The long-term objective of this program is to investigate molecular mechanisms of transducin (Gt) signaling in the vertebrate photoreceptor cells. The properties and regulation of rod transducin Gt1 during phototransduction are understood to a far greater extent than those of cone transducin Gt2. It remains unknown whether the conserved sequence differences in rod and cone transducin-a (Gat1) subunits translate into the functional differences that contribute the remarkably distinct physiology of rods and cones. We hypothesize that because of conserved differences within the Gat N-termini and the interdomain interfaces, cone Gt2 is activated by photoexcited pigment with a lower efficiency than rod Gt1,and the Gat2-mediated stimulation of cGMP-phoshodiesterase 6 is less potent in comparison to the effector enzyme activation in rods. In order to test this hypothesis, we will carry out detailed characterization and mutational analysis of Gat2 and heterotrimeric cone transducin Gat2?3?8. The proposed analysis will be based on a newly established procedure for bacterial expression of human Gat2. These studies will advance our understanding of the molecular mechanisms that distinguish phototransduction in cones and rods. Another obscure aspect of transducin biology and signaling involves the mechanisms of its bi-directional transport between the inner and outer segments in rods, the determinants of light-dependent compartmentalization, and mobility on photoreceptor membranes. We will explore the roles of transducin/rhodopsin interactions and lipid modifications in transducin targeting, membrane mobility and interdisc transfer using transgenic Xenopus laevis expressing mutant EGFP-fused Gat1 subunits in rod photoreceptors. The mutant Gat1 models will be examined with EGFP imaging, immunofluorescence, and Fluorescence Recovery After Photobleaching (FRAP) analysis of lateral and longitudinal diffusion. The proposed research will provide important insights into transport and mobility of peripheral membrane proteins in photoreceptor cells.
Photoreceptor GTP-binding proteins, transducins, are the key signaling molecules in vision. Functional properties of cone transducin and the differences in signaling of cone and rod transducins are largely unknown. The proposed studies will yield a new level of understanding the function and regulation of cone transducin and advance our understanding of the molecular mechanisms responsible for the markedly distinct physiology of cones and rods. This research will also provide important insights into the transport and mobility of transducin in photoreceptor cells.
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