Biomedical subjects

K Palczewski

Publications and source records attributed to K Palczewski.

At least 127 records · Page 7Linked to original sources

PubMed1991

The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase.

Light-dependent deactivation of rhodopsin as well as homologous desensitization of beta-adrenergic receptors involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase (RK) and beta-adrenergic receptor kinase (beta ARK), respectively. We report here the cloning of a complementary DNA for RK. The deduced amino acid sequence shows a high degree of homology to beta ARK. In a phylogenetic tree constructed by comparing the catalytic domains of several protein kinases, RK and beta ARK are located on a branch close to, but separate from the cyclic nucleotide-dependent protein kinase and protein kinase C subfamilies. From the common structural features we conclude that both RK and beta ARK are members of a newly delineated gene family of guanine nucleotide-binding protein (G protein)-coupled receptor kinases that may function in diverse pathways to regulate the function of such receptors.

Amino Acid Sequence↗

PubMed1991

Role of the carboxyl-terminal region of arrestin in binding to phosphorylated rhodopsin.

The structural and functional properties of arrestin were studied by subjecting the protein to limited proteolysis. Limited proteolysis by trypsin cleaves arrestin (48 kDa), producing 20-25-kDa fragments. Prior to this stage of proteolysis, trypsin produced 46.6-, 45.4-, and 42-kDa fragments. Structural analysis of the proteolytic fragments demonstrated major cleavage at the carboxyl terminus, indicating that the carboxyl terminus is highly exposed. We found that forms of arrestin truncated at their carboxyl terminus maintained their functional properties and bound to phosphorylated rhodopsin. Native arrestin binds only to photoexcited phosphorylated rhodopsin, whereas the truncated arrestin binds to phosphorylated rhodopsin independent of its exposure to light. The truncated forms of arrestin were separated from native arrestin by a chromatographic procedure and subsequently characterized in functional studies. The binding of the truncated forms of arrestin to phosphorylated photoexcited rhodopsin is more tight than the binding of native arrestin as determined by a direct binding assay and the phosphodiesterase assay. We suggest that the acidic carboxyl-terminal region of arrestin may act as a regulator for light-dependent binding to phosphorylated rhodopsin.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1991

Mechanism of rhodopsin kinase activation.

The role of the cytoplasmic loops and C-terminal region of bovine rhodopsin (Rho) in binding and activating rhodopsin kinase was investigated. The ability of various enzymatically truncated forms of photolyzed rhodopsin (Rho*) to stimulate rhodopsin kinase activity was quantified. Following endopeptidase Asp-N cleavage of all phosphorylation sites on the C-terminal, the resulting truncated Rho* (329G-Rho*) was not phosphorylated by rhodopsin kinase. This suggests that rhodopsin kinase only phosphorylates C-terminal sites of Rho*. However 329G-Rho* could bind rhodopsin kinase and stimulate phosphorylation of exogenous peptide. Kinase stimulation was investigated for other truncated forms of Rho* in which the C-terminal region was either partially or completely eliminated, and the V-VI loop was either cleaved or left intact (339K-Rho*, 341E239E-Rho*, 329G239E-Rho*, 327P240S-Rho*). Results suggest that the V-VI loop is crucial for kinase binding (similar to the binding of GT). Mastoparan, a model peptide for G-protein-coupled receptors, was found to stimulate rhodopsin kinase in a mechanism similar to that of truncated Rho*. We conclude that rhodopsin kinase binds to the cytoplasmic loops of Rho* to cause a stimulation of its catalytic activity.

Amino Acid Sequence↗

PubMed1991

Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase.

The beta-adrenergic receptor kinase (beta-ARK) phosphorylates G protein coupled receptors in an agonist-dependent manner. Since the exact sites of receptor phosphorylation by beta-ARK are poorly defined, the identification of substrate amino acids that are critical to phosphorylation by the kinase are also unknown. In this study, a peptide whose sequence is present in a portion of the third intracellular loop region of the human platelet alpha 2-adrenergic receptor is shown to serve as a substrate for beta-ARK. Removal of the negatively charged amino acids surrounding a cluster of serines in this alpha 2-peptide resulted in a complete loss of phosphorylation by the kinase. A family of peptides was synthesized to further study the role of acidic amino acids in peptide substrates of beta-ARK. By kinetic analyses of the phosphorylation reactions, beta-ARK exhibited a marked preference for negatively charged amino acids localized to the NH2-terminal side of a serine or threonine residue. While there were no significant differences between glutamic and aspartic acid residues, serine-containing peptides were 4-fold better substrates than threonine. Comparing a variety of kinases, only rhodopsin kinase and casein kinase II exhibited significant phosphorylation of the acidic peptides. Unlike beta-ARK, RK preferred acid residues localized to the carboxyl-terminal side of the serine. A feature common to beta-ARK and RK was a much greater Km for peptide substrates as compared to that for intact receptor substrates.

Amino Acid Sequence↗

PubMed1991

Regulation of rhodopsin kinase by autophosphorylation.

Rhodopsin kinase (RK) catalyzes the phosphorylation of rhodopsin (Rho) as one of the steps in quenching photoactivated Rho. In this work, we investigated the autophosphorylation of RK and how it affects the interaction between RK and Rho. RK undergoes intramolecular phosphorylation, resulting in the incorporation of three or four phosphates per RK molecule. Phosphorylated RK subsequently is a substrate for protein phosphatases 2A and 2B. We isolated three forms of RK based on their differential interactions with heparin-Sepharose. Fully phosphorylated RK (alpha-RK) binds tightly to Rho but has significantly lower affinity to phosphorylated Rho, whereas unphosphorylated RK (gamma-RK) binds avidly to both forms of Rho. The heterogenous intermediately phosphorylated RK (beta-RK) was not studied. Our data support the hypothesis that RK dissociates from Rho when both Rho and RK become phosphorylated, thereby allowing the binding of arrestin to phosphorylated Rho. These results suggest that autophosphorylation plays an important role in regulating the binding of RK to Rho and that the binding sites of RK and arrestin overlap at least partially.

Animals↗

PubMed1991

Studies of ligand binding to arrestin.

A striking homology is observed between the regions 70-83 and 361-374 of the sequence of bovine arrestin and the calcium-binding loops of calmodulin and troponin C. However, the predicted alpha-helices flanking the calcium-binding site in calmodulin and troponin C are not present in arrestin. Direct measurements therefore were made in order to assess whether arrestin can bind calcium. We found that arrestin does not bind Ca2+ at physiological ionic strength, as determined by equilibrium dialysis, gel filtration, and fluorescence spectroscopy. Rapid and quantitative precipitation of arrestin occurs with Tb3+. The precipitation is reversed by EDTA and blocked by Mg2+ but not by Ca2+. Prompted by several reports, we also investigated whether nucleotides bind to arrestin. Neither ATP nor GTP binds under the conditions tested. Binding of arrestin to photolyzed, phosphorylated rhodopsin also does not influence the binding of calcium or nucleotides.

Amino Acid Sequence↗

PubMed1991

Elevated level of protein phosphatase 2A activity in retinas of rd mice.

Phosphorylation of rhodopsin is not detectable in vitro in the retina of the rd mouse. We investigated the enzymatic system responsible for this abnormality by measuring the levels of rhodopsin kinase and protein phosphatase 2A in normal (rd/+) and diseased (rd/rd) mouse retinas of several ages. For each enzyme, we developed micro assays that were suitable for measuring enzyme activity in one-half mouse retina. Our results indicate that rhodopsin kinase activity is identical in rd/+ and rd/rd retinas until post-natal day 11, and it decreases thereafter in the rd/rd retina, correlating with the loss of rod photoreceptors that occurs in this tissue. Protein phosphatase 2A has a constant level of activity in rd/+ retinas from ages 5 to 32 days but it is higher than normal in rd/rd retinas from post-natal days 5 to 10. It then decreases to levels that are comparable to those in rd/+ retina. Although the rd/rd extract contains the elevated protein phosphatase 2A activity, when rd/rd and rd/+ retinal extracts are each subjected to gel filtration, the elution profiles of protein phosphatase 2A activity appear to be quantitatively identical. This apparent loss of rd/rd phosphatase activity suggests a difference in the regulatory behavior of the enzyme in the normal and degenerative retinas. Thus, the failure to detect in vitro phosphorylation of rhodopsin in the rd/rd retina seems to result from the elevated level of protein phosphatase 2A activity which could more rapidly remove the phosphate from phosphorylated rhodopsin. Since protein phosphatase 2A is a ubiquitous enzyme with broad specificity, an elevation in its activity also could affect other protein phosphorylations.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1991

Anti-rhodopsin monoclonal antibodies of defined specificity: characterization and application.

A panel of anti-bovine rhodopsin monoclonal antibodies (MAbs) of defined site-specificity has been prepared and used for functional and topographic studies of rhodopsins. In order to select these antibodies, hybridoma supernatants that contained anti-rhodopsin antibodies have been screened by enzyme-linked immunosorbent assay (ELISA) in the presence of synthetic peptides from rhodopsin's cytoplasmic regions. We selected for antibodies against predominantly linear determinants (as distinct from complex assembled determinants) and have isolated antibodies that recognize rhodopsin's amino terminus, its carboxyl terminus, as well as the hydrophilic helix-connecting regions 61-75, 96-115, 118-203, 230-252 and 310-321. Detailed specificities have been further determined by using a series of overlapping peptides and chemically modified rhodopsins as competitors. A group of seven antibodies with epitopes clustered within the amino terminal region of rhodopsin and a group of 15 antibodies with epitopes within the carboxyl terminal region are described. These MAbs have high affinities for rhodopsin with Kas in the range of 10(8)-10(10) M-1. Some MAbs specific for the carboxyl and amino terminal regions were used to compare these bovine rhodopsin sequences to those of different vertebrates. The MAbs cross-reacted with the different species tested to different extents indicating that there is some similarity in the sequences of these regions. However, some differences in the sequences were indicated by a reduced or absent cross-reactivity with some MAbs. In membrane topographic studies the MAbs showed both the presence and the accessibility of rhodopsin sequences 330-348, 310-321 and 230-252 on the cytoplasmic surface of the disk membrane. Similarly, sequences 1-20 and 188-203 were shown to reside on the lumenal surface of the disk and to be accessible to a macromolecular (antibody) probe. Antibodies directed against rhodopsin's carboxyl terminal sequence did not bind well to highly phosphorylated rhodopsin. Similarly, these antibodies as well as those against the V-VI loop inhibited phosphorylation of rhodopsin. Antibody A11-82P, specific for phosphorylated rhodopsin, recognized rhodopsin containing two or more phosphates and inhibited its further phosphorylation.

Animals↗

PubMed1991

G-protein-coupled receptor kinases.

Rhodopsin kinase and the beta-adrenergic receptor kinase (beta ARK) catalyse the phosphorylation of the activated forms of the G-protein-coupled receptors, rhodopsin and the beta 2-adrenergic receptor (beta 2AR), respectively. The interaction between receptor and kinase is independent of second messengers and appears to involve a multipoint attachment of kinase and substrate with the specificity being restricted by both the primary amino acid sequence and conformation of the substrate. Kinetic, functional and sequence information reveals that rhodopsin kinase and beta ARK are closely related, suggesting they may be members of a family of G-protein-coupled receptor kinases.

Animals↗

PubMed1991

Properties of solubilized and reconstituted A1 adenosine receptors from bovine brain.

A simple method for solubilization and reconstitution of the A1 adenosine receptor from bovine brain is presented. Solubilization with CHAPS-phosphatidylcholine (CHAPS/PC) mixture did not alter the binding properties of the A1 adenosine receptor antagonist [3H]-DPCPX. The solubilized receptors were chromatographed on hydroxyapatite or DEAE-cellulose to remove native membrane lipids and part of non-receptor proteins. Elution of the receptor fractions was obtained from DEAE-cellulose column with a linear gradient of KCl (0-0.4 M). The fractions corresponding to the peak of [3H]-DPCPX binding activity were then reconstituted in phosphatidylcholine by dialysis. The reconstituted receptor retained all the binding characteristics and the same rank order of competition potency (R-PIA greater than S-PIA greater than NECA) as the native receptor, although its thermal stability was remarkably reduced. The binding of [3H]-DPCPX to A1 adenosine receptors was increased by GTP, probably as result of interactions with coeluted G-proteins.

Adenosine↗

PubMed1991

A photoreceptor calcium binding protein is recognized by autoantibodies obtained from patients with cancer-associated retinopathy.

Cancer-associated retinopathy (CAR), a paraneoplastic syndrome, is characterized by the degeneration of retinal photoreceptors under conditions where the tumor and its metastases have not invaded the eye. The retinopathy often is apparent before the diagnosis of cancer and may be associated with autoantibodies that react with specific sites in the retina. We have examined the sera from patients with CAR to further characterize the retinal antigen. Western blot analysis of human retinal proteins reveals a prominent band at 26 kD that is labeled by the CAR antisera. Antibodies to the 26-kD protein were affinity-purified from complex CAR antisera and used for EM-immunocytochemical localization of the protein to the nuclei, inner and outer segments of both rod and cone cells. Other antibodies obtained from the CAR sera did not label photoreceptors. Using the affinity-purified antibodies for detection, the 26-kD protein, designated p26, was purified to homogeneity from the outer segments of bovine rod photoreceptor cells by Phenyl-Sepharose and ion exchange chromatography. Partial amino acid sequence of p26 was determined by gas phase Edman degradation and revealed extensive homology with a cone-specific protein, visinin. Based upon structural relatedness, both the p26 rod protein and visinin are members of the calmodulin family and contain calcium binding domains of the E-F hand structure.

Amino Acid Sequence↗

PubMed1990

Phosphorylation of iodopsin, chicken red-sensitive cone visual pigment.

The amino acid sequence has been determined for the carboxyl-terminal 41 amino acids of chicken red-sensitive cone pigment, iodopsin. This sequence is distinct from but structurally homologous to that of other visual pigments. It contains a region rich in the hydroxy amino acids serine and threonine. In the related rod cell visual pigment, rhodopsin, such serines and threonines have previously been identified as sites for phosphorylation by rhodopsin kinase. Phosphorylation of photolyzed rhodopsin serves to terminate its ability to function in visual transduction as an activator of G-protein. We have purified and reconstituted both chicken rhodopsin and chicken iodopsin and shown them to be phosphorylated by bovine rhodopsin kinase. Chicken iodopsin has a Km and Vmax similar to but distinguishably different from that for bovine rhodopsin. These results, in conjunction with other data, suggest that visual pigments in cone cells, upon absorption of light, undergo functional processes similar to those of the visual pigments in rod cells.

Amino Acid Sequence↗

PubMed1990

Nucleoside inhibitors of rhodopsin kinase.

The specificity of the ATP-binding site of rhodopsin kinase was studied with adenosine analogues that are competitive inhibitors. Systematic changes in the ribose ring (position 5') and the purine ring (positions 2, 6, 7, 8, and 9) and determination of the inhibitory properties of these analogues lead to the following conclusions: (1) The N6 nitrogen in the purine ring is essential for binding at the active site, which may explain the marked preference for ATP rather than GTP as substrate. (2) The configuration of the sugar moiety is critical for the binding. (3) Positions 2, 3, and 8 of the purine ring, as well as the polyphosphate chain, play a minor role in substrate recognition by rhodopsin kinase. (4) ATP gamma S is a good substrate for rhodopsin kinase (thus rhodopsin phosphorothioate, a phosphatase-resistant product, can be formed in order to study the role of phosphorylation in rod outer segments). Pyrrolopyrimidine derivatives are very potent inhibitors of rhodopsin kinase. The Ki of one of these, sangivamycin, is 180 nM. Sangivamycin in solution assumes the anti conformation, as determined by nuclear Overhauser measurement. These measurements show that the most potent inhibitors of rhodopsin kinase, sangivamycin and toyocamycin, occur in solution preferentially in the anti conformation. Many nucleotides and nucleosides tested that are not inhibitors are syn, and many that are inhibitors form a mixture of syn and anti. The hypothesis that inhibitors may have a conformation intermediate between syn and anti was strengthened by testing a cyclic nucleoside locked in an anti conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1990

X-ray crystal structure of sangivamycin, a potent inhibitor of protein kinases.

The X-ray crystal structure of sangivamycin, a potent nucleoside inhibitor of protein kinases, has been determined. Sangivamycin crystallizes from water with its purine ring in a conformation anti to its ribose sugar. Such an anti conformation has been detected in solution for sangivamycin and other potent protein kinase inhibitors and appears to correlate with inhibitor potency [(1990) Biochemistry (in press)]. An intramolecular hydrogen bond between purine ring substituents is detected in the X-ray structure and may be an important structural feature of sangivamycin related to its degree of inhibition of rhodopsin kinase and of protein kinases C and A.

Crystallography↗

PubMed1990

Synthetic phosphopeptides are substrates for casein kinase II.

Casein kinase II is a protein serine/threonine kinase that exhibits a preference for acidic substrates. Previous studies have demonstrated that a glutamic acid 3 amino acids C-terminal (+3) to a serine or threonine is required for phosphorylation. To examine the ability of phosphoserine and phosphothreonine residues to serve as specificity determinants for casein kinase II, phosphopeptides containing either of these phosphoamino acids in the +3 position were synthesized and tested as substrates. Phosphopeptides containing phosphoserine in the +3 position were readily phosphorylated. In contrast, corresponding phosphothreonine-containing peptides were very poorly phosphorylated. These results imply that prior phosphorylation of substrate proteins on serine, but not threonine residues, may II.

Amino Acid Sequence↗

PubMed1990

Molecular, enzymatic and functional properties of rhodopsin kinase from rat pineal gland.

Rhodopsin kinase activity from rat pineal gland and from rat retina are indistinguishable, based upon determination of a variety of enzymatic and molecular properties. Both activities are independent of calcium, cyclic nucleotides, and calmodulin. Both are activated by spermine and inhibited by adenosine and some rhodopsin kinase specific adenosine derivatives such as sangivamycin. The Km's for rhodopsin, ATP, and GTP are indistinguishable for the protein kinase in extracts from the retina and from the pineal gland. The apparent molecular weight of the kinase from both sources, as determined by gel filtration and autoradiography of the 32P-labeled autophosphorylated kinase, is about 70 kDa. Rhodopsin kinase activity from pineal binds in a light-dependent manner to rhodopsin in rod outer segments as does the enzyme from retina. Monoclonal antibodies against bovine rhodopsin were used in an immunochemical study that identified a rhodopsin-immunoreactive protein in rat pineal gland and retina. Using an ELISA we demonstrated the presence of a rhodopsin-immunoreactive protein in rat pineal gland equivalent to 0.075 pmol rhodopsin per gland. Frog pineal organ (Rana catesbiana) contains 33 times more of this rhodopsin-like protein than does rat pineal gland.

Animals↗

PubMed1989

Substrate recognition determinants for rhodopsin kinase: studies with synthetic peptides, polyanions, and polycations.

Rhodopsin kinase phosphorylates serine- and threonine-containing peptides from bovine rhodopsin's carboxyl-terminal sequence. Km's for the peptides decrease as the length of the peptide is increased over the range 12-31 amino acids, reaching 1.7 mM for peptide 318-348 from the rhodopsin sequence. The Km for phosphorylation of rhodopsin is about 10(3) lower than that for the peptides, which suggests that binding of rhodopsin kinase to its substrate, photolyzed rhodopsin, involves more than just binding to the carboxyl-terminal peptide region that is to be phosphorylated. A synthetic peptide from the rhodopsin sequence that contains both serines and threonines is improved as a substrate by substitution of serines for the threonines, suggesting that serine residues are preferred as substrates. Analogous 25 amino acid peptides from the human red or green cone visual pigment, a beta-adrenergic receptor, or M1 muscarinic acetylcholine receptors are better substrates for bovine rhodopsin kinase than is the peptide from bovine rhodopsin. An acidic serine-containing peptide from a non-receptor protein, alpha s1B-casein, is also a good substrate for rhodopsin kinase. However, many basic peptides that are substrates for other protein kinases--histone IIA, histone IIS, clupeine, salmine, and a neurofilament peptide--are not phosphorylated by rhodopsin kinase. Polycations such as spermine or spermidine are nonessential activators of phosphorylation of rhodopsin or its synthetic peptide 324-348. Polyanions such as poly(aspartic acid), dextran sulfate, or poly(adenylic acid) inhibit the kinase. Poly(L-aspartic acid) is a competitive inhibitor with respect to rhodopsin (KI = 300 microM) and shows mixed type inhibition with respect to ATP.

Amino Acid Sequence↗

PubMed1989

Regulation of rhodopsin dephosphorylation by arrestin.

We have characterized the opsin phosphatase activities in extracts of rod outer segments and determined their relationship to known protein phosphatases. The opsin phosphatase activity in the extracts was not due to protein phosphatases 1, 2B, or 2C because it was neither stimulated by Mg2+ or Ca2+/calmodulin nor inhibited by protein phosphatase inhibitors-1 or -2. Opsin phosphatase activity in rod outer segment extracts was potently inhibited by okadaic acid (IC50 approximately 10 nM), a preferential inhibitor of protein phosphatase 2A. Moreover, during chromatography on DEAE-Sepharose, the opsin phosphatase activity co-eluted with three peaks of protein phosphatase 2A activity, termed protein phosphatases 2A0, 2A1, and 2A2. The opsin phosphatase activity of each peak was stimulated by polylysine, a known activator of protein phosphatase 2A. Finally, treatment of rod outer segment extracts with 80% ethanol at room temperature converted the activity from a high molecular weight form characteristic of the protein phosphatase 2A0, 2A1, and 2A2 species to a low molecular weight form characteristic of the protein phosphatase 2A catalytic subunit. We conclude that protein phosphatase 2A is likely to be the physiologically relevant rhodopsin phosphatase. The 48-kDa rod outer segment protein arrestin (S-antigen) was found to inhibit the dephosphorylation of freshly photolyzed rhodopsin by protein phosphatase 2A but did not inhibit the dephosphorylation of unbleached rhodopsin. Arrestin has no effect on the dephosphorylation of phorphorylase a, indicating that the effect was substrate-directed. It appears that dephosphorylation of the photoreceptor protein phosphorhodopsin occurs only after decay of the photoactivated protein and that this may be regulated in vivo by arrestin. The binding of arrestin to photolyzed phosphorylated rhodopsin, i.e. the binding of a regulatory protein to a protein phosphatase substrate to form a complex resistant to dephosphorylation represents a novel mechanism for the regulation of protein phosphatase 2A.

Animals↗