To elucidate the molecular mechanisms of the phototransduction process, we propose to study conformational changes induced by light in rhodopsin, transducin and cGMP phosphodiesterase, and transient interactions between them in the course of phototransduction. Regulation of these interactions by phosphorylation/dephosphorylation reactions of accessory proteins will also be investigated. Several novel methodologies recently developed in this laboratory will be used to explore these questions. Specifically, the following questions will be addressed. 1) What determinants on rhodopsin are responsible for interacting with and activating transducin? 2) What are the sequelae on transducin of rhodopsin activation, and what are the relative roles of the alpha, beta and gamma subunits in this process? 3) How does transducin in turn activate cGMP phosphodiesterase, and what region of the alpha subunit is responsible? 4) What is the role of cyclic nucleotide-dependent phosphorylation of transducin accessory proteins in the regulation of phototransduction? To determine surfaces of interaction between these proteins, cross-linking studies followed by analysis with site-directed antibody probes to determine the makeup of the cross-linked species. Then the complexes will be cleaved by specific proteases, purified by reverse phase HPLC and sequenced. Synthetic peptides corresponding to sites of interaction between the proteins will be used to block the interaction between these proteins in vitro. Studies with peptide analogs will determine the important structural features of the interaction domains, then structural studies of the peptides will be used to determine their three-dimensional structure. To test how close transducin approaches to the retinal binding pocket of rhodopsin the novel method of photosensitized activation of the photoaffinity label 5-iodo [125I]-naphthalene-1-azide (125I-INA) will be used. These studies will probe the accessibility of protein surfaces under various conditions and the effects of such perturbations on protein function will help to delineate the relationship between structure and function on the target proteins. It is expected that these new approaches to the study of protein structure, function and interactions applied to the vertebrate photoreceptor system will provide new insight into the molecular details of phototransduction and other signal transduction processes. The information obtained in these studies about the mechanisms of visual transduction may be pertinent for understanding malfunctions in diseases which provoke retinal degenerations, and for targeting medical interventions for such diseases.
Showing the most recent 10 out of 40 publications
