Localization and expression of CaBP1/caldendrin in the mouse brain.

Ca(2+) binding protein 1 (CaBP1) and caldendrin are alternatively spliced variants of a subfamily of CaBPs with high homology to calmodulin. Although CaBP1 and caldendrin regulate effectors including plasma membrane and intracellular Ca(2+) channels in heterologous expression systems, little is known about their functions in vivo. Therefore, we generated mice deficient in CaBP1/caldendrin expression (C-KO) and analyzed the expression and cellular localization of CaBP1 and caldendrin in the mouse brain. Immunoperoxidase labeling with antibodies recognizing both CaBP1 and caldendrin was absent in the brain of C-KO mice, but was intense in multiple brain regions of wild-type mice. By Western blot, the antibodies detected two proteins that were absent in the C-KO mouse and consistent in size with caldendrin variants originating from alternative translation initiation sites. By quantitative PCR, caldendrin transcript levels were far greater than those for CaBP1, particularly in the cerebral cortex and hippocampus. In the frontal cortex but not in the hippocampus, caldendrin expression increased steadily from birth. By double-label immunofluorescence, CaBP1/caldendrin was localized in principal neurons and parvalbumin-positive interneurons. In the cerebellum, CaBP1/caldendrin antibodies labeled interneurons in the molecular layer and in basket cell terminals surrounding the soma and axon initial segment of Purkinje neurons, but immunolabeling was absent in Purkinje neurons. We conclude that CaBP1/caldendrin is localized both pre- and postsynaptically where it may regulate Ca(2+) signaling and excitability in select groups of excitatory and inhibitory neurons.

Gene transfer mediated by recombinant baculovirus into mouse eye.

PURPOSE: To determine the efficiency of baculoviruses (BVs) to transfer recombinant genes in vivo into murine ocular tissues. METHODS: Recombinant (r)BVs carrying fluorescent protein (FP) cDNA under the control of cytomegalovirus (CMV) immediate early promoter were constructed. Initially, cultured HEK293 and ARPE19 cells were infected with these rBVs and analyzed for efficiency and stability of transgene expression. The rBV-CMV green (G)FP was also injected into the intravitreal and subretinal space of mouse eye. Mice were periodically analyzed to determine the efficiency and stability of expression by histologic examination under fluorescence microscopy. The effect of rBV-CMV-GFP on the physiology of the retina was analyzed by electroretinography. RESULTS: cDNAs encoding fluorescent proteins were efficiently transduced in HEK293 and ARPE19 cells in vitro. GFP expression in vivo was observed exclusively in retinal pigment epithelial (RPE) cells after subretinal injections. Intravitreal injections of rBV resulted in GFP expression in the corneal endothelium, lens, RPE, and retina. GFP expression was observed for up to 14 days after injection. The infiltration of macrophages, observed 2 days after injection in the area of GFP transduction, had dissipated by day 8 after injection. No alteration in ERG responses was observed 6 weeks after injection of rBV-CMV-GFP. CONCLUSIONS: BV efficiently transduces cultured RPE cells and many cell types in vivo in the eye, including endothelial, epithelial, and neuronal cells. BV may be a useful vector for transferring genes in cultured cells and in vivo into ocular tissue.

Cloning and characterization of a human beta,beta-carotene-15,15'-dioxygenase that is highly expressed in the retinal pigment epithelium.

Retinoids play a critical role in vision, as well as in development and cellular differentiation. beta,beta-Carotene-15,15'-dioxygenase (Bcdo), the enzyme that catalyzes the oxidative cleavage of beta,beta-carotene into two retinal molecules, plays an important role in retinoid synthesis. We report here the first cloning of a mammalian Bcdo. Human BCDO encodes a protein of 547 amino acid residues that demonstrates 68% identity with chicken Bcdo. It is expressed highly in the retinal pigment epithelium (RPE) and also in kidney, intestine, liver, brain, stomach, and testis. The gene spans approximately 20 kb, is composed of 11 exons and 10 introns, and maps to chromosome 16q21-q23. A mouse orthologue was also identified, and its predicted amino acid sequence is 83% identical with human BCDO. Biochemical analysis of baculovirus expressed human BCDO demonstrates the predicted beta,beta-carotene-15,15'-dioxygenase activity. The expression pattern of BCDO suggests that it may provide a local supplement to the retinoids available to photoreceptors, as well as a supplement to the retinoid pools utilized elsewhere in the body. In addition, the finding that many of the enzymes involved in retinoid metabolism are mutated in retinal degenerations suggests that BCDO may also be a candidate gene for retinal degenerative disease.

Pan1b (17betaHSD11)-enzymatic activity and distribution in the lung.

We describe a new member of the 17beta-hydroxysteroid dehydrogenase group of enzymes. Human Pan1b displays greatest activity with 5alpha-androstan-3alpha,17beta-diol (3alpha-Diol) as substrate, suggesting that it may be important in androgen metabolism. Enzymic activity was non-saturable with 3alpha-Diol but saturable with retinoids, although retinoids were not metabolized. Immunohistochemical studies on 10% formalin fixed and paraffin embedded sections of human tissues showed that Pan1b was present in acini and ciliated epithelia of the lung. In the fetus immuno reactivity was present in ciliated epithelia throughout gestation and staining appeared to be stronger in the second half of pregnancy. Pan1b was also expressed in the nonpigmented epithelium of the ciliary body, and in adrenocortical tumor cells. Although 3alpha-Diol is generally considered a degradation product of androgen metabolism it could have its own biological function. Pan1b may be an important modulator of the endocrine, or intracrine activity of this steroid.

Ca(2+)-binding proteins in the retina: from discovery to etiology of human disease(1).

Examination of the role of Ca(2+)-binding proteins (CaBPs) in mammalian retinal neurons has yielded new insights into the function of these proteins in normal and pathological states. In the last 8 years, studies on guanylate cyclase (GC) regulation by three GC-activating proteins (GCAP1-3) led to several breakthroughs, among them the recent biochemical analysis of GCAP1(Y99) mutants associated with autosomal dominant cone dystrophy. Perturbation of Ca(2+) homeostasis controlled by mutant GCAP1 in photoreceptor cells may result ultimately in degeneration of these cells. Here, detailed analysis of biochemical properties of GCAP1(P50L), which causes a milder form of autosomal dominant cone dystrophy than constitutive active Y99C mutation, showed that the P50L mutation resulted in a decrease of Ca(2+)-binding, without changes in the GC activity profile of the mutant GCAP1. In contrast to this biochemically well-defined regulatory mechanism that involves GCAPs, understanding of other processes in the retina that are regulated by Ca(2+) is at a rudimentary stage. Recently, we have identified five homologous genes encoding CaBPs that are expressed in the mammalian retina. Several members of this subfamily are also present in other tissues. In contrast to GCAPs, the function of this subfamily of calmodulin (CaM)-like CaBPs is poorly understood. CaBPs are closely related to CaM and in biochemical assays CaBPs substitute for CaM in stimulation of CaM-dependent kinase II, and calcineurin, a protein phosphatase. These results suggest that CaM-like CaBPs have evolved into diverse subfamilies that control fundamental processes in cells where they are expressed.

Isomerization of all-trans-retinol to cis-retinols in bovine retinal pigment epithelial cells: dependence on the specificity of retinoid-binding proteins.

In the retinal rod and cone photoreceptors, light photoactivates rhodopsin or cone visual pigments by converting 11-cis-retinal to all-trans-retinal, the process that ultimately results in phototransduction and visual sensation. The production of 11-cis-retinal in adjacent retinal pigment epithelial (RPE) cells is a fundamental process that allows regeneration of the vertebrate visual system. Here, we present evidence that all-trans-retinol is unstable in the presence of H(+) and rearranges to anhydroretinol through a carbocation intermediate, which can be trapped by alcohols to form retro-retinyl ethers. This ability of all-trans-retinol to form a carbocation could be relevant for isomerization. The calculated activation energy of isomerization of all-trans-retinyl carbocation to the 11-cis-isomer was only approximately 18 kcal/mol, as compared to approximately 36 kcal/mol for all-trans-retinol. This activation energy is similar to approximately 17 kcal/mol obtained experimentally for the isomerization reaction in RPE microsomes. Mass spectrometric (MS) analysis of isotopically labeled retinoids showed that isomerization proceeds via alkyl cleavage mechanism, but the product of isomerization depended on the specificity of the retinoid-binding protein(s) as evidenced by the production of 13-cis-retinol in the presence of cellular retinoid-binding protein (CRBP). To test the influence of an electron-withdrawing group on the polyene chain, which would inhibit carbocation formation, 11-fluoro-all-trans-retinol was used in the isomerization assay and was shown to be inactive. Together, these results strengthen the idea that the isomerization reaction is driven by mass action and may occur via carbocation intermediate.

Stereoisomeric specificity of the retinoid cycle in the vertebrate retina.

Understanding of the stereospecificity of enzymatic reactions that regenerate the universal chromophore required to sustain vision in vertebrates, 11-cis-retinal, is needed for an accurate molecular model of retinoid transformations. In rod outer segments (ROS), the redox reaction involves all-trans-retinal and pro-S-NADPH that results in the production of pro-R-all-trans-retinol. A recently identified all-trans-retinol dehydrogenase (photoreceptor retinol dehydrogenase) displays identical stereospecificity to that of the ROS enzyme(s). This result is unusual, because photoreceptor retinol dehydrogenase is a member of a short chain alcohol dehydrogenase family, which is often pro-S-specific toward their hydrophobic alcohol substrates. The second redox reaction occurring in retinal pigment epithelium, oxidation of 11-cis-retinol, which is largely catalyzed by abundantly expressed 11-cis-retinol dehydrogenase, is pro-S-specific to both 11-cis-retinol and NADH. However, there is notable presence of pro-R-specific activities. Therefore, multiple retinol dehydrogenases are involved in regeneration of 11-cis-retinal. Finally, the cellular retinaldehyde-binding protein-induced isomerization of all-trans-retinol to 11-cis-retinol proceeds with inversion of configuration at the C(15) carbon of retinol. Together, these results provide important additions to our understanding of retinoid transformations in the eye and a prelude for in vivo studies that ultimately may result in efficient pharmacological intervention to restore and prevent deterioration of vision in several inherited eye diseases.

Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin.

Five members of a novel Ca(2+)-binding protein subfamily (CaBP), with 46-58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca(2+)-binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca(2+) coordination and the fact that their central alpha-helixes are extended by one alpha-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca(2+)-mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.

Molecular mechanism of visual transduction.

Our vision renders an incredible wealth of information about the external environment presented in the form of light of different wavelengths and intensities. To operate in a wide range of light intensities, our visual system has developed several mechanisms that allow an adjustment of its sensitivity to light. Immense progress has been made in understanding how light is captured and activates visual phototransduction cascade within photoreceptor cells; however, much less is known about desensitization. It has been known for some time, that many of these processes rely on Ca2+ as the principal modifier of phototransduction. Ca(2+)-binding proteins (CBPs) are specifically poised to take advantage of transient changes in [Ca2+] to act as enzymatic regulators. Some other CBPs are capable of changing the intracellular Ca2+ buffering capacity. Various retinal CBP proteins have been identified, including recoverin, GCAP1, GCAP2, GCAP3, GCIP, CBP1, CBP3 and CBP4. Although these numerous CBPs were identified, functions can be ascribed to only a few of them. Recently, genetic, physiological and biochemical analyses of retinal diseases have yielded additional insights into the role of many phototransduction proteins, including CBPs. Understanding the properties and the functions of these CBPs will pave the way for a more complete picture of visual transduction and accompanying desensitization processes.

Molecular characterization of a third member of the guanylyl cyclase-activating protein subfamily.

The mammalian retina contains at least two guanylyl cyclases (GC1 and GC2) and two guanylyl cyclase-activating proteins (GCAP1 and GCAP2). Here we present evidence of the presence of a new photoreceptor-specific GCAP, termed GCAP3, which is closely related to GCAP1. The sequence similarity of GCAP3 with GCAP1 and GCAP2 is 57 and 49%, respectively. Recombinant GCAP3 and GCAP2 stimulate GC1 and GC2 in low [Ca2+]free and inhibit GCs when [Ca2+]free is elevated, unlike GCAP1, which only stimulates GC1. GCAP3 is encoded by a distinct gene present in other mammalian species but could not be detected by genomic Southern blotting in rodents, amphibians, and lower vertebrates. The intron/exon arrangement of the GCAP3 gene is identical to that of the other GCAP genes. While the GCAP1 and GCAP2 genes are arranged in a tail-to-tail array on chromosome 6p in human, the GCAP3 gene is located on 3q13.1, suggesting an ancestral gene duplication/translocation event. The identification of multiple Ca2+-binding proteins that interact with GC is suggestive of complex regulatory mechanisms for photoreceptor GC.

Identification of a guanylyl cyclase-activating protein-binding site within the catalytic domain of retinal guanylyl cyclase 1.

Regulation of cAMP and cGMP production is a fundamental step in a broad range of signal transduction systems, including phototransduction. To identify regions within photoreceptor guanylyl cyclase 1 (GC1) that interact with GC-activating proteins (GCAPs), we synthesized the intracellular fragment of GC1, residues 491-1110, as a set of 15 amino acid long, partially overlapping peptides on the surface of individual pins arranged in a microtiter plate format. This pin assay identified 8 peptides derived from different regions of the GC1 intracellular domain that bind GCAPs. Peptide variants containing these sequences were synthesized as free peptides and tested for their ability to inhibit GC1 stimulation by GCAPs. A free peptide,968GTFRMRHMPEVPVRIRIG, from the catalytic domain of GC1 was the strongest inhibitor of GCAP1/GCAP2-mediated activation. In native GC1, this polypeptide fragment is likely to form a loop between alpha-helix 3 and beta-strand 4. When this region in GC1 was replaced by the corresponding sequence of GCAP-insensitive GC type A, GCAPs did not stimulate the GC1 mutant. The corresponding loops in related adenylyl cyclase (AC) are involved in the activating and inhibiting interactions with Gs alpha and Gi alpha, respectively. Thus, despite interacting with different activating proteins, both AC and GC activity may be modulated through their respective regions within catalytic domains.

Molecular characterization of a novel short-chain dehydrogenase/reductase that reduces all-trans-retinal.

The reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigments. We report here the cloning of a dehydrogenase, retSDR1, that belongs to the short-chain dehydrogenase/reductase superfamily and localizes predominantly in cone photoreceptors. retSDR1 expressed in insect cells displayed substrate specificities of the photoreceptor all-trans-retinol dehydrogenase. Homology modeling of retSDR1 using the carbonyl reductase structure as a scaffold predicted a classical Rossmann fold for the nucleotide binding, and an N-terminal extension that could facilitate binding of the enzyme to the cell membranes. The presence of retSDR1 in a subset of inner retinal neurons and in other tissues suggests that the enzyme may also be involved in retinol metabolism outside of photoreceptors.

Guanylate-cyclase-inhibitory protein is a frog retinal Ca2+-binding protein related to mammalian guanylate-cyclase-activating proteins.

Two guanylate-cyclase-activating proteins (GCAP) encoded by a tail-to-tail gene array have been characterized in the mammalian retina. Using frog retina as a model, we obtained evidence for the presence of a photoreceptor Ca2+-binding protein closely related to GCAP. This protein (206 amino acids) does not stimulate guanylate cyclase (GC) in low [Ca2+], but inhibits GC in high [Ca2+], and is therefore termed guanylate-cyclase-inhibitory protein (GCIP). Sequence analysis indicates that GCIP and GCAP1 and GCAP2 have diverged substantially, but conserved domains present in all vertebrate GCAP are present in GCIP. Moreover, partial characterization of the GCIP gene showed that the positions of two introns in the GCIP gene are identical to positions of corresponding introns of the mammalian GCAP gene array. As to the major differences between GCIP and GCAP, the fourth EF hand Ca2+-binding motif of GCIP is disabled for Ca2+ binding, and GCIP does not stimulate GC. Monoclonal and polyclonal antibodies raised against recombinant GCIP identified high levels of GCIP in the inner segments, somata and synaptic terminals of frog cone photoreceptors. The results suggest that GCIP is a Ca2+-binding protein of the GCAP/recoverin subfamily. Its localization in frog cones closely resembles that of GC in mammalian cones. GCIP inhibits GC at high free [Ca2+], competing with GCAP1 and GCAP2 for GC regulatory sites.

Changes in biological activity and folding of guanylate cyclase-activating protein 1 as a function of calcium.

Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca2+-binding protein, activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca2+-binding proteins from the calmodulin superfamily, the Ca2+-free form of GCAP1 stimulates the effector enzyme. In this study, we analyzed the Ca2+-dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca2+-sensitive modulation of GC1 activity. The change from a Ca2+-bound to a Ca2+-free form of GCAP1 increased susceptibility of Ca2+-free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca2+-bound form, only the N-terminus (myristoylated Gly2-Lys9) and C-terminus (171-205 fragment) of GCAP1 are removed by trypsin, while in the Ca2+-free form, GCAP1 is readily degraded to small fragments. Successive inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca2+-dependent inactivation of GCAP1. GCAP1(E75D,E111D,E155D) mutant did not bind Ca2+ and stimulated GC1 in a [Ca2+]-independent manner. GCAP1 and GCAP2, but not S-100beta, a high [Ca2+]free activator of GC1, competed with the triple mutant at high [Ca2+]free, inhibiting GC1 with similar IC50's. These competition results are consistent with comparable affinities between GC1 and GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca2+ binding and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein from the activator to the inhibitor of GC1.

Natural antiestrogen receptor autoantibodies in man with estrogenic activity in mammary carcinoma cell culture: study of their mechanism of action; evidence for involvement of estrogen-like epitopes.

We previously reported that human natural autoantibodies enriched in antiestrogen receptor Ig (IgGs) display estrogenic activity in MCF-7 mammary carcinoma cells. In this study, we investigated IgGs' mechanism of action. We showed that: 1) IgGs Fab fragments (which contain only one antigen binding site) induced an estrogenic response in MCF-7 cells, producing estrogen receptor (ER) down-regulation and an increase in progesterone receptor concentration; 2) IgGs specifically inhibited MCF-7 cell surface labeling with fluorescent estradiol (E2)-BSA conjugates; 3) this inhibition of E2-BSA binding to membrane estrogen binding sites was largely caused by natural anti-E2-BSA antibodies (Ab) selectively associated with the natural anti-ER Ab within IgGs; 4) furthermore, these natural anti-E2-BSA Ab accounted for most of IgGs estrogenic activity in cell culture; 5) however, when incubated with cytosolic ER, they did not behave like estrogens, but they decreased ER hormone binding capacity; and 6) although IgGs stimulated cAMP production, their anti-E2-BSA Ab subpopulation did not. In conclusion, the estrogenic activity of IgGs does not involve Ab mimicking E2 molecular configuration or ligand-independent cAMP mediated pathways, membrane Fc receptors, and membrane receptor cross-linking mechanisms. On the contrary, IgGs seem to function by neutralizing estrogen-like epitopes, associated with ER-related peptides, which might inhibit ER activation.

Localization of guanylate cyclase-activating protein 2 in mammalian retinas.

Guanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca2+, a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC in bovine rod outer segment homogenates. These results suggest that although GCAP1 is involved in the Ca2+-sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons.

Molecular cloning and localization of rhodopsin kinase in the mammalian pineal.

Several retinal photoreceptor proteins involved in phototransduction have also been found in the mammalian pineal. This study demonstrates that rat and human pineals express protein kinases that are identical to the corresponding rod photoreceptor rhodopsin kinases. The deduced amino acid sequence of rat and human rhodopsin kinases have 84% sequence similarity to the earlier reported sequence of the bovine retinal enzyme, with complete conservation of the topological regions containing the position of the catalytic domain and sites of posttranslational modifications. Rat pineal also expresses rod opsin and putative blue cone opsin. Using immunocytochemistry, rod opsin and rhodopsin kinase were found to be co-localized in pinealocytes in the human tissue. These data demonstrate that the mammalian pineal contains light-sensitive opsins and a kinase involved in their inactivation. These findings correlate with an earlier report that neonatal rats show extraretinal light sensitivity, and suggest that a functional photoreceptive system may be present in the adult mammalian pineal.