Recoverin Gene Research Articles

Interaction of G protein-coupled receptor kinases and recoverin isoforms is determined by localization in zebrafish photoreceptors

The zebrafish retina expresses four recoverin genes (rcv1a, rcv1b, rcv2a and rcv2b) and four opsin kinase genes (grk1a, grk1b, grk7a and grk7b) coding for recoverin and G protein-coupled receptor kinase (opsin kinase) paralogs, respectively. Both protein groups are suggested to form regulatory complexes in rod and cone outer segments, but at present, we lack information about co-localization of recoverin and opsin kinases in zebrafish retinae and which protein-protein interacting pairs form. We analyzed the distribution and co-localization of recoverin and opsin kinase expression in the zebrafish retina. For this purpose, we used custom-tailored monospecific antibodies revealing that the amount of recoverin paralogs in a zebrafish retina can differ by more than one order of magnitude with the highest amount for recoverin 1a and 2b. Further, immunohistochemical labelling showed presence of recoverin 1a in all rod cell compartments, but it only co-localized with opsin kinase 1a in rod outer segments. In contrast, recoverin 2b was only detected in double cones and co-localized with opsin kinases 1b, 7a and 7b. Further, we investigated the interaction between recoverin and opsin kinase variants by surface plasmon resonance spectroscopy indicating interaction of recoverin 1a and recoverin 2b with all opsin kinases. However, binding kinetics for recoverin 1a differed from those observed with recoverin 2b that showed slower association and dissociation processes. Our results indicate diverse recoverin and opsin kinase properties due to differential expression and interaction profiles.

Effect of let-7a overexpression on the differentiation of conjunctiva mesenchymal stem cells into photoreceptor-like cells.

Objective(s):MicroRNAs (miRNAs) could regulate many cellular processes such as proliferation and differentiation. let-7a miRNA is one of the key regulators in the developmental transition of retinal progenitor cells into differentiated cells. Current evidence suggests that mesenchymal stem cells (MSCs) can isolate from various tissues such as bone marrow and conjunctiva. In this study, we investigated the effect of let-7a overexpression on induced differentiation of conjunctiva mesenchymal stem cells (CJMSCs) into photoreceptor-like cells.Materials and Methods:After isolation and characterization, CJMSCs were transduced with lentiviruses containing let-7a or empty vector. The effect of let-7a overexpression on expression of photoreceptor-specific markers was evaluated by quantitative real-time PCR (RT-qPCR) after 28 and 42 days of transduction.Results:The relative expression of rhodopsin and recoverin genes was evaluated by RT-qPCR in let-7a overexpressing cells, control vector transduced cells and untransduced CJMSCs (control cells). Our results indicated that following overexpression of let-7a, after 28 and 42 days of transduction, significant up-regulation in the expression of recoverin (574.7 and 43.9 folds) and rhodopsin (3334.7 and 53.1 folds) were observed, respectively.Conclusion:Our findings indicate that overexpression of let-7a microRNA can increase the expression of photoreceptor-specific genes in CJMSCs. Moreover, it is prospective that let-7a overexpression can use as an alternative protocol for the differentiation of mesenchymal stem cells into photoreceptors. It seems that the effect of let-7a on the differentiation of CJMSCs into photoreceptors is also time-dependent.

The cancer-retina antigen recoverin as a potential biomarker for renal tumors.

The renal cell carcinoma is the ninth most common cancer with an increasing occurrence and mortality. Recoverin is the first retina-specific photoreceptor protein that was shown to undergo aberrant expression, due to its promoter demethylation, as a cancer-retina antigen in a number of malignant tumors. In this work, we demonstrated that recoverin is indeed expressed in 68.4% of patients with different subtypes of renal cell carcinoma, and this expression has tendency to correlate with tumor size. Interestingly, 91.7% of patients with the benign renal tumor, oncocytoma, express recoverin as well in their tumor. Epigenetic analysis of the recoverin gene promoter revealed a stable mosaic methylation pattern with the predominance of the methylated state, with the exception of -80 and 56 CpG dinucleotides (CpGs). While the recoverin expression does not correlate withoverall survival of the tumor patients, the methylation of the recoverin gene promoter at -80 position is associated with better overall survival of the patients. This work is the first report pointing towards the association of overall survival of renal cell carcinoma (RCC) patients with promoter methylation of a cancer-retina antigen. Taken together, these data allow to consider recoverin as a potential therapeutic target and/or marker for renal tumors.

Aberrant Stratifin Overexpression Is Regulated by Tumor-Associated CpG Demethylation in Lung Adenocarcinoma

We previously have shown the aberrant overexpression of stratifin (SFN, 14-3-3 ς) in lung adenocarcinoma. Although SFN is known to facilitate tumor cell proliferation, the mechanism that underlies its aberrant expression has remained unclear. SFN, the downstream target of p53, often has been reported to be hypermethylated and subsequently silenced in certain cancers; however, its hypomethylation-linked reactivation has not yet been validated. In this study, we investigated the DNA methylation status of the SFN promoter region using 8 lung cancer cell lines and 32 specimens of adenocarcinoma tissue. Real-time methylation-specific PCR analysis showed that although both normal lung tissue and adenocarcinoma in situ bore a completely methylated SFN promoter, the promoter region in almost all invasive adenocarcinomas was at least partially methylated. The expression of SFN and its level of methylation were correlated strongly. Furthermore, statistical analysis revealed that the level of methylation became reduced with progression of the pathologic stage, although no clear relationship between methylation level and p53 abnormality was found. These results suggest that methylation-related silencing of SFN occurs in both normal lung tissues and adenocarcinoma in situ, and that demethylation of the SFN promoter participates in the aberrant expression of SFN in invasive adenocarcinoma cells, independently of p53 alteration. This novel finding might be informative for clarifying the mechanism that underlies the progression of early lung adenocarcinoma.

Aberrant demethylation of the recoverin gene is involved in the aberrant expression of recoverin in cancer cells

The Ca(2+) -binding protein recoverin is normally specific for the retina. Recoverin aberrantly expressed in lung and melanoma tumors can trigger the host immune response followed by the development of a paraneoplastic neurological syndrome represented by cancer- and melanoma-associated retinopathy, respectively. The mechanisms, underlying the aberrant expression of recoverin in tumor cells, have remained unknown. The data obtained in this study suggest that (i) DNA methylation participates in the repression of synthesis of mRNA for recoverin in normal tissues and (ii) aberrant hypomethylation of the recoverin gene region, overlapping the promoter up-stream of the first exon and the first exon itself, is involved in the aberrant expression of recoverin in tumor cells.

Recoverin Binds Exclusively to an Amphipathic Peptide at the N Terminus of Rhodopsin Kinase, Inhibiting Rhodopsin Phosphorylation without Affecting Catalytic Activity of the Kinase

Recoverin is a calcium-dependent inhibitor of rhodopsin kinase. It prevents premature phosphorylation of rhodopsin until the opening of cGMP-gated ion channels causes a decrease in intracellular calcium levels, signaling completion of the light response. This calcium depletion causes release of recoverin from rhodopsin kinase, freeing the kinase to phosphorylate rhodopsin and to terminate the light response. Previous studies have shown that recoverin is able to bind to a region at the N terminus of rhodopsin kinase. In this study we map this interaction interface, showing that residues 1-15 of the kinase form the interaction site for recoverin binding. Mutation of hydrophobic residues in this region have the greatest effect on the interaction. The periodic nature of these residues suggests that they lie along one face of an amphipathic helix. We show that this region is essential for recoverin binding, as a catalytically active kinase lacking these residues is unable to bind recoverin. In addition, we show that neither the N-terminal deletion nor the presence of recoverin inhibits the overall catalytic activity of the kinase, as measured by light-independent autophosphorylation. Finally, we observe that a kinase mutant lacking the N-terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin. Taken together, these data support a model in which recoverin prevents rhodopsin phosphorylation by sterically blocking a region of kinase essential for its interaction with rhodopsin, thereby preventing recognition of rhodopsin as a kinase substrate.

Functional analysis of the human recoverin gene promoter

Purpose. Following photoactivation of rhodopsin, recoverin inhibits rhodopsin kinase activity in retinal photoreceptors by reducing the binding of arrestin to rhodopsin and therefore prolonging the termination of the phototransduction cascade. To identify potential cis -elements that may be involved in understanding the mechanisms that determine the cellspecific expression of recoverin in retinal and cancerous cells, the promoter region of the human recoverin gene was studied in cultured human Y79 retinoblastoma cells. Methods. A 2.5 kb EcoRI fragment of a 9.4 kb cosmid that contains the 5' non-coding region of the human recoverin gene was sequenced and cloned into an expression vector upstream of a luciferase gene. Cultured Y79 human retinoblastoma cells were transfected with DNA constructs using lipofection. Deletion mutants were generated by site-directed mutagenesis, cloned into the expression vector, and transfected into Y79 cells. Reporter gene activity was measured with a luciferase assay, and normalized to ß-beta-galactosidase activity resulting from a co-transfected SV-ß-galactosidase SV40 vector. Results. Reporter gene expression in the transfected Y79 cells demonstrated an increase in activity between 232 and 620 bp from the translational start site of the recoverin gene. There was a decrease in the reporter gene expression between 900 and 1200 bp from the start site, followed by an increase between 1200 and 1440 bp. Conclusions. This analysis suggests that there are cis -acting elements in the 5' non-coding region of the recoverin gene that are involved in the activation and suppression of gene expression.

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).

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.

Evaluation of the human gene encoding recoverin in patients with retinitis pigmentosa or an allied disease.

Purpose. To determine whether defects in the human recoverin gene cause retinitis pigmentosa (RP) or an allied disease such as Usher syndrome, Leber congenital amaurosis, or the Bardet-Biedl syndrome. Methods. Single-strand conformation polymorphism analysis and direct genomic sequencing techniques were used to screen 596 unrelated patients, comprising 167 patients with dominant RP, 168 with recessive RP, and 261 with an allied disease. Results. Four sequence variants were discovered. The first was a missense change (Ala200Thr) found in one family with autosomal dominant RP and in one family with autosomal recessive RP; it did not segregate with disease. The second was a silent, single-base variation affecting codon Ser24 with a minor allele frequency of approximately 0.5%. The third was a silent, single-base variation affecting codon Va1122. The fourth was a single-nucleotide substitution in intron 2, 11 bp upstream of exon 3. Conclusions. The authors found no evidence that mutations in the recoverin gene are a cause of RP or another of the hereditary retinal diseases studied. The human phenotype associated with mutations of the recoverin gene remains unknown.

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.

Exclusion of ROM-1 and recoverin genes in 16 families affected with ADRP

Exclusion of ROM-1 and recoverin genes in 16 families affected with ADRP

P 370 Exclusion of ROM-1 and recoverin genes in 16 families affected with ADRP

P 370 Exclusion of ROM-1 and recoverin genes in 16 families affected with ADRP

Evidence against involvement of recoverin in autosomal recessive retinitis pigmentosa in 42 Spanish families.

Autosomal recessive retinitis pigmentosa (ARRP) is a degenerative disease of photoreceptors in which defects in the genes encoding rhodopsin, the beta subunit of rod phosphodiesterase (PDEB) and, recently, in the gene for rod cGMP-gated channel, have been reported. However, detailed genetic involvement has not been ascertained in the great majority of cases. Recoverin, another member of the light transduction pathway, is a candidate gene for ARRP. We report the first analyses of the involvement of the recoverin gene (RCV1) in 42 Spanish ARRP families. Linkage and homozygosity studies with an intragenic polymorphism and the close markers D17S945 and D17S786 ruled out RCV1 as the cause of ARRP in 38 pedigrees. In the four remaining families, single strand conformation polymorphism analysis of the recoverin-coding region detected no mutations in the parents or in the affected members. These results strongly suggest that mutations in the RCV1 gene are not responsible for ARRP in these families.

Chromosomal assignment of the human gene for the cancer-associated retinopathy protein (recoverin) to chromosome 17p13.1.

The gene for the mouse recoverin protein (23 kDa photoreceptor-specific protein, S-modulin, or the Cancer-Associated Retinopathy protein) was recently assigned to mouse chromosome 11, closely linked to trp53. In this paper, the human gene for recoverin was localized to human chromosome 17 by Southern analysis of restriction digests of the DNA from mouse/human somatic cell hybrids. Using a 7 kb subclone of the human recoverin gene, a positive fluorescence in situ hybridization signal was demonstrated near the terminus of the short arm of chromosome 17 at position p13.1. The mapping of recoverin to this region of human chromosome 17, which contains a number of cancer-related loci, suggests a possible mechanism by which cancer-associated retinopathy occurs in humans.

Chromosomal assignment of the recoverin gene and cancer-associated retinopathy

The deduced amino acid sequence of the recently cloned mouse 23kD photoreceptor cell-specific protein showed it to be identical to the recoverin protein and the CAR (cancer-associated retinopathy) protein. DNA sequence variants were found in the mouse recoverin gene (Rcvrn), and segregation analysis of restriction fragment length variants in recombinant inbred strains of mice assigned Rcvrn to mouse Chromosome (Chr) 11, between Sparc (3.7 map units) and Zfp-3 (2.3 map units). These results demonstrate a close linkage of recoverin to the tumor suppressor gene, Trp53. On the basis of these data, knowledge of the function of recoverin, and the characteristics of CAR, an experimentally testable model is presented to explain the molecular basis for CAR.

Isolation of human retinal genes: Recoverin cDNA and gene

A human retina cDNA library enriched for retina-specific clones was prepared by subtraction with a non-retina population of cDNA in combination with polymerase chain reaction (PCR) amplifications. A highly retina-specific cDNA clone (1190 bp) was obtained through this library encoding a 200 amino acid protein with three calcium binding sites and 87% homology to the bovine photoreceptor protein, recoverin, which has been shown to mediate the recovery of the dark current after photoactivation, and 58% homology to the calcium-binding chick cone protein, visinin. Analysis of the gene indicated a 9-10 kb single-copy gene with at least three exons and two introns. The three exons contained the entire coding sequence, and all of the calcium-binding EF-hand regions were in putative exon 1. The recoverin gene was mapped to human chromosome 17 by hybridization to a panel of human-rodent hybrid DNAs.