Publications

• Non-visual GRKs: are we seeing the whole picture?

  Willets J.M., Challiss R.A., Nahorski S.R.

  G-protein-coupled receptor kinases (GRKs) comprise a family of seven mammalian serine/threonine protein kinases that phosphorylate and regulate agonist-occupied or constitutively active G-protein-coupled receptors (GPCRs). Studies of the details and consequences of these mechanisms have focused he ... >> More

  G-protein-coupled receptor kinases (GRKs) comprise a family of seven mammalian serine/threonine protein kinases that phosphorylate and regulate agonist-occupied or constitutively active G-protein-coupled receptors (GPCRs). Studies of the details and consequences of these mechanisms have focused heavily on the original beta-adrenoceptor kinase (beta-ARK) family (GRK2 and GRK3) and, in particular, on phosphorylation-dependent recruitment of adaptor proteins such as the beta-arrestins. However, recent work has indicated roles for the other, non-visual GRKs (GRK4, GRK5 and GRK6) and has revealed potential phosphorylation-independent regulation of GPCRs by GRK2 and GRK3. In this article, we review this newer information and attempt to put it into context with GRKs as physiological regulators that could be appropriate targets for future pharmacological intervention. << Less

  Trends Pharmacol Sci 24:626-633(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Rhodopsin kinase: expression in baculovirus-infected insect cells, and characterization of post-translational modifications.

  Cha K., Bruel C., Inglese J., Khorana H.G.

  Structure-function studies of rhodopsin kinase (RK; EC 2.7.1.125) require a variety of mutants. Therefore, there is need for a suitable system for the expression of RK mutant genes. Here we report on a study of expression of the RK gene in baculovirus-infected Sf21 cells and characterization of th ... >> More

  Structure-function studies of rhodopsin kinase (RK; EC 2.7.1.125) require a variety of mutants. Therefore, there is need for a suitable system for the expression of RK mutant genes. Here we report on a study of expression of the RK gene in baculovirus-infected Sf21 cells and characterization of the enzyme produced as purified to near homogeneity. Particular attention has been paid to the post-translational modifications, autophosphorylation and isoprenylation, found in the native bovine RK. The protein produced has been purified using, successively, heparin-Sepharose, Mono Q, and Mono S FPLC (fast protein liquid chromatography) and was obtained in amounts of about 2 mg from 1 liter of cell culture. The enzyme from the last step of purification was obtained in two main fractions that differ in the level of phosphorylation. The protein peak eluted first carries two phosphate groups per protein, whereas the second protein peak is monophosphorylated. Further, while both peaks are isoprenylated, the isoprenyl groups consist of mixtures of C5, C10, C15, and C20 isoprenyl moieties. From these results, we conclude that the above expression system is suitable for some but not all aspects of structure-function studies. << Less

  Proc Natl Acad Sci U S A 94:10577-10582(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Light-dependent phosphorylation of rhodopsin. Purification and properties of rhodopsin kinase.

  Shichi H., Somers R.L.

  J Biol Chem 253:7040-7046(1978) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of the autophosphorylation sites in rhodopsin kinase.

  Palczewski K., Buczylko J., van Hooser P., Carr S.A., Huddleston M.J., Crabb J.W.

  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-Sephar ... >> More

  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. << Less

  J. Biol. Chem. 267:18991-18998(1992) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of human GRK7 as a potential cone opsin kinase.

  Chen C.-K., Zhang K., Church-Kopish J., Huang W., Zhang H., Chen Y.-J., Frederick J.M., Baehr W.

  <h4>Purpose</h4>Homozygous inactivation of the mouse gene for GRK1 (G protein-coupled receptor kinase 1, or rhodopsin kinase) causes severe defects in the recovery of cone phototransduction. However, electroretinographic (ERG) analyses of human oguchi patients with defective GRK1 alleles showed no ... >> More

  <h4>Purpose</h4>Homozygous inactivation of the mouse gene for GRK1 (G protein-coupled receptor kinase 1, or rhodopsin kinase) causes severe defects in the recovery of cone phototransduction. However, electroretinographic (ERG) analyses of human oguchi patients with defective GRK1 alleles showed normal or slightly abnormal photopic responses. It remains unclear why the loss of GRK1 yields such different phenotypes in the recovery of mouse and human cones. We examined the localization and enzyme activity of GRK7, the human ortholog of the seventh member of the GRK family, in an attempt to understand its potential role in photopic vision.<h4>Methods</h4>Bioinformatic approaches were used to identify the human GRK7 gene. Human and bovine GRK7 cDNAs were isolated by RT-PCR. Recombinant GRK7, expressed in insect cells, was used to phosphorylate activated rhodopsin. Antibodies raised against GRK7 peptides were used to examine the retina specific expression of GRK7 by immunoblotting and its subcellular localization by immunocytochemistry.<h4>Results</h4>The human GRK7 gene is located on chromosome 3q21, spans at least 10 Kb and consists of 4 exons. In human, GRK7 is expressed exclusively in the retina and is found in all retinal neurons, and specifically, in cone outer segments. Recombinant human GRK7 catalyzes rhodopsin phosphorylation in a light dependent manner. We provide evidence that GRK1 and GRK7 are co-expressed in human cones. In contrast, mouse GRK7 is expressed in many tissues including retina where photoreceptors apparently do not express GRK7.<h4>Conclusions</h4>The presence of GRK7 in human, but not in mouse, cone outer segments suggests that GRK7 may function to provide the normal photopic vision reported by oguchi patients with a defective GRK1 gene. The absence of GRK7 expression in cone outer segments of mice is consistent with the notion that mouse cones rely solely on GRK1 to shutoff cone visual pigments. << Less

  Mol. Vis. 7:305-313(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• GRK1 and GRK7: unique cellular distribution and widely different activities of opsin phosphorylation in the zebrafish rods and cones.

  Wada Y., Sugiyama J., Okano T., Fukada Y.

  Retinal cone cells exhibit distinctive photoresponse with a more restrained sensitivity to light and a more rapid shutoff kinetics than those of rods. To understand the molecular basis for these characteristics of cone responses, we focused on the opsin deactivation process initiated by G protein- ... >> More

  Retinal cone cells exhibit distinctive photoresponse with a more restrained sensitivity to light and a more rapid shutoff kinetics than those of rods. To understand the molecular basis for these characteristics of cone responses, we focused on the opsin deactivation process initiated by G protein-coupled receptor kinase (GRK) 1 and GRK7 in the zebrafish, an animal model suitable for studies on retinal physiology and biochemistry. Screening of the ocular cDNAs identified two homologs for each of GRK1 (1A and 1B) and GRK7 (7-1 and 7-2), and they were classified into three GRK subfamilies, 1 A, 1B and 7 by phylogenetic analysis. In situ hybridization and immunohistochemical studies localized both GRK1B and GRK7-1 in the cone outer segments and GRK1A in the rod outer segments. The opsin/GRKs molar ratio was estimated to be 569 in the rod and 153 in the cone. The recombinant GRKs phosphorylated light-activated rhodopsin, and the Vmax value of the major cone subtype, GRK7-1, was 32-fold higher than that of the rod kinase, GRK1A. The reinforced activity of the cone kinase should provide a strengthened shutoff mechanism of the light-signaling in the cone and contribute to the characteristics of the cone responses by reducing signal amplification efficiency. << Less

  J. Neurochem. 98:824-837(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization and chromosomal localization of the gene for human rhodopsin kinase.

  Khani S.C., Abitbol M., Yamamoto S., Maravic-Magovcevic I., Dryja T.P.

  G-protein-dependent receptor kinases (GRKs) play a key role in the adaptation of receptors to persistent stimuli. In rod photoreceptors rhodopsin kinase (RK) mediates rapid desensitization of rod photoreceptors to light by catalyzing phosphorylation of the visual pigment rhodopsin. To study the st ... >> More

  G-protein-dependent receptor kinases (GRKs) play a key role in the adaptation of receptors to persistent stimuli. In rod photoreceptors rhodopsin kinase (RK) mediates rapid desensitization of rod photoreceptors to light by catalyzing phosphorylation of the visual pigment rhodopsin. To study the structure and mechanism of GRKs in human photoreceptors, we have isolated and characterized cDNA and genomic clones derived from the human RK locus using a bovine rhodopsin kinase cDNA fragment as a probe. The RK locus, assigned to chromosome 13 band q34, is composed of seven exons that encode a protein 92% identical in amino acid sequence to bovine rhodopsin kinase. The marked difference between the structure of this gene and that of another recently cloned human GRK gene suggests the existence of a wide evolutionary gap between members of the GRK gene family. << Less

  Genomics 35:571-576(1996) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and characterization of rhodopsin kinase.

  Palczewski K., McDowell J.H., Hargrave P.A.

  Rhodopsin kinase was purified to near homogeneity by affinity binding to light-exposed rod cell outer segment membranes, followed by DEAE-cellulose and hydroxyapatite chromatography. This resulted in a 1055-fold purification of highly active rhodopsin kinase with an overall recovery of 19%. Rhodop ... >> More

  Rhodopsin kinase was purified to near homogeneity by affinity binding to light-exposed rod cell outer segment membranes, followed by DEAE-cellulose and hydroxyapatite chromatography. This resulted in a 1055-fold purification of highly active rhodopsin kinase with an overall recovery of 19%. Rhodopsin kinase is a single polypeptide chain with Mr = 67,000-70,000 as determined by gel filtration and SDS-PAGE. The kinetic parameters of the enzyme for freshly bleached rhodopsin are Km = 4 microM and Vmax = 700 nmol/min/mg whereas for ATP Km = 2 microM (which is a low value for kinases generally, and about 20 times lower than comparable measurements for a kinase of a similar type, the beta-adrenergic-receptor kinase (Benovic, J.L., Mayor, F. Jr., Staniszewski, C., Lefkowitz, R.J., and Caron, M.G. (1987) J. Biol. Chem. 262, 9026-9032). GTP, on the other hand, is a very poor substrate (Km = 1 mM, Vmax = 10 nmol/min/mg). Rhodopsin kinase is competitively inhibited by adenosine and its mono- and diphosphate derivatives, but not by most other adenosine derivatives. Based upon measurements with 28 nucleotide derivatives, the ATP-binding site of rhodopsin kinase appears to have more specific requirements than that for other kinases. Compounds such as cGMP, inositol trisphosphate, and others that change concentration during exposure of rod cells to light have only minor inhibitory effects on the kinase activity, with the exception of inositol monophosphate, which can activate the kinase about 20% at 50-100 microM. Rhodopsin kinase has been difficult to store with retention of activity, but can be successfully stored frozen at -20 degrees C in 20% adonitol. << Less

  J Biol Chem 263:14067-14073(1988) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Light-stimulated phosphorylation of rhodopsin in the retina: the presence of a protein kinase that is specific for photobleached rhodopsin.

  Weller M., Virmaux N., Mandel P.

  A protein kinase has been extracted from bovine rod outer segments by a mild procedure. The enzyme acts specifically on photobleached, not unbleached, rhodopsin and will not catalyze the phosphorylation of histones, phosvitin, or casein. We propose the name "opsin kinase" for the enzyme, which is ... >> More

  A protein kinase has been extracted from bovine rod outer segments by a mild procedure. The enzyme acts specifically on photobleached, not unbleached, rhodopsin and will not catalyze the phosphorylation of histones, phosvitin, or casein. We propose the name "opsin kinase" for the enzyme, which is not affected by cyclic nucleotides but which is inhibited by theophylline. Preparations of purified rod outer segments, however, appear to contain only low concentration of opsin phosphatase activity. << Less

  Proc Natl Acad Sci U S A 72:381-385(1975) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Light-dependent phosphorylation of rhodopsin by beta-adrenergic receptor kinase.

  Benovic J.L., Mayor F. Jr., Somers R.L., Caron M.G., Lefkowitz R.J.

  The structural components involved in transduction of extracellular signals as diverse as a photon of light impinging on the retina or a hormone molecule impinging on a cell have been highly conserved. These components include a recognition unit or receptor (for example, the beta-adrenergic recept ... >> More

  The structural components involved in transduction of extracellular signals as diverse as a photon of light impinging on the retina or a hormone molecule impinging on a cell have been highly conserved. These components include a recognition unit or receptor (for example, the beta-adrenergic receptor (beta AR) for catecholamines or the 'light receptor' rhodopsin), a guanine nucleotide regulatory or transducing protein, and an effector enzyme (for example, adenylate cyclase or cyclic GMP phosphodiesterase). Molecular cloning has revealed that the beta AR shares significant sequence and three-dimensional homology with rhodopsin. The function of the beta AR is diminished by exposure to stimulatory agonists, leading to desensitization. Similarly, 'light adaptation' involves decreased coupling of photoactivated rhodopsin to cGMP phosphodiesterase activation. Both forms of desensitization involve receptor phosphorylation. The latter is mediated by a unique protein kinase, rhodopsin kinase, which phosphorylates only the light-bleached form of rhodopsin. An analogous enzyme (termed beta AR kinase or beta ARK) phosphorylates only the agonist-occupied beta AR. We report here that beta ARK is also capable of phosphorylating rhodopsin in a totally light-dependent fashion. Moreover, rhodopsin kinase can phosphorylate the agonist-occupied beta AR. Thus the mechanisms which regulate the function of these disparate signalling systems also appear to be similar. << Less

  Nature 321:869-872(1986) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase.

  Chen C.K., Burns M.E., Spencer M., Niemi G.A., Chen J., Hurley J.B., Baylor D.A., Simon M.I.

  Phosphorylation is thought to be an essential first step in the prompt deactivation of photoexcited rhodopsin. In vitro, the phosphorylation can be catalyzed either by rhodopsin kinase (RK) or by protein kinase C (PKC). To investigate the specific role of RK, we inactivated both alleles of the RK ... >> More

  Phosphorylation is thought to be an essential first step in the prompt deactivation of photoexcited rhodopsin. In vitro, the phosphorylation can be catalyzed either by rhodopsin kinase (RK) or by protein kinase C (PKC). To investigate the specific role of RK, we inactivated both alleles of the RK gene in mice. This eliminated the light-dependent phosphorylation of rhodopsin and caused the single-photon response to become larger and longer lasting than normal. These results demonstrate that RK is required for normal rhodopsin deactivation. When the photon responses of RK-/-rods did finally turn off, they did so abruptly and stochastically, revealing a first-order backup mechanism for rhodopsin deactivation. The rod outer segments of RK-/-mice raised in 12-hr cyclic illumination were 50% shorter than those of normal (RK+/+) rods or rods from RK-/-mice raised in constant darkness. One day of constant light caused the rods in the RK-/-mouse retina to undergo apoptotic degeneration. Mice lacking RK provide a valuable model for the study of Oguchi disease, a human RK deficiency that causes congenital stationary night blindness. << Less

  Proc. Natl. Acad. Sci. U.S.A. 96:3718-3722(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.