The overall molecular mechanisms by which a light signal is transduced into a decrease in neurotransmitter release from the photoreceptor are now largely understood, at least in general terms. Most of the proteins involved in these processes are known and mechanisms by which they are controlled have been elucidated. One of the more important components of this transduction cascade are the photoreceptor phosphodiesterases (PDEs) that are activated in response to the light signal. However, it is this part of the system that is perhaps the least well understood. In particular, many of the molecular aspects about the location and nature of the interactions among the PDE subunits are unknown. One subunit in particular, the 15 kDa PDEdelta subunit, has not even been assigned a function. One of the major reasons for this lack of knowledge is the fact that only a few of the subunits can be isolated from the native enzyme without denaturation. Moreover, most have not been expressed individually from recombinant sources and therefore the whole complex has not been fully and functionally reconstituted. This application proposes to do just that. In particular, the characterization, expression, and reconstitution of the rod and cone delta subunits with the native enzyme will be addressed. These studies will lead to identification and characterization of the sites on the delta subunit and on the PDEs that interact. Similar studies for identification and characterization of the interaction sites of cone transducin with the rod and cone PDEs are also proposed. We will use deletion, point mutation, and reconstitution analysis of the subunits expressed in baculovirus to study the structural and functional relationships of the subunits. We will also define the exact boundaries of the interaction sites by use of synthetic peptides to the site. Physiologically, it is very likely that the molecular mechanisms that allow rods and cones to respond and adapt differently to light are due at least in part to differences in the interactions of the various protein components of the visual transduction cascade. As such it is important to understand the roles for all of the polypeptides in the cascade and also to understand how they differ in rods and cones. It also seems likely that just as cloning and characterization of the PDE beta subunits have now led to an understanding at the molecular level for several types of retinal degeneration, that understanding the roles played by other polypeptides in the cascade will also be found to have clinical importance.
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