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Molecular mechanisms of Beta-adrenergic receptor function and regulation

Objective

Production of the protein components of the beta-adrenergic receptor system and reconstitution in phospholipid vesicles. Generation of antibodies to these proteins. Development of 3-dimensional models of the receptor and definition of the ligand binding and the G-protein-coupling sites. Identification of limiting steps in the activation and inactivation of the receptor system. Development of compounds that interfere with these limiting steps.
Receptors, many G-protein subunits, and several isoforms of regulatory proteins (receptor kinases, arrestins, phosducins) were produced by recombinant techniques, purified and reconstituted. Antibodies were generated against the receptors, the kinases, arrestins and phosducin. 3-D models of the beta2-adrenergic receptor and other related receptors were generated and used to direct mutagenesis for the definition of the ligand binding and the G-protein coupling domains. Several critical domains in the receptor were identified and characterized: Agonist recognition domain, G-protein-coupling domain, (constitutive) activation domain.The contributions of these domains (alone or in combination) to ligand binding and receptor activation were determined. Biophysical models of the activation energy of receptors and of receptor-G-protein-complexes were developed on the basis of a series of such experiments. The interaction between receptor kinases and receptors was studied in several respects: First, the process of receptor phosphorylation and desensitization was identified and characterized for a number of G-protein-coupled receptors. Second, the dynamics of subcellular distribution of the kinases were studied. Association of the kinases with membranes were found to be a critical means of kinase regulation. Third, it was discovered that the activity as well as the subcellular distribution of these kinases are altered following phosphorylation by protein kinase C. Fourth, a role for these kinases in the process of receptor internalization was described. Phosducin and a homologous protein were identified and characterized as regulators of G-protein-function. The interaction site was mapped and its 3D structure determined by NMR. An assay system was developed to search for compounds modulating kinase activity as a drug target. Cooperation with a pharmaceutical company was begun to adapt and use such tests for screening in drug development.
MAJOR SCIENTIFIC BREAKTHROUGHS:
3-D models of the receptor and the ligand binding site and verification of the models by site-directed mutagenesis. Development of models of the receptor activation process by analysis of combinations of receptor mutants and by molecular dynamics. Generalization of the role of G-protein-coupled receptor kinases to multiple receptors. Discovery of mechanisms controlling activity and subcellular localization of these kinases. Characterization of phosducins as G-protein regulators and elucidation of the 3D-structure of the interaction domain.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Ludwig-Maximilians-Universität München
Address
Würmtalstraße 221
81375 München
Germany

Participants (5)

Bayerische Julius-Maximilians-Universität Würzburg
Germany
Address
Versbacher Straße
97078 Würzburg
Istituto Superiore di Sanitá
Italy
Address
Viale Regina Elena 299
00161 Roma
Rijksuniversiteit Leiden
Netherlands
Address
55,Einsteinweg
2333 AA Leiden
UNIVERSIDAD AUTONOMA DE MADRID
Spain
Address
Univrsidad Autonoma De Madrid Cantoblanco
28049 Canto-blanco - Madrid
Université de Lausanne
Switzerland
Address
27,Rue Du Bugnon
1005 Lausanne