This proposal focuses on molecular mechanisms of signal transduction by G-protein coupled receptors (GPCR). Mammalian rhodopsin is the prototypic receptor in this family and is the major focus of the work herein proposed. A unifying hypothesis is proposed that in all GPCR, the molecular mechanisms of signal transduction (after light activation in rhodopsin and after ligand binding in GPCR in general) are conserved and similar. In rhodopsin, the first consequence of light-induced retinal isomerization has been established to be movements of helices in the transmembrane domain. These movements induce a specific conformational change in the cytoplasmic face. The tertiary structure changes involved have previously been studied by this PI by biochemical (chemical reactivity, accessibility and proximity relationships between amino acids) and by spin labeling and EPR spectroscopy. These will now be studied more precisely by NMR. Both, solution NMR (19F and TROSY,) and solid state NMR will be used. Large scale expressions of proteins which are necessary for NMR experiments will be carried out by further investigation of HEK293 stable cell lines and by the Drosophila expression system. Protein-protein interactions leading to sensitization and desensitization are at the heart of signal transduction by rhodopsin. Detailed kinetic studies of interactions between light-activated rhodopsin, transducin and rhodopsin kinase will be carried out by the techniques of surface plasmon resonance using a Biacore instrument. A further focus in studies of protein-protein interactions is to define the interacting sites and amino acid sequences by covalent cross-linking methods. The interacting regions will be identified by mass spectrometric methods. Finally, it is now feasible to test the unifying hypothesis proposed for signal transduction by studying helix movements on ligand binding in two members of GPCR, alpha1 adrenergic receptor and beta2 adrenergic receptor.
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