The long-term objective of the proposed program is to investigate molecular mechanisms underlying signaling via the photoreceptor G protein, transducin (Gt), in the vertebrate visual transduction cascade. The role and general regulation of Gt during the activation and turnoff phases in visual excitation are well understood. However, our understanding of the molecular details and mechanisms of Gt interactions with photoexcited rhodopsin (R*), cGMP-phosphodiesterase (PDE) and visual regulator of G protein signaling, RGS9, remains largely incomplete. The interface of interaction between Gt and R* will be examined using photo crosslinking of Gt-alpha mutants with a single reactive cysteine residue or specific Gt-alpha peptide probes to R* followed by identification of the crosslinked sites. A second approach to probing the R*/Gt interface will involve generation of chimeric Gs-alpha/Gt-alpha proteins. Competition between rhodopsin and synthetic peptides corresponding to selected regions of rhodopsin for binding to Gs-alpha/Gt-alpha chimeric proteins will reveal the point-to-point interaction regions on R* and Gt. Results of the crosslinking experiments and peptide competition studies will guide the site-directed mutagenesis of R*. Rhodopsin mutants expressed in HEK-293 cells will be analyzed for interaction with Gt-alpha to identify critical R* residues. Gs-alpha/Gt-alpha chimeras will also be analyzed for binding to and activation by R*. The goals of this analysis are to both elucidate the role of Gt-alpha regions known to be involved in activation by R* and to identify novel Gt-alpha site(s) and residues that may interact with rhodopsin. The detailed mapping of the effector interface on Gt will be accomplished by scanning mutagenesis of the switch I and the helical domain of Gt-alpha. Comprehensive analysis of the effector interface on Gt will provide valuable insights into the mechanism of PDE activation by Gt and the role of the Gt-alpha helical domain in effector regulation. Photoreceptor GTPase activating protein (GAP), RGS9, stimulates GTP hydrolysis of Gt-alpha thus accelerating the turnoff phase in the visual cascade. Our analysis of Gt-alpha regulation by RGS9 will include examination of phosphorylation and other potential posttranslational modifications of RGS9. The functional role of the association of RGS9 with the G-beta5L subunit for Gt-alpha GTPase activity will be explored. Overall, these studies will help to achieve a better understanding of signaling mechanisms in the visual cascade as well as in other G protein mediated systems and will provide information relevant to retinal diseases and diseases of G protein function in general.
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