Intracellular membrane surfaces play an essential role in bringing together protein components of signal transduction pathways. The focus of this project is on the critical role of disk membranes in mediating amplified signal transduction in rod photoreceptor cells of the mammalian retina. Th molecular mechanisms by which the GTP-binding protein transducin activates cGMP phosphodiesterase on the membrane surface during the light response will be determined. Stoichiometry and kinetics will be determined for formation of complexes of protein subunits on the phospholipid surface usin both native membranes and well defined synthetic membranes. Spectroscopic techniques using specifically labelled protein subunits will be combined with light scattering, centrifugation, gel filtration, and enzymatic assays to monitor formation of these complexes. The molecular basis will be determined for the existence of a transducin subpopulation that efficiently activates the phosphodiesterase but requires detergent concentrations sufficient to dissolve transmembrane proteins to be extracted from membranes. This protein will be purified to homogeneity, its chemical structure will be analyzed, and it will be reconstituted with membranes so its interactions with cGMP phosphodiesterase and other disk membrane proteins can be studied. The role of covalently attached lipids in transducin function will be studied, particularly that of the N-myristoyl group that is present in some but not all transducin alpha subunits. The molecular mechanisms of transducin myristoylation and its regulation will be explored. The role of membrane interactions in the deactivation phase of the light responses will also be studied, with an emphasis on the GTPase-dependent inactivation of cGMP phosphodiesterase. These studies will help to elucidate not only key features of visual transduction, but also common features of G protein-mediated pathways that are more difficult to study in membranes coupling G proteins to receptors for hormones and other extracellular signals. Understanding the role of signal-transducing membranes, and of protein modifications that regulate proteins' interactions with these membranes, is essential to understanding the normal functioning of signal transduction pathways, the disruption of these pathways that can occur in disease, as well as the potential consequences of drugs that affect these interactions.
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