Human Recoverin Research Articles

G-Protein-Coupled Receptors Mediate Modulations of Cell Viability and Drug Sensitivity by Aberrantly Expressed Recoverin 3 within A549 Cells

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.

Experimental Insight into the Structural and Functional Roles of the 'Black' and 'Gray' Clusters in Recoverin, a Calcium Binding Protein with Four EF-Hand Motifs.

Recently, we have found that calcium binding proteins of the EF-hand superfamily (i.e., a large family of proteins containing helix-loop-helix calcium binding motif or EF-hand) contain two types of conserved clusters called cluster I (‘black’ cluster) and cluster II (‘grey’ cluster), which provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domains. Cluster I is more conservative and mostly incorporates aromatic amino acids, whereas cluster II includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. Recoverin is EF-hand Ca2+-binding protein containing two ‘black’ clusters comprised of F35, F83, Y86 (N-terminal domain) and F106, E169, F172 (C-terminal domain) as well as two ‘gray’ clusters comprised of F70, Q46, F49 (N-terminal domain) and W156, K119, V122 (C-terminal domain). To understand a role of these residues in structure and function of human recoverin, we sequentially substituted them for alanine and studied the resulting mutants by a set of biophysical methods. Under metal-free conditions, the ‘black’ clusters mutants (except for F35A and E169A) were characterized by an increase in the α-helical content, whereas the ‘gray’ cluster mutants (except for K119A) exhibited the opposite behavior. By contrast, in Ca2+-loaded mutants the α-helical content was always elevated. In the absence of calcium, the substitutions only slightly affected multimerization of recoverin regardless of their localization (except for K119A). Meanwhile, in the presence of calcium mutations in N-terminal domain of the protein significantly suppressed this process, indicating that surface properties of Ca2+-bound recoverin are highly affected by N-terminal cluster residues. The substitutions in C-terminal clusters generally reduced thermal stability of recoverin with F172A (‘black’ cluster) as well as W156A and K119A (‘gray’ cluster) being the most efficacious in this respect. In contrast, the mutations in the N-terminal clusters caused less pronounced differently directed changes in thermal stability of the protein. The substitutions of F172, W156, and K119 in C-terminal domain of recoverin together with substitution of Q46 in its N-terminal domain provoked significant but diverse changes in free energy associated with Ca2+ binding to the protein: the mutant K119A demonstrated significantly improved calcium binding, whereas F172A and W156A showed decrease in the calcium affinity and Q46A exhibited no ion coordination in one of the Ca2+-binding sites. The most of the N-terminal clusters mutations suppressed membrane binding of recoverin and its inhibitory activity towards rhodopsin kinase (GRK1). Surprisingly, the mutant W156A aberrantly activated rhodopsin phosphorylation regardless of the presence of calcium. Taken together, these data confirm the scaffolding function of several cluster-forming residues and point to their critical role in supporting physiological activity of recoverin.

Cancer-Associated Retinopathy in Non-Hodgkin Lymphoma: The Challenge of Treatment

Abstract Introduction Cancer-associated retinopathy (CAR) syndrome is rare paraneoplastic visual syndrome, usually associated with small-cell lung carcinoma. We report, to our knowledge, the first case in the English-language literature of CAR syndrome occurring in a patient with non-Hodgkin lymphoma (NHL) treated with intravenous immunoglobulin (IVIG). Case Report A 62-year-old male was diagnosed with stage IV B-cell follicular NHL treated with 6 cycles of R-CHOP (rituximab plus cyclophosphamide/doxorubicin/vincristine/prednisone). Five weeks after the last cycle, he presented with progressive decrease of vision in both eyes, flickering lights, and night blindness. On examination, corrected visual acuity was 20/80 and 20/100 in the right and left eye, respectively, and color vision was diminished bilaterally. Fundus examination revealed pallor of the optic disks while visual field examination showed profound field constriction. A multifocal electroretinogram revealed dysfunction of both rods and cones characteristic of diffuse photoreceptor degeneration that is supporting the diagnosis of CAR syndrome. Magnetic resonance imaging of the head and orbits showed no signs of metastases or other abnormalities. Western blot analysis of the patient's serum showed a positive antibody reaction with 23-kD component coinciding with the molecular mass of human recoverin. The patient was started on oral prednisone 60 mg daily. Three weeks later he still complained of decreased vision in both eyes. IVIG (400 mg/kg/day) was given for 4 doses. Following the second dose, the patient showed signs of improvement. Four weeks after finishing the full treatment, visual acuity improved to 20/40 in both the right and left eye, with no relapse during the 8-month follow-up period. Discussion To our knowledge, there has been only 1 previous report (in the French-language literature) of CAR syndrome in lymphoma patients. Matus et al described 2 NHL patients with symptoms of CAR, which was confirmed by the presence of anti-recoverin antibodies. Both cases were treated by corticosteroids with only minimal response. Since CAR syndrome is a rare condition, no specific treatment has been shown to be effective. Treating the underlining malignancy has not been shown to result in better visual outcomes, and immunosuppression with oral or intravenous corticosteroid exhibits only a transient or undetectable response. Furthermore, plasmapheresis has not been effective. Conclusion CAR may occur in NHL. IVIG may be an effective option for treating these patients, especially if started early enough before permanent retinal damage occurs.

Establishment of a novel small cell lung carcinoma cell line with specific recoverin expression from a patient with cancer-associated retinopathy

We analysed the biologic properties of a small cell lung carcinoma cell line (designated KK0206) established from a patient with SCLC who had cancer-associated retinopathy (CAR). Morphological and immunohistochemical studies showed that KK0206 cells have features of the classic type of SCLC. KK0206 cells grew in suspension, forming relatively small clumps of cells with a doubling time of 72 h. On light microscopy, the cells were relatively small with little cytoplasm. On immunohistochemistry using anti-bovine recoverin rabbit antibody, the cells were intensely positive for recoverin. In addition, they were positive for NSE, Ki-67, and TP53. They also expressed human recoverin, a photoreceptor protein, whose presence was confirmed by RT-PCR analysis with cDNA sequencing and Western blot analysis. The point mutation of their TP53 gene (exon 156) was detected as well. The present study demonstrates that human recoverin is expressed in SCLC cells cultured from an anti-recoverin antibody-negative patient with CAR. KK0206 might be important for further research on SCLC related retinopathy.

Clinicopathological features of gastric cancer cases and aberrantly expressed recoverin.

Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions toward recoverin, a retina-specific calcium binding protein, and other retinal antigens aberrantly expressed in tumor cells lead to the degeneration of retinal photoreceptor cells. Recently we reported that aberrant expression of recoverin was identified in more than 50% of tumor cells and their cell lines from several kinds of cancers, including gastric cancer, lung carcinoma, and other cancers. To elucidate the clinicopathological significance of aberrantly expressed recoverin in cancer cells, we performed immunocytochemical analysis using a monoclonal antibody against human recoverin. Within 18 patients with different clinical stages (I-IV) of gastric cancer, the aberrant expression of recoverin in tumor cells was recognized in 6 out of 18 patients (2 out of 2 stage IA, 1 out of 1 stage IB, 2 out of 3 stage II, 0 out of 7 stage IIIA, 0 out of 1 stage IIIB, and 1 out 4 stage IV). The present data are consistent in part with the previous observations that recoverin-expressing cancer cells induced tumor immunity and provide a favorable prognosis for primary cancer in CAR patients.

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.

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.

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.

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.

Characterization of a small-cell-lung-carcinoma cell line from a patient with cancer-associated retinopathy

We examined the biologic properties of a small-cell-lung-carcinoma (SCLC) cell line (designated MN-1112) established from a patient with SCLC who showed paraneoplastic retinopathy syndrome. Morphologic and immunocytochemical analyses showed that MN-1112 cells possess features of the classic type of SCLC. MN-1112 cells grew in suspension forming relatively large clumps of cells with a doubling time of 72 hr. By light-microscope examination, the cells were relatively small and had scanty cytoplasm. The cells produced NSE, ACTH and CK (BB isozyme); they also expressed recoverin, a novel photoreceptor protein, detected by Northern-blot and Western-immunoblot analysis using human-recoverin-specific DNA probe and anti-bovine-recoverin polyclonal antibody. This report shows that human recoverin is expressed in cultured SCLC cells. Our results support the hypothesis that, in cancer-associated retinopathy (CAR) patients, auto-immune antibody targeting for ectopic recoverin in SCLC is initially produced and cross-reacts with the retinal protein, resulting in the retinal degeneration that occurs in CAR patients.

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.

Heterogeneous N-acylation is a tissue- and species-specific posttranslational modification.

Heterogeneous N-terminal glycine acylation recently has been reported for two proteins involved in visual signal transduction. Similar N-acylations have typically involved only myristate; however, none of the previously examined proteins were isolated from retinas. To determine whether heterogeneous N-acylation is tissue-specific or protein sequence-specific, the N-terminal modifications of the catalytic subunit of cAMP-dependent protein kinase, partially purified from bovine retinas, heart, and brain tissues, were characterized. Using tandem mass spectrometry and liquid chromatography coupled directly to an electrospray mass spectrometer, we found only myristate at the N termini of catalytic subunits from brain and heart tissue, whereas the N termini of the retina-derived subunits were heterogeneously acylated in a manner similar to recoverin and transducin. Thus it appears that the nature of N-terminal glycine acylation is determined by the cell or tissue type in which it is located, and not by the sequence of the modified protein. We also examined the N-acylation of recoverin purified from human retinas, as well as transducin purified from frog retinas, to determine if heterogeneous acylation of retinal proteins is a uniquely bovine phenomenon. Interestingly, human recoverin was modified by the same family of fatty acids found on the bovine retinal proteins, while frog transducin was modified homogeneously not with myristate, but with a doubly unsaturated (C14:2) fatty acyl group.

Expression of Recoverin mRNA in the Human Retina: Localization by In Situ Hybridization

The localization of mRNA encoding recoverin in the human retina was examined by in situ hybridization. The riboprobe utilized in this study was transcribed from a cDNA clone containing the complete coding region of human recoverin. The riboprobe hybridized extensively with the photoreceptor inner segments, and the outer nuclear layer. Specific hybridization was also seen in a subpopulation of cells in the inner nuclear layer, and occasionally in cells of the ganglion cell layer (GCL). Recoverin-like immunoreactivity was observed in all photoreceptors, and in a subpopulation or bipolar cells. Very robust immunolabeling was observed in a small population of cells in the GCL. This study suggests that recoverin, or a recoverin-like protein, is produced by at least three different cell types in the human retina: photoreceptors, bipolars and rare cells in the ganglion cell layer.

Dinucleotide repeat polymorphism at the human recoverin RCVI gene locus on chromosome 17p.

Dinucleotide repeat polymorphism at the human recoverin RCVI gene locus on chromosome 17p.