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K Palczewski

Publications and source records attributed to K Palczewski.

At least 109 records · Page 6Linked to original sources
PubMed1994

Inositolhexakisphosphate (InsP6): an antagonist of fibroblast growth factor receptor binding and activity.

Fibroblast growth factors (FGF), which have been implicated in tumor cell growth and angiogenesis, have biological activities that appear to be mediated by both heparinlike extracellular matrix sites and transmembrane tyrosine kinase receptor sites. In the present study, we demonstrated that inositolhexakisphosphate (InsP6) inhibits basic FGF (bFGF) binding to heparin. Our spectrofluorometric analyses demonstrated that InsP6 not only bound to bFGF, presumably within the bFGF heparin-binding domain, but also protected bFGF from degradation by trypsin. Also, InsP6 inhibited the cellular binding of bFGF and other fibroblast growth factor family members such as acidic FGF (aFGF) and K-FGF in a saturable and dose-dependent manner. Furthermore, concentrations as low as 100 microM InsP6 inhibited bFGF-induced DNA synthesis in AKR-2B fibroblasts, as well as the growth of bFGF- and K-FGF-transfected NIH/3T3 cells. Together, these results indicate that InsP6 may serve as a useful antagonist of FGF activity.

Animals↗
PubMed1994

Molecular cloning and characterization of retinal photoreceptor guanylyl cyclase-activating protein.

Guanylyl cyclase-activating protein (GCAP) is thought to mediate Ca(2+)-sensitive regulation of guanylyl cyclase (GC), a key event in recovery of the dark state of rod photoreceptors following light exposure. Here, we characterize GCAP from several vertebrate species by molecular cloning and provide evidence that GCAP contains a heterogeneously acylated N-terminal region that interacts with GC. Vertebrate GCAPs consist of 201-205 amino acids, and sequence analysis indicates the presence fo three EF hand Ca(2+)-binding motifs. These results establish that GCAP is a novel photoreceptor-specific member of a large family of Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-sensitive activation of GC.

Amino Acid Oxidoreductases↗
PubMed1993

Interaction between photoactivated rhodopsin and its kinase: stability and kinetics of complex formation.

Rhodopsin phosphorylation is a key event in the deactivation of this G-protein-coupled receptor. Rhodopsin kinase mediates the reaction and requires specific cytoplasmic loop domains on rhodopsin, distinct from the sites of phosphorylation, for binding and activation. In this study, we characterize the formation of a stable complex between photolyzed rhodopsin in native washed disk membranes and its kinase. Centrifugation of the membranes after illumination and subsequent polyacrylamide gel electrophoresis demonstrates light-dependent binding of rhodopsin kinase to the membranes. A real-time monitor for the transition of the solubilized kinase into the bound state is provided by flash-induced light-scattering binding signals. The complex has the following characteristics: (i) the on-rate of the reaction rises in linear proportion to the concentrations of both the kinase and photoactivated rhodopsin; kinetic analysis yields a bimolecular rate constant of kon = 0.5-1 microM-1s-1. (ii) The dissociation constant of the complex is 0.3 < KD < 0.5 microM in the absence of ATP, but with ATP, it decreases by at least a factor of 10; however, phosphorylation of rhodopsin or (auto)phosphorylation of rhodopsin kinase leads to destabilization of the complex. (iii) In contrast to the binding of arrestin and transducin, the binding of rhodopsin kinase to photoactivated rhodopsin does not stabilize the metarhodopsin II photoproduct; however, rhodopsin kinase competes with the G-protein transducin for binding to photoactivated rhodopsin. Extrapolation of the kinetic parameters to cellular concentrations at room temperature suggests that free competitive binding of the kinase would strongly inhibit the G-protein activation process after a few hundred catalytic cycles.

Animals↗
PubMed1993

Assays of retinoid dehydrogenases by phase partition.

Two modifications of an extraction assay for retinoid dehydrogenases are described. The first method involves the transfer of tritium from carbon-15 of [15-3H]-retinol or [15-3H]retinaldehyde to NAD, whereas in the second method, tritium from [3H]NADPH is transferred to all-trans-retinaldehyde. Since both versions of the assay involve the interconversion of water-soluble and -insoluble tritium-labeled compounds, a simple phase partition is sufficient to separate labeled products from labeled reactants. The assays are shown to provide reliable estimations of the reaction progress for three retinoid dehydrogenases of the visual system when compared to HPLC analysis of retinoid products or gel filtration analysis of pyridine nucleotide. The assays will be useful in studying retinoid dehydrogenases from other tissues and in principle can be modified for other dehydrogenase reactions with water-insoluble substrates.

Alcohol Oxidoreductases↗
PubMed1993

The kinetics of multiphosphorylation of rhodopsin.

Rhodopsin kinase catalyzes the incorporation of up to seven phosphates into the carboxyl terminal region of freshly bleached rhodopsin. In order to study the mechanism of this reaction, we have separated different phosphorylated species of rhodopsin using Mono P FPLC chromatofocusing chromatography. The purity of the isolated species of rhodopsin was determined by isoelectric focusing. Separation yielded two forms of monophosphorylated and two diphosphorylated species of rhodopsin. Other species, containing up to five phosphates, were not fully separated. The phosphorylated forms of rhodopsin were characterized by competition enzyme-linked immunosorbent assay and immunoblotting using anti-rhodopsin site-specific monoclonal antibodies. A combination of the above methods allowed quantitative determination of the formation of different phosphorylated species of rhodopsin. A computer model for the consecutive time course of rhodopsin phosphorylation was developed and employed to characterize this reaction. Our data suggest that the rate of incorporation of the first phosphates into rhodopsin is slower than the rate of formation of more highly phosphorylated species. These data are supported by results showing that some monophosphorylated synthetic peptides are phosphorylated significantly faster than control unphosphorylated peptides.

Amino Acid Sequence↗
PubMed1993

Sequential phosphorylation of rhodopsin at multiple sites.

Photolyzed rhodopsin is phosphorylated at multiple serine and threonine residues during the quenching of phototransduction. Sites of phosphorylation by rhodopsin kinase have been localized to the C-terminal region of rhodopsin, but no information was available on the kinetics and identity of phosphorylated residues. To determine the kinetics of phosphorylation at specific residues, the phosphorylated C-terminal peptide of rhodopsin (330DDEASTTVSKTETSQVAPA) obtained by proteolysis of rhodopsin with endoproteinase Asp-N was subjected to further subdigestion followed by electrospray mass spectrometry. Analysis of monophosphorylated peptide revealed that the major initial phosphorylation site is 338Ser. The analysis of di- and triphosphorylated peptides indicated that 343Ser or 336Thr residues are subsequent phosphorylation sites. These three residues, located in the C-terminal region of rhodopsin, are likely to be key phosphorylation sites of rhodopsin during the quenching of phototransduction. Identification of the kinetics of phosphorylation will facilitate understanding the functional significance of rhodopsin phosphorylation at multiple sites and the mechanism of rhodopsin kinase action.

Amino Acid Sequence↗
PubMed1993

Beta-arrestin and arrestin are recognized by autoantibodies in sera from multiple sclerosis patients.

Multiple sclerosis (MS), one of the most common chronic neurologic diseases, is characterized by the presence of multiple plaques of demyelination throughout the central nervous system. Although the etiology of the disease has not been established, it is believed to involve autoimmune mechanisms. We have examined sera from patients with MS for the presence of antibodies to antigens from brain and retina. Immunoblot analysis of soluble fraction of proteins from bovine brain revealed a prominent band at 45 kDa stained with sera of 8-14 patients with MS. In two patients with MS, serum antibody titers during relapse were higher compared with those when the patients were in remission. These antibodies were undetectable in cerebrospinal fluid of our MS patients and additionally were absent in sera of patients with other neurological diseases and normal control subjects. Furthermore, immunoblot analysis of the soluble fraction from bovine retinal rod outer segments revealed a prominent protein band at 48 kDa stained with MS sera. This antigen was purified to homogeneity from bovine retinal outer segments and identified as arrestin. Additionally, sera from MS patients reacted with purified beta-arrestin 1, a 45-kDa protein homologous to arrestin that is found in various tissues. Using limited proteolysis of arrestin and a competitive ELISA test with a synthetic peptide, we identified the recognition site(s) for antibodies in sera of MS patients at a dominant immunogenic site on arrestin located at the C-terminal region of the molecule. We suggest that the presence of circulating antibodies reactive with beta-arrestin or arrestin may be related to the course of MS progression.

Amino Acid Sequence↗
PubMed1993

Basic fibroblast growth factor expression is required for clonogenic growth of human glioma cells.

Basic fibroblast growth factor (bFGF) is a heparin-binding protein, expressing potent mitogenic and angiogenic properties. Elevated levels of bFGF have been identified in human gliomas and glioma cell lines, suggesting that bFGF expression is involved in the aberrant growth patterns associated with these tumors. In the present study, the influence of bFGF on additional parameters of glioma cell malignancy was evaluated utilizing three distinct methods to suppress bFGF expression or activity including antisense oligonucleotide primers, a neutralizing monoclonal antibody or an inhibitor of the agonist action of bFGF: (1) The addition of 30 microM bFGF-specific antisense oligonucleotide primer to the human glioma cell line SNB-19 resulted in a 55% inhibition in colony formation in soft agar. This effect was dose-dependent and specific, as sense strand primer was ineffective in suppressing growth. In addition to exhibiting fewer colonies, antisense treatment significantly altered colony morphology. (2) SNB-19 cell growth in culture was suppressed in the presence of a neutralizing bFGF-specific monoclonal antibody. (3) Inositolhexakisphosphate, a newly identified antagonist of FGF binding and activity, suppressed SNB-19 cell growth in soft agar culture. These results demonstrate that bFGF may regulate glioma growth and progression independent of its role in tumor angiogenesis and that bFGF release or secretion may be required for these actions.

Astrocytes↗
PubMed1993

Identification of the N-terminal region in rhodopsin kinase involved in its interaction with rhodopsin.

Upon illumination rhodopsin kinase (RK) phosphorylates the visual pigment, rhodopsin, in a reaction that is thought to terminate in part the biochemical events that follow photon absorption. In this paper, RK was studied to assign functional regions to the primary structure of the enzyme. Peptides derived from the sequence of RK were used to prepare site-specific anti-peptide antibodies against: 1) the N-terminal region located between residues 17 and 34, which contains an autophosphorylation site; 2) the Lys/Arg-rich region corresponding to residues 216-237 near the catalytic domain; 3) the region located between residues 483 and 497, which encompasses the major autophosphorylation sites; and 4) the C-terminal region located between residues 539 and 556, close to the isoprenylation site of RK. Antibodies also were raised against purified RK. Application of the antibodies directed against the N-terminal domain blocks RK activity toward Rho*, but has no affect on the phosphorylation of a synthetic peptide substrate. Additionally, a significant portion of the inhibitory effect seen with an antibody directed against whole RK could be reversed by the peptide derived from the N-terminal region. We conclude that the N-terminal region of RK contains a sequence involved in the recognition of photolyzed Rho. Furthermore, the inhibition of RK activity eliminates the effect of ATP during the inactivation of cGMP phosphodiesterase, implying that RK is a necessary component of a cascade of reactions involved in the quenching of phototransduction. Light microscopic immunocytochemistry using these antibodies revealed that RK was localized to the rod and cone outer segments of human and bovine retinas.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

The effect of recoverin-like calcium-binding proteins on the photoresponse of retinal rods.

The rod photoresponse is triggered by an enzyme cascade that stimulates cGMP hydrolysis. The resulting fall in cGMP leads to a decrease in Ca2+, which promotes photoresponse recovery by activating guanylate cyclase, causing cGMP resynthesis. In vitro biochemical studies suggest that Ca2+ activation of guanylate cyclase is medicated by recoverin, a 26 kd Ca(2+)-binding protein. To evaluate this, exogenous bovine recoverin and two other homologous Ca(2+)-binding proteins from chicken and Gecko retina were dialyzed into functionally intact Gecko rods using whole-cell recording. All three proteins prolonged the rising phase of the photoresponse without affecting the kinetics of response recovery. These results suggest that recoverin-like proteins affect termination of the transduction cascade, rather than mediate Ca(2+)-sensitive activation of guanylate cyclase.

Animals↗
PubMed1993

Recoverin, but not visinin, is an autoantigen in the human retina identified with a cancer-associated retinopathy.

PURPOSE: We investigated the hypothesis that visinin, a cone-specific protein first characterized in chicken retina, is a cone homologue of recoverin and may be the cancer-associated retinopathy (CAR) autoantigen in human cone cells. METHODS: Visinin was purified from chicken retinas and tested for binding by CAR antisera. In addition, antibodies specific to visinin were used immunocytochemically and for Western analysis to determine whether visinin is present in human or bovine retinas. Anti-peptide antibodies against recoverin were used immunocytochemically to localize recoverin to mammalian cone cells. RESULTS: CAR antisera recognized recoverin but not visinin. Furthermore, visinin could not be detected in mammalian retinas by immunocytochemical methods or by attempts to purify the protein. In contrast to visinin, antibodies specific for different regions of the recoverin molecule stained both rod and cone cells in the human retina. CONCLUSIONS: Visinin is not the CAR autoantigen in human cone cells. Differences between recoverin and visinin probably reflect species differences rather than rod-cone differences. Recoverin, or a nearly identical molecule, is present in mammalian cones and likely is the cone cell CAR autoantigen.

Amino Acid Sequence↗
PubMed1992

Identification of the autophosphorylation sites in rhodopsin kinase.

Rhodopsin kinase (RK) is a second-messenger-independent protein kinase that is involved in deactivation of photolyzed rhodopsin (Rho*). We have developed a significantly improved method for isolation of RK based on the specific interactions of phosphorylated forms of the enzyme with heparin-Sepharose. Conversion of the dephosphorylated form of RK to the fully phosphorylated enzyme leads to specific elution of the kinase from the resin. Limited proteolysis of RK with endoproteinase Asp-N removes the phosphorylation sites. Peptides containing the autophosphorylation sites were isolated by reverse-phase high performance liquid chromatography and analyzed by Edman degradation and tandem mass spectrometry. The derived amino acid sequence of the peptide containing the major autophosphorylation site yielded the following sequence: DVGAFS488T489VKGVAFEK, where Ser488 and Thr489 are phosphorylated. Additionally, a minor autophosphorylation site was identified at Ser21. A 15-residue peptide (DVGAFSTVKGVAFEK) encompassing the major autophosphorylation site was synthesized and used for phosphorylation and inhibition studies. In contrast to many other protein kinases, the low catalytic activity of RK toward its autophosphorylation site peptide and the poor inhibitory properties of this peptide suggest unique properties of this member of the family of G protein-coupled receptor kinases.

Amino Acid Sequence↗
PubMed1992

The role of arrestin and retinoids in the regeneration pathway of rhodopsin.

Phototransduction results from a cascade of reactions that culminate in a neuronal signal. Photoisomerization of rhodopsin's chromophore, 11-cis-retinal to all-trans-retinal, leads to the formation of the activated photoproduct metarhodopsin II (Meta II). Subsequently, Meta II initiates the excitation events by activating many copies of the rod cell-specific G-proteins (Gt or transducin). To terminate the signal, the long-lived Meta II must be quenched. Deactivation of Meta II involves phosphorylation by rhodopsin kinase followed by the binding of arrestin. In order to recycle rhodopsin for phototransduction, arrestin must dissociate, and the chromophore must be replaced. In this study, we show that the reduction of the photolyzed chromophore all-trans-retinal to all-trans-retinol is essential for recycling photoactivated rhodopsin. Once this reduction has occurred, the arrestin blockade of the receptor is removed, the chromophore site becomes accessible for regeneration, and the phosphates can be hydrolyzed. If the reduction does not occur, we demonstrate that free all-trans-retinal can react with the apoprotein to form pseudo-photoproducts that are spectrally identical to the photoinduced metarhodopsin species (Meta I/II/III). The Meta II-like product, M380, interacts tightly with arrestin and kinase, however, it does not measurably interact with Gt. The persistent blockade by arrestin and the low affinity for Gt together prevent activation of the visual cascade. Therefore, any insufficiency in the reduction of all-trans-retinal to all-trans-retinol may lead to the accumulation of M380-arrestin in situ, which may effect adaptational processes.

Adenosine Triphosphate↗
PubMed1992

Structural properties of arrestin studied by chemical modification and circular dichroism.

A unique conformation of arrestin is crucial for its interaction with phosphorylated photolyzed rhodopsin. Conformational changes in arrestin were investigated using chemical modification and circular dichroism. We studied the kinetics of sulfhydryl modification of bovine arrestin in order to determine whether its conformation is altered by the presence of ligands or salts at different ionic strengths. We found that all three cysteines (stoichiometry was 2.7 +/- 0.06 3-carboxy-4-nitrophenyl sulfide (NbS)/arrestin) are accessible for modification by NbS2. Under pseudo-first-order conditions (30-100-fold excess of NbS2 over arrestin), the modifications of the 3 cysteines are indistinguishable. At higher concentrations of NbS2 (150-300-fold excess), the pseudo-first-order plot is not linear, and the reaction can be resolved into two processes that involve two classes of sulfhydryl groups. Addition of CaCl2, MgCl2, inorganic phosphate, MgATP, or MgGTP had little effect on the rate of modification of the cysteine residues; however, heparin and inositol hexakisphosphate, which have been shown to induce conformational changes in arrestin, block modification of one sulfhydryl group of arrestin and accelerate the modification of the remaining two. Analysis of CD spectra revealed that arrestin has virtually no alpha-helical structure, about 40% beta-structure, about 18% beta-turns, and about 40% other structure. The CD spectrum for arrestin did not change in the presence of heparin. These studies suggest that arrestin exists in equilibrium between two or more conformational states. However, it is proposed that conversion between these conformations occur without altering significantly the secondary structure of arrestin.

Adenosine Triphosphate↗
PubMed1992

The influence of arrestin (48K protein) and rhodopsin kinase on visual transduction.

The shutoff of the phototransduction cascade in retinal rods requires the inactivation of light-activated rhodopsin. The underlying mechanisms were studied in functionally intact detached rod outer segments by testing the effect of either sangivamycin, an inhibitor of rhodopsin kinase, or phytic acid, an inhibitor of 48K protein binding to phosphorylated rhodopsin, on light responses recorded in whole-cell voltage clamp. The results suggest that isomerized rhodopsin is inactivated fully by multiple phosphorylation and that the binding of 48K protein accelerates recovery by quenching partially phosphorylated rhodopsin. Higher concentrations of sangivamycin cause changes in the light response that cannot be explained by selective inhibition of rhodopsin kinase and suggest that other protein kinases are needed for normal rod function.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1991

Binding of inositol phosphates to arrestin.

Arrestin binds to phosphorylated rhodopsin in its light-activated form (metarhodopsin II), blocking thereby its interaction with the G-protein, transducin. In this study, we show that highly phosphorylated forms of inositol compete against the arrestin-rhodopsin interaction. Competition curves and direct binding assays with free arrestin consistently yield affinities in the micromolar range; for example, inositol 1,3,4,5-tetrakisphosphate (InP4) and inositol hexakisphosphate (InP6 bind to arrestin with dissociation constants of 12 microM and 5 microM, respectively. Only a small control amount of inositol phosphates is bound, when arrestin interacts with phosphorylated rhodopsin. This argues for a release of bound inositol phosphates by interaction with rhodopsin. Transducin, rhodopsin kinase, or cyclic GMP phosphodiesterase are not affected by inositol phosphates. These observations open a new way to purify arrestin and to inhibit its interaction with rhodopsin. Their physiological significance deserves further investigation.

Animals↗
PubMed1991

Phosphorylated rhodopsin and heparin induce similar conformational changes in arrestin.

Photoactivated rhodopsin is quenched upon its phosphorylation in the reaction catalyzed by rhodopsin kinase and the subsequent binding of a regulatory protein, arrestin. We have found that heparin and other polyanions compete with photoactivated, phosphorylated rhodopsin to bind arrestin (48-kDa protein, S-antigen). This is shown (a) by the suppression of stabilized metarhodopsin II; (b) by changes in the digestion of arrestin in the presence of heparin; and (c) by the restoration of arrestin-quenched phosphodiesterase activity. When bound to arrestin, heparin also mimics phosphorylated rhodopsin by similarly exposing arrestin to limited proteolysis. We conclude that heparin and rhodopsin have similar means of binding to arrestin, and we propose a cationic region of arrestin (beginning with Lys163 of the bovine sequence) as the interaction site. In agreement with previous kinetic data we interpret the results in terms of a binding conformation of arrestin which is stabilized by rhodopsin or heparin and is open to proteolytic attack.

Amino Acid Sequence↗