Objetivo G protein coupled receptors (GPCRs) are membrane proteins, which mediate a number of physiological signal transduction processes. Currently, about 60% of known pharmacological targets belong to the GPCR superfamily. Rhodopsin is a member of this super family of receptors, which initiates visual transduction. This signalling process comprises biochemical pathways underlying the complex sensorial phenomenon of vision. Activation of rhodopsin by light is caused by the cis-trans summarisation of its 11-cis-retinal chromophore upon light absorption. This initial ultra fast chemical event promotes a conformational change in rhodopsin, which allows it to bind and to activate a G protein called Gt, or transducin. Activation of Gt, in turn, is followed by a cascade of enzymatic reactions resulting in hyperpolarization of photoreceptor cell membranes. Much attention has been devoted to this activation mechanism, while the inactivation mechanism of photoactivated rhodopsin by phosphorylation by its kinase, GRK-1, is still less understood. In particular, the structural determinants in the cytoplasmic domain of rhodopsin responsible for GRK-1 binding and activation are still not determined in detail; the same is true for the domains and specific amino acid residues of GRK-1 responsible for interaction with and phosphorylation of rhodopsin. Furthermore, kinetic information about the receptor-kinase enzymatic reaction is still lacking. This knowledge is crucial for understanding the rhodopsin inactivation process and the role of the receptor-kinase interactions (as well as other involved key players like Ca2+) in retinal degenerations, particularly Retinitis Pigmentosa. Rhodopsin phosphorylation by its kinase GRK-1 is also a critical step in the desensitization process and plays a role in the fine regulation of the physiological process of light adaptation. In the present project, key amino acid residues and structural elements in the cytoplasmic regions of rhodopsin (including membrane boundaries) crucial for rhodopsin phosphorylation by and interaction with GRK-1 will be elucidated. To this aim, rhodopsin mutants, including those related to the retinal degenerative disease Retinitis Pigmentosa, in these domains would be produced. The rhodopsin mutants will be structurally and biochemically characterized, and the levels of their phosphorylation by GRK-1 will be compared to reveal amino acid residues in rhodopsin crucial for its recognition by GRK-1. The phenomenon of constitutive phosphorylation of rhodopsin will also be investigated. This will be followed by a detailed study of the different kinetic parameters, with the aim to obtain a complete mechanistic description of the receptor-kinase interaction (both binding and dissociation), will be undertaken. This will be done using a novel spectroscopic technique, surface plasmon resonance spectroscopy (SPR-spectroscopy). In these experiments, both non-phosphorylated and phosphorylated rhodopsin of a wild type. Programa(s) IC-INTAS - International Association for the promotion of cooperation with scientists from the independent states of the former Soviet Union (INTAS), 1993- Tema(s) OPEN - OPEN Call Convocatoria de propuestas Data not available Régimen de financiación Data not available Coordinador Universitat Autònoma de Barcelona, Fac. Medicina Aportación de la UE Sin datos Dirección Campus Bellaterra 08193 Bellaterra (Barcelona) España Ver en el mapa Coste total Sin datos Participantes (4) Ordenar alfabéticamente Ordenar por aportación de la UE Ampliar todo Contraer todo Forschungszentrum Jülich, Institute for Biological Information Processing Alemania Aportación de la UE Sin datos Dirección ` 52425 Jülich Ver en el mapa Coste total Sin datos Institute for Biological Instrumentation Rusia Aportación de la UE Sin datos Dirección Pushchino region 142292 Pushchino Ver en el mapa Coste total Sin datos M. V. Lomonosov Moscow State University Rusia Aportación de la UE Sin datos Dirección Laboratory Building A 119992 Moscow Ver en el mapa Coste total Sin datos Universitat Politècnica de Catalunya España Aportación de la UE Sin datos Dirección Colom 1 08222 Terrassa Ver en el mapa Coste total Sin datos
