMK autophosphorylation, mediated by their visinin-like domain. In addition, a typical CaM antagonist, trifluoperazine, had no effect on the CCaMK and PpCaMK autophosphorylation. Repaglinide appears to be the first antagonist of NCS proteins and visinin-like domain-bearing enzymes. It may serve as a useful tool for evaluating the physiological functions of the NCS protein family. In addition, since repaglinide selectively targets NCS proteins among the EF-hand Ca2+-binding proteins, it is a potential lead compound for the development of more potent NCS antagonists.

Animals↗
PubMed1997

Recoverin inhibits the phosphorylation of dark-adapted rhodopsin more than it does that of bleached rhodopsin: a possible mechanism through which rhodopsin kinase is prevented from participation in a side reaction.

In its resting state rhodopsin kinase is present in an inactive from and is activated after interaction with light-activated rhodopsin (Rho*). The activated rhodopsin kinase then phosphorylates Rho* but is also able to catalyse the phosphorylation of dark-adapted rhodopsin. A consequence of the latter behaviour of the activated kinase is that at low levels of bleach a large number of phosphoryl groups are incorporated per mol of Rho*. Recoverin- and Ca2+-dependent inhibition of rhodopsin kinase was found to be inversely related to the extent of bleaching; the lower the fraction of rhodopsin bleached, the greater the inhibition. The IC50 of recoverin is approx. 1 microM at a 0.2% level of bleach and about 5 microM in a fully bleached sample. The inhibitory effect of recoverin was studied separately on the phosphorylation of rhodopsin and Rho*. The formation of phosphorylated rhodopsin was inhibited 4.5-fold more strongly than that of phosphorylated Rho*. These results are interpreted to suggest that one of the roles of the recoverin-dependent regulation of the activity of rhodopsin kinase is to prevent the enzyme from participating in the unwanted phosphorylation of dark-adapted rhodopsin, directing it to fulfil its 'correct' function of quenching the transduction activity of Rho*.

Calcium↗
PubMed1998

Synaptic clustering of GABA(C) receptor rho-subunits in the rat retina.

Polyclonal antibodies which recognize the rho-subunits of the GABA(C) receptor were applied to sections of the rat retina. Strong punctate immunoreactivity was found in the inner plexiform layer (IPL), which was shown by electron microscopy to represent a clustering of the GABA(C) receptors at synaptic sites. During postnatal development diffuse rho-immunoreactivity was first observed at postnatal day P3. Distinct labelling of bipolar cells appeared at P7 and punctate, synaptic labelling was observed at P10. In order to show that the rho-immunoreactive puncta coincide with the axons of bipolar cells, double immunostainings of retinal sections with an antiserum against syntaxin 3 and with the rho-antiserum were performed. The experiments showed that rho-immunoreactive puncta are preferentially located on the axon terminals of rod and cone bipolar cells. In order to determine whether GABA(C) receptor rho-subunits coassemble with GABA(A) receptor subunits, double-labelling experiments were performed with subunit specific antisera. Punctate, putative synaptic clustering was observed with all antisera applied, however, GABA(C) receptor expressing puncta did not coincide with GABA(A) receptor containing puncta. This suggests that there are no synaptic GABA receptors in the retina in which GABA(A) and GABA(C) receptor subunits are coassembled. Similar double-labelling experiments were also performed to find out whether GABA(C) receptors and glycine receptors are colocalized. They were clustered at different synapses. This suggests that synaptic GABA(C) receptors consist of rho-subunits and are not coassembled with GABA(A)- or glycine-receptor subunits.

Animals↗
PubMed2003

The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retina.

Connexin 36 (Cx36) is a channel-forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double-labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells.

Animals↗
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

[Carcinoma-associated retinopathy: a review with clinical examples].

Cancer-associated retinopathy (CAR) is a rare paraneoplastic syndrome. In this survey we report about two further patients with CAR, who were referred to the University Eye Hospital of Tuebingen within a few months. The most common primary tumor associated with CAR is small cell carcinoma of the lung. Case reports about rhabdomyosarcoma, carcinoma of the endometrium, prostate and mamma were also described. The exact pathogenesis of CAR is still unknown. Specific autoantibodies were found against the photoreceptor protein recovering (23-kd retinal CAR antigen). However, this reaction is not present in all patients, and probably other antigens are also involved. Most of the patients experience symptoms of CAR before the primary tumor is detected. Besides glare sensitivity and flashing lights, a rapidly progressive, often asymmetric visual loss may occur. Although paracentral and mid-peripheral scotomas can be found frequently, visual field defects are often quite heterogeneous. Typically, the responses in the electroretinogram (ERG) are markedly reduced, but normal ERGs were also described. The fundus picture in CAR shows sheathing of the retinal vessels, narrowing of the arterioles and clumbing of the retinal pigment epithelium. The prognosis is poor. Frequently there is progression to bilateral loss of vision within a few months. Treatment of the primary tumor does not seem to alter the ocular prognosis. Systemic corticosteroids may be helpful in some patients. Nevertheless, no proven therapeutic regimen is currently available.

Aged↗
PubMed1992

Cloning, expression, and crystallization of recoverin, a calcium sensor in vision.

Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range. We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library. The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter. A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture). The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97). Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum. The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies. The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A. These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A. The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.

Amino Acid Sequence↗