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PubMed2005

Iris-derived cells from adult rodents and primates adopt photoreceptor-specific phenotypes.

PURPOSE: The purpose of this study was to investigate the effects of various genes related to photoreceptor development on rodent and primate iris cells and the potential of iris cells as donor cells for retinal transplantation. METHODS: Adult rat and monkey iris tissue were cultured in serum-free medium containing basic fibroblast growth factor. Gene deliveries of Crx, Nrl, NeuroD and some combinations (Crx-Nrl, Crx-NeuroD) were performed with recombinant retrovirus. Immunocytochemistry, Western blot analysis, RT-PCR, and intracellular recording were used to examine the expression of photoreceptor-specific phenotypes in the iris-derived cells after gene transfer, . Coculture of the iris-derived cells with embryonic retinal explant was conducted, to investigate the potential integration of these cells in coculture conditions. RESULTS: Misexpression of Crx induced adult rat iris cells to express several photoreceptor-specific antigens and transcripts, such as rhodopsin, recoverin, cGMP-gated channel, arrestin, interphotoreceptor retinal-binding protein, rhodopsin kinase, and NeuroD. In primates, a combination of Crx and NeuroD was needed to induce monkey iris-derived cells to adopt photoreceptor-specific phenotypes. Furthermore, the photoreceptor-like cells derived from both rat- and primate-iris tissues showed rod photoreceptor-specific electrophysiological response to light stimuli after Crx and Crx-NeuroD gene transfer, respectively. The results further showed that iris-derived cells integrated in the developing host retina in coculture conditions. CONCLUSIONS: Adult iris-derived cultured cells of both rodents and primates expressed photoreceptor-specific phenotypes by inductions of transcription factors. These iris-derived photoreceptor-like cells have electrophysiological characteristics of rod photoreceptors. Furthermore, they can integrate in the developing retina under coculture conditions.

Animals↗
PubMed2007

Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate.

PURPOSE: To characterize the potential of newborn retinal stem cells (RSCs) isolated from the radial glia population to integrate the retina, this study was conducted to investigate the fate of in vitro expanded RSCs transplanted into retinas devoid of photoreceptors (adult rd1 and old VPP mice and rhodopsin-mutated transgenic mice) or partially degenerated retina (adult VPP mice) retinas. METHODS: Populations of RSCs and progenitor cells were isolated either from DBA2J newborn mice and labeled with the red lipophilic fluorescent dye (PKH26) or from GFP (green fluorescent protein) transgenic mice. After expansion in EGF+FGF2 (epidermal growth factor+fibroblast growth factor), cells were transplanted intravitreally or subretinally into the eyes of adult wild-type, transgenic mice undergoing slow (VPP strain) or rapid (rd1 strain) retinal degeneration. RESULTS: Only limited migration and differentiation of the cells were observed in normal mice injected subretinally or in VPP and rd1 mice injected intravitreally. After subretinal injection in old VPP mice, transplanted cells massively migrated into the ganglion cell layer and, at 1 and 4 weeks after injection, harbored neuronal and glial markers expressed locally, such as beta-tubulin-III, NeuN, Brn3b, or glial fibrillary acidic protein (GFAP), with a marked preference for the glial phenotype. In adult VPP retinas, the grafted cells behaved similarly. Few grafted cells stayed in the degenerating outer nuclear layer (ONL). These cells were, in rare cases, positive for rhodopsin or recoverin, markers specific for photoreceptors and some bipolar cells. CONCLUSIONS: These results show that the grafted cells preferentially integrate into the GCL and IPL and express ganglion cell or glial markers, thus exhibiting migratory and differentiation preferences when injected subretinally. It also appears that the retina, whether partially degenerated or already degenerated, does not provide signals to induce massive differentiation of RSCs into photoreceptors. This observation suggests that a predifferentiation of RSCs into photoreceptors before transplantation may be necessary to obtain graft integration in the ONL.

Animals↗
PubMedDate not supplied

Intraocular transplantation of E1A-immortalized retinal precursor cells.

The purpose of this study was to examine the effect of the ocular environment on the survival, tumorigenicity, and phenotypic marker expression of immortalized retinal precursor cells transplanted into immunocompetent adult and neonatal Sprague-Dawley rats. EIA-NR.3, a rat immortalized retinal precursor cell culture, was used as an inexhaustible source of experimental graft material. These cells were prelabeled with the fluorescent marker dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and transplanted intravitreally (50,000 cells per microL) into 11 adult and 31 neonatal Sprague-Dawley rat eyes. At 1 mo posttransplant, animals were sacrificed and retinal tissue sections examined histologically for the presence of grafted cells, signs of tumor formation, and retinal phenotypic marker expression. No obvious signs of tumor formation or rejection were seen in a total of 42 eyes in the immunocompetent hosts. Our results indicate that EIA-NR.3 cells survive at least 1 month in vivo, and can migrate from the vitreous into neuroretinal cell layers. Subpopulations of surviving grafted cells were seen to express photoreceptor markers rhodopsin and recoverin comparably between in vitro and in vivo conditions. However, the number of cells immunoreactive for vimentin and E1A decreased significantly under in vivo conditions. This report represents the first experimental intravitreal transplantation of E1A-immortalized retinal precursor cells into adult and neonatal rats. The intraocular location and environment appears to affect phenotypic expression of surviving grafted cells, especially with respect to vimentin and E1A expression. The fact that E1A-NR.3 cells survived intraocularly at least 1 mo without tumor formation suggests that the cells may continue to be useful for further in vivo studies of experimental retinal transplantation, and effects of histological location on retinal cell phenotype and histogenesis in immunocompetent hosts.

Affinity Labels↗
PubMedDate not supplied

Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells.

Retinal progenitor cells (RPCs) are multipotent central nervous system precursors that give rise to all of the cell types of the retina during development. Several groups have reported that mammalian RPCs can be isolated and expanded in culture and can differentiate into retinal neurons upon grafting to the mature, diseased eye. However, cell delivery and survival remain formidable obstacles to application of RPCs in a clinical setting. Because biodegradable polymer/progenitor constructs have been shown to be capable of tissue generation in other compartments, we evaluated the survival, migration, and differentiation of RPCs delivered on PLLA/PLGA polymer substrates to the mouse subretinal space and compared these results to conventional injections of RPCs. Polymer composite grafts resulted in a near 10-fold increase in the number of surviving cells after 4 weeks, with a 16-fold increase in cell delivery. Grafted RPCs migrated into the host retina and expressed the mature markers neurofilament-200, glial fibrillary acidic protein, protein kinase C-alpha, recoverin, and rhodopsin. We conclude that biodegradable polymer/progenitor cell composite grafts provide an effective means of increasing progenitor cell survival and overall yield when transplanting to sites within the central nervous system such as the retina.

Absorbable Implants↗
PubMed2006

High yield of cells committed to the photoreceptor fate from expanded mouse retinal stem cells.

The purpose of the present work was to generate, from retinal stem cells (RSCs), a large number of cells committed toward the photoreceptor fate in order to provide an unlimited cell source for neurogenesis and transplantation studies. We expanded RSCs (at least 34 passages) sharing characteristics of radial glial cells and primed the cells in vitro with fibroblast growth factor (FGF)-2 for 5 days, after which cells were treated with the B27 supplement to induce cell differentiation and maturation. Upon differentiation, cells expressed cell type-specific markers corresponding to neurons and glia. We show by immunocytochemistry analysis that a subpopulation of differentiated cells was committed to the photoreceptor lineage given that these cells expressed the photoreceptor proteins recoverin, peripherin, and rhodopsin in a same ratio. Furthermore, cells infected during the differentiation procedure with a lentiviral vector expressing green fluorescent protein (GFP) under the control of either the rhodopsin promoter or the interphotoreceptor retinoid-binding protein (IRBP) promoter, expressed GFP. FGF-2 priming increased neuronal differentiation while decreasing glia generation. Reverse transcription-polymerase chain reaction analyses revealed that the differentiated cells expressed photoreceptor-specific genes such as Crx, rhodopsin, peripherin, IRBP, and phosphodiesterase-alpha. Quantification of the differentiated cells showed a robust differentiation into the photoreceptor lineage: Approximately 25%-35% of the total cells harbored photoreceptor markers. The generation of a significant number of nondifferentiated RSCs as well as differentiated photoreceptors will enable researchers to determine via transplantation studies which cells are the most adequate to integrate a degenerating retina.

Animals↗
PubMed2006

A comparison of neural differentiation and retinal transplantation with bone marrow-derived cells and retinal progenitor cells.

Retinal progenitor cells (RPCs) are immature precursors that can differentiate into retinal neurons, including photoreceptors. Recently, it has been reported that bone marrow-derived cells may also be capable of differentiation into cells of central nervous system lineage, including retinal neurons. We compared these two cell types to evaluate their potential as a source of cells for retinal transplantation. Marrow stromal cells (MSCs) and macrophages were isolated from enhanced green fluorescence protein mice. MSCs were cultured with brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor to induce neuronal differentiation. RPCs were cultured under the same conditions or with 10% fetal bovine serum. Neuronal marker expression was examined and compared between MSCs and RPCs. MSCs, macrophages, and RPCs were also cultured with explanted retinas from rhodopsin knockout mice to study their potential for retinal integration. MSCs expressed neuronal and retina-specific markers by reverse transcription-polymerase chain reaction and immunocytochemistry. Both types of cells migrated into retinal explants and expressed neurofilament 200, glial fibrillary acidic protein, protein kinase C-alpha, and recoverin. RPCs expressed rhodopsin, a photoreceptor marker we never detected in MSCs. A majority of bone marrow derived-macrophages differentiated into cells that resembled microglia, rather than neural cells, in the explanted retina. This study shows that RPCs are likely to be a preferred cell type for retinal transplantation studies, compared with MSCs. However, MSCs may remain an attractive candidate for autologous transplantation.

Animals↗
PubMed2006

Evaluation of a comparative pathogenesis between cancer-associated retinopathy in humans and sudden acquired retinal degeneration syndrome in dogs via diagnostic imaging and western blot analysis.

OBJECTIVE: To evaluate dogs with sudden acquired retinal degeneration syndrome (SARDS) for evidence of pituitary gland, adrenal gland, and pulmonary neoplasia and antiretinal antibodies and to evaluate dogs with neoplasia for antiretinal antibodies. ANIMALS: 57 clinically normal dogs, 17 with SARDS, and 53 with neoplasia. PROCEDURE: Thoracic radiography, ultrasonography of adrenal glands, and contrast-enhanced computed tomography of pituitary glands were performed in 15 dogs with SARDS. Western blot analysis was performed on sera of all dogs; recoverin (23 kd) and arrestin (48 kd) retinal antibodies were used as positive controls. RESULTS: Neoplasia was not detected via diagnostic imaging in dogs with SARDS. Western blot analysis revealed bands in all dogs ranging from > 48 to < 23 kd. Prominent bands with equivalent or greater density than 1 or both positive controls at the 1:1,000 dilution, and present at the 1:3,000 dilution, were detected in 28% of clinically normal dogs, 40% of dogs with neoplasia, and 41% of dogs with SARDS. No bands in dogs with SARDS had a consistent location of immune activity, and none were detected at the 23-kd site. The area around the 48-kd site had increased immune activity in all 3 groups. CONCLUSIONS AND CLINICAL RELEVANCE: The etiology of SARDS in dogs does not appear to be similar to cancer-associated retinopathy in humans on the basis of absence of differential antibody activity against retinal proteins. Although dogs with SARDS often have clinical signs compatible with hyperadrenocorticism, neoplasia of the adrenal glands, pituitary gland, or lungs was not detected.

Acute Disease↗
PubMedDate not supplied

Identification of neuronal calcium sensor (NCS-1) possibly involved in the regulation of receptor phosphorylation.

Persistent stimulation of G protein-coupled receptors by agonists leads rapidly to reduced responses, a phenomenon described as desensitization. It involves primarily the phosphorylation of receptor sites by specific kinases of the G protein-coupled receptor kinase (GRK) family. The beta-adrenergic receptor kinase 1 (GRK2) desensitizes agonist-activated beta2-adrenergic receptors, whereas rhodopsin kinase (GRK1) phosphorylates and inactivates photon-activated rhodopsin. Little is known about the role of calcium in desensitization. Here we report the characterization of a novel neuronal calcium sensor (NCS) named NCS-1 possibly involved in the regulation of receptor phosphorylation. NCS-1 is a new member of the EF-hand superfamily, which includes calmodulin, troponin C, parvalbumin, and recoverins. By Northern analysis and in situ hybridization, we discovered that NCS-1 is specifically expressed in the central and peripheral nervous systems. Chick NCS-1 has 72% of amino acid identity with Drosophila frequenin, a protein found in the nervous system and at the motor nerve terminals of neuromuscular junctions. By analogy with the reported function for two other members of the NCS family, we discuss whether G protein-coupled receptors or GRKs are the targets of neuronal calcium sensors.

Amino Acid Sequence↗
PubMed2000

Distinct ionotropic GABA receptors mediate presynaptic and postsynaptic inhibition in retinal bipolar cells.

Ionotropic GABA receptors can mediate presynaptic and postsynaptic inhibition. We assessed the contributions of GABA(A) and GABA(C) receptors to inhibition at the dendrites and axon terminals of ferret retinal bipolar cells by recording currents evoked by focal application of GABA in the retinal slice. Currents elicited at the dendrites were mediated predominantly by GABA(A) receptors, whereas responses evoked at the terminals had GABA(A) and GABA(C) components. The ratio of GABA(C) to GABA(A) (GABA(C):GABA(A)) was highest in rod bipolar cell terminals and variable among cone bipolars, but generally was lower in OFF than in ON classes. Our results also suggest that the GABA(C):GABA(A) could influence the time course of responses. Currents evoked at the terminals decayed slowly in cell types for which the GABA(C):GABA(A) was high, but decayed relatively rapidly in cells for which this ratio was low. Immunohistochemical studies corroborated our physiological results. GABA(A) beta2/3 subunit immunoreactivity was intense in the outer and inner plexiform layers (OPL and IPL, respectively). GABA(C) rho subunit labeling was weak in the OPL but strong in the IPL in which puncta colocalized with terminals of rod bipolars immunoreactive for protein kinase C and of cone bipolars immunoreactive for calbindin or recoverin. These data demonstrate that GABA(A) receptors mediate GABAergic inhibition on bipolar cell dendrites in the OPL, that GABA(A) and GABA(C) receptors mediate inhibition on axon terminals in the IPL, and that the GABA(C):GABA(A) on the terminals may tune the response characteristics of the bipolar cell.

Animals↗
PubMed2000

Immunocytochemical analysis of the mouse retina.

Transgenic mice provide a new approach for studying the structure and function of the mammalian retina. In the past, the cellular organization of the mammalian retina was investigated preferentially in primates, cats, and rats but rarely in mice. In the current study, the authors applied 42 different immunocytochemical markers to sections of the mouse retina and studied their cellular and synaptic localization by using confocal microscopy. The markers applied were from three major groups: 1) antibodies against calcium-binding proteins, such as calbindin, parvalbumin, recoverin, or caldendrin; 2) antibodies that recognize specific transmitter systems, such as glycine, gamma-aminobutyric acid, or acetylcholine; and 3) antibodies that recognize transmitter receptors and show their aggregation at specific synapses. Only a few markers labeled only one cell type: Most antibodies recognized specific groups of neurons. These were analyzed in more detail in double-labeling experiments with different combinations of the antibodies. In light of their results, the authors offer a list of immunocytochemical markers that can be used to detect possible changes in the retinal organization of mutant mice.

Animals↗
PubMed2000

Localization of tubby-like protein 1 in developing and adult human retinas.

PURPOSE: To localize tubby-like protein 1 (TULP1) in developing and adult human retinas. METHODS: TULP1 was localized by immunofluorescence microscopy in human retinas, aged 8.4 fetal weeks to adult. TULP1-positive cells were identified by double labeling with antibodies specific for cones, rods, and astrocytes. RESULTS: In adult retinas, anti-TULP1 labels cone and rod inner segments, somata, and synapses; outer segments are TULP1-negative. A few inner nuclear and ganglion cells are weakly TULP1-positive. In fetal retinas, cells at the outer retinal border are TULP1-positive at 8.4 weeks. At 11 weeks, the differentiating central cones are strongly TULP1-reactive and some are positive for blue cone opsin. At 15.4 weeks, all central cones are strongly positive for TULP1 and many are reactive for red/green cone opsin. At 17.4 weeks, central rods are weakly TULP-reactive. In peripheral retina at 15.4 weeks to 1 month after birth, displaced cones in the nerve fiber layer are positive for TULP1, recoverin, and blue cone opsin. Some ganglion cells are weakly reactive for TULP1 at 11 weeks and later, but astrocytes and the optic nerve are TULP1-negative at all ages examined. CONCLUSIONS: The finding of TULP1 labeling of cones before they are reactive for blue or red/green cone opsin suggests an important role for TULP1 in development. TULP1 expression in both developing and mature cones and rods is consistent with a primary photoreceptor defect in retinitis pigmentosa (RP) caused by TULP1 mutations. Weak TULP1-immunolabeling of some inner retinal neurons in developing and adult retinas suggests that optic disc changes in patients with RP who have TULP1 mutations may be primary as well as secondary to photoreceptor degeneration.

Adult↗
PubMedDate not supplied

[Use of method of protein engineering in studying calcium-binding proteins].

Major results of the use of protein engineering methods in studies of calcium-binding proteins with the highest affinity for calcium and known three-dimensional structure (parvalbumin, calmodulin, troponin C, calbindin, recoverin, alpha-lactalbumin, and others) are presented. Specific features of recombinant calcium-binding proteins are discussed. Experiments with genetic introduction of fluorescent probes, tryptophan and tyrosine, into proteins are overviewed. Effects of mutations in different parts of protein molecules (calcium-binding loops, hydrophobic core, and others) on their structure and properties and attempts of creation of artificial calcium-binding sites are discussed.

Calcium-Binding Proteins↗
PubMed2003

Differentiation of marrow stromal cells into photoreceptors in the rat eye.

Retinal degenerations and dystrophies are the major causes of genetically inherited blindness that are characterized by the apoptotic death of the photoreceptor cell layer of the retina. To date, no treatment exists for these diseases and only recently have they been considered as candidates for gene and stem cell therapies. Here we report the ability of adult CD90+ marrow stromal cells (MSCs) to be induced by activin A, taurine, and EGF into cells (20-32%) expressing photoreceptor-specific markers rhodopsin, opsin, and recoverin in vitro. CD90+ cells were either transduced with recombinant adeno-associated virus expressing green fluorescent protein (GFP) or bromodeoxyuridine (BrdU) labeled and then injected into the subretinal space of adult Royal College of Surgeons rats. Fundus photography and angiography showed no adverse effects of CD90+ MSC transplantation. GFP-expressing cells or BrdU-positive cells covered approximately 30% of the entire retinal area. By 2 weeks after injection, CD90+ MSCs integrated into the host retina, forming structures similar to the photoreceptor layer and expressed a photoreceptor-specific marker. No teratoma formation was observed in the recipient retina. The subretinally delivered CD90+ MSCs did not stain for proliferating cell nuclear antigen, indicating that they primarily undergo differentiation rather than proliferation. In addition, we established that transplanted cells can attract synaptic vesicles and hence are potentially capable of signal transduction. This study demonstrates for the first time the partial differentiation of adult CD90+ MSCs into photoreceptors in vitro and in vivo. Our results establish a proof of concept for CD90+ MSC differentiation with autologous transplantation, which may provide a promising therapeutic strategy for the treatment of some forms of genetically inherited retinal degenerations.

Animals↗
PubMed1992

[Transduction of the visual signal in retinal cells].

Transduction of a visual signal is a complex process. It involves photochemical, enzymatic and ionic reactions. An electrophysiological response is generated on absorption of a photon by a photoreceptor cell's pigment molecule, then propagates to the synapses. The first photochemical reaction, isomerization of retinal, in vertebrates occurs in the photoreceptor cells--rods and cones--of the retina, so changes conformation and activity of a pigment-bound protein, rhodopsin, in membranes of intracellular discs. Rhodopsin becomes enzymatically active and catalyses the activation by GTP of a great number of transducins, which in turn activate cGMP phosphodiesterase. This enzymatic chain propagates and greatly enhances hydrolysis of cytoplasmic cGMP. One photon incites hydrolysis of 10(5) cGMP molecules in 100 ms. Local cGMP decrease frees it from specific binding sites in cytoplasm occupied by proteinaceous canals in cell membrane around the activated disc. In darkness high cGMP concentration, hence binding, kept canals open, maintaining high cellular cation permeability, especially to Na+, and a strong cellular depolarization. Ca2+ influx, also allowed, balanced Na+ movement. Canal closure induces local hyperpolarization, the first electrophysiological response, which propagates through the cell to synaptic contacts. It also lowers intracellular Ca2+ concentration which initiates cGMP synthesis--from GTP by a guanylate cyclase controlled negatively by recoverin, a calcium-dependent protein--to restore cGMP towards at rest level. Although all macromolecules involved in this now fairly complete scheme have been isolated and characterized, cloned and sequenced, no three-dimensional structure has yet been established. The proteins are membrane-bound rather than in independent crystal form, which renders such structural studies difficult.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

[Proteome analysis of autoantibodies in sera of patients with cancer].

Circulating autoantibodies are useful diagnostic markers of cancers and autoimmune diseases. Research over the past decade has resulted in some reports on the presence of autoantibodies against disease-related proteins such as annexin-I & II, recoverin and protein gene product 9.5 in the sera of patients with lung cancer, and also against calreticulin and alpha-enolase in autoimmune diseases. In this study, we first identified the a-enolase autoantibody in the sera of patients with lung adenocarcinoma by proteomics-based analysis. The comparison of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)/western blot (WB)/ECL detection revealed distinct distributions of antibodies in the sera of lung adenocarcinoma, tuberculosis and healthy subjects which reacted with soluble proteins derived from the adenocarcinoma A549 cell line. We found 16 spots in patients with adenocarcinoma by 2D-PAGE/WB/ECL detection and identified alpha-enolase, chaperonin, and other autoantibodies in the adenocarcinoma patients' sera. The specificities of an antibody against alpha-enolase was preliminarily observed in sera from 3 of 5 patients with adenocarcinoma, 0 of 10 patients with tuberculosis and 0 of 10 healthy subjects. In conclusion, we first identified alpha-enolase autoantibody in sera of lung adenocarcinoma and the autoantibody was seemed to be a specific marker of the lung adenocarcinoma. In addition, we also identified various autoantibodies in esophageal cancer, hepatocellular carcinoma, and non-Hodgkin's lymphoma. Moreover, we tried to identify the corresponding antigen of an unknown anti-cytoplasmic autoantibody, and an anti-red blood cell antibody by proteomics-based analysis. These antibodies might become new diagnosis markers.

Adenocarcinoma↗
PubMed1995

Forskolin and camptothecin induce a 30 kDa protein associated with melatonin production in Y79 human retinoblastoma cells.

The synthesis of melatonin in Xenopus retinas, chick and quail retinal cell cultures, and Y79 human retinoblastoma cells is stimulated by cAMP through a protein synthesis-dependent mechanism. In Y79 retinoblastoma cells, combined treatment with the RNA synthesis inhibitor camptothecin and agents that elevate cAMP, such as forskolin, causes a synergistic elevation of melatonin. Using two-dimensional gel analysis we have identified a 30 kDa cytosolic protein (p30) whose radiolabeling was consistently increased in parallel with increases in arylalkylamine N-acetyltransferase activity and melatonin production that were induced by forskolin and/or camptothecin. Pulse-chase experiments suggest that the elevation in radiolabeling of p30 is due to increased synthesis. Three candidate proteins found in the mammalian pineal, protein 14-3-3, malate dehydrogenase, and recoverin, do not comigrate with p30.

Camptothecin↗
PubMed1994

Molecular characterization of human and mouse photoreceptor guanylate cyclase-activating protein (GCAP) and chromosomal localization of the human gene.

Guanylate cyclase-activating protein (GCAP) is a novel Ca(2+)-binding protein that stimulates synthesis of cGMP in photoreceptors. Molecular cloning of human and mouse GCAP cDNA revealed that the known mammalian GCAPs are more than 90% similar, consist of 201-205 amino acids, and contain three identically conserved EF hand Ca2+ binding sites. The sequence homology with recoverin, a related photoreceptor Ca(2+)-binding protein, is less than 35%. In situ hybridization in primate retinas shows that the GCAP gene is expressed exclusively in photoreceptor inner segments. To investigate the GCAP gene structure, we probed 10 eucaryotic genomic DNAs with a bovine GCAP cDNA under stringent conditions. The results demonstrate that the GCAP gene has been well conserved during evolution of vertebrate species and that each gene is most likely present as a single copy. By genomic cloning, polymerase chain reaction, mapping, and direct sequencing, we show that the human GCAP gene spans approximately 6 kilobases of genomic DNA, and consists of four exons (> 250, 146, 94, and 800 base pairs) separated by three introns (4.5 kilobases, 370 base pairs, and 347 base pairs). Using human/hamster hybrid panels and fluorescent in situ hybridization, the GCAP gene was localized to the short arm of chromosome 6 (p21.1).

Amino Acid Sequence↗
PubMed1993

Myristoylation of hippocalcin is linked to its calcium-dependent membrane association properties.

Hippocalcin, a recently identified Ca(2+)-binding protein of the recoverin family exclusively expressed in the hippocampus, has a primary structure containing three putative Ca(2+)-binding sites (EF-hands) and a possible NH2-terminal myristoylation site. 45Ca blots demonstrated that every three EF-hand domains, expressed as fusion proteins in Escherichia coli, bind Ca2+, indicating that hippocalcin binds 3 mol of Ca2+/mol of protein. To determine whether hippocalcin is myristoylated, hippocalcin mRNA was translated in vitro in the presence of [3H]myristic acid. 3H label was resistant to hydroxylamine treatment, and replacement of NH2-terminal glycine with alanine prevented 3H label incorporation, indicating that in vitro translated hippocalcin covalently bound [3H]myristic acid at the NH2-terminal glycine. In vitro translated hippocalcin is quantitatively myristoylated, as evidenced by an electrophoretic mobility shift of [35S]methionine-labeled protein on two-dimensional gels. Native hippocalcin comigrated precisely with the in vitro translated hippocalcin on two-dimensional gels, suggesting that native hippocalcin is myristoylated. Native and in vitro translated hippocalcins, but not non-myristoylated mutagenic (Gly1-Ala1) hippocalcin, displayed Ca(2+)-dependent membrane association, indicating that myristoylation participates in its Ca(2+)-dependent membrane association properties. In vitro translated hippocalcin bound to phospholipid vesicles somewhat, however, phospholipid association was insufficient for its membrane association properties, suggesting that the NH2-terminal myristoyl moiety on hippocalcin interacts with lipid bilayers and facilitates interaction with other membrane proteins.

Alanine↗