Phosphorylation of rhodopsin by specific protein kinase, rhodopsin kinase is a part of the visual transduction system. This phosphorylation following light absorption is one mechanism of shutting off the excitation process. Recently, a number of other receptor proteins that operate via guanine nucleotide binding protein have been found to be homologous to rhodopsin and have been shown to undergo phosphorylation. The phosphorylation causes heterologous and homologous desensitization. I will focus on phosphorylation of rhodopsin kinase by autophosphorylation and phosphorylation of the enzyme by other protein kinases, since the reaction is widely known to regulate the activity and function of many other enzymes, including protein kinases. I am going to look at the reaction conditions in vitro and identify the reaction in vivo. Also, I would like to find a dephosphorylation mechanism for rhodopsin kinase. Properties of phosphorylated rhodopsin kinase such as interaction with ROS membrane enzyme activity, interaction with nucleotides, etc. will be compared with that of the native enzyme. Three proteins, transducin, 48k protein and rhodopsin kinase are known to interact with photo-excited rhodopsin, but no kinetic data are available. It is not clear how activation of transducin precedes phosphorylation of rhodopsin by the kinase. I propose appropriate kinetic and binding studies for determination of the competition between these proteins. I will extend these studies to interaction of these proteins with non-phosphorylated rhodopsin as well as with different phosphorylated species of rhodopsin. Rhodopsin kinase activity has been detected in the pineal gland and in retinoblastoma cells. It is unknown whether these activities are a response to rhodopsin kinase or to a protein kinase with similar specificities. It is also unclear what role the activity has, particularly in pineal gland, and in the original precursor of the retinoblastoma cells. Properties of these enzymes will be compared with those of retina RK by evaluation of kinetic properties and by immunological methods, using MAb to RK. These monoclonal antibodies also can be used for studies of the related protein kinases from the family of receptors operating via G- protein (adrenergic and muscarinic cholinergic receptors, thylakoid proteins). Since the rhodopsin kinase sequence is unknown, I propose to determine the sequence of functionally important parts of the protein, which opens the possibility of fully sequencing the kinase using cDNA techniques. This part of my studies, together with partial limited proteolysis, affinity labeling and fluorescent analysis, should give detailed information about the sequence of phosphorylation and autophosphorylation sites, localization of the active site, and confirmation of the domain structure of the kinase.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29EY008061-04
Application #
3465722
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1990-02-01
Project End
1992-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Good Samaritan Hosp & Medical Center(Prtlnd,OR)
Department
Type
DUNS #
City
Portland
State
OR
Country
United States
Zip Code
97210
Jastrzebska, Beata; Chen, Yuanyuan; Orban, Tivadar et al. (2015) Disruption of Rhodopsin Dimerization with Synthetic Peptides Targeting an Interaction Interface. J Biol Chem 290:25728-44
Chen, Yuanyuan; Jastrzebska, Beata; Cao, Pengxiu et al. (2014) Inherent instability of the retinitis pigmentosa P23H mutant opsin. J Biol Chem 289:9288-303
Tu, Xiongying; Palczewski, Krzysztof (2014) The macular degeneration-linked C1QTNF5 (S163) mutation causes higher-order structural rearrangements. J Struct Biol 186:86-94
Mustafi, Debarshi; Kikano, Sandra; Palczewski, Krzysztof (2014) Serial block face-scanning electron microscopy: a method to study retinal degenerative phenotypes. Curr Protoc Mouse Biol 4:197-204
Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof (2014) Structural approaches to understanding retinal proteins needed for vision. Curr Opin Cell Biol 27:32-43
Chen, Yuanyuan; Tang, Hong; Seibel, William et al. (2014) Identification and characterization of novel inhibitors of Mammalian aspartyl aminopeptidase. Mol Pharmacol 86:231-42
Palczewski, Krzysztof; Kiser, Philip D (2013) Biochemistry. As good as chocolate. Science 340:562-3
Jastrzebska, Beata; Orban, Tivadar; Golczak, Marcin et al. (2013) Asymmetry of the rhodopsin dimer in complex with transducin. FASEB J 27:1572-84
Palczewski, Krzysztof; Orban, Tivadar (2013) From atomic structures to neuronal functions of g protein-coupled receptors. Annu Rev Neurosci 36:139-64
Jastrzebska, Beata; Salom, David; Jin, Hui et al. (2013) Expression of mammalian G protein-coupled receptors in Caenorhabditis elegans. Methods Enzymol 520:239-56

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