While the basic features of visual transduction in rod photoreceptors are generally known, the protein-protein interactions and allosteric regulation that occur during activation and deactivation of the photoreceptor phosphodiesterase (PDE6) are poorly understood. Further, the biochemical mechanisms underlying light adaptation of the mammalian photoresponse are not well delineated, but there is strong evidence that additional mechanisms of PDE6 regulation play a role in photoresponse desensitization. These serious gaps in knowledge of the molecular mechanism of visual signaling need to be remedied to fully understand how alterations in PDE6 or its binding partners can lead to visual dysfunction and retinal disease. The long-term goal is to elucidate the multiple mechanisms controlling PDE6 activity during excitation, recovery, and adaptation of the photoresponse in retinal photoreceptors. The overall objective of this application is to define the biochemical and structural progression that starts with light-induced activation of PDE6 holoenzyme by transducin, followed by the events responsible for PDE6 recovery to the dark-adapted state and its desensitization during light adaptation. The unifying hypothesis is that the inhibitory ?-subunt of PDE6 (P?) is responsible for mediating allosteric interactions that occur between the PDE6 catalytic subunits and transducin as well as with other regulatory proteins. The experimental basis for this hypothesis relies on recent work showing that P? interacts with several structurally and functionally distinct domains of the PDE6 catalytic subunits. Furthermore, the linearly extended conformation of P? bound to the PDE6 catalytic dimer provides multiple sites of interaction with activated transducin, as well as with another PDE6 binding partner, GARP2.
In Aim 1, the sequence of steps leading to transient activation of PDE6 holoenzyme subsequent to binding of transducin will be defined. The goal of Aim 2 is to determine the allosteric mechanisms by which the active lifetime of PDE6 is controlled.
Aim 3 will define the topological relationship of known PDE6-interacting proteins and the mechanism(s) by which they modulate PDE6 activity. This innovative integration of biochemical pathway information with the structural alterations in the PDE6 signaling complex during the photoresponse will significantly increase our understanding of the phototransduction pathway in rods and cones. This new knowledge of the sequence of steps in the activation, inactivation, and adaptation of the macromolecular complex of PDE6 and its interacting partners is a prerequisite for predicting and treating retinal degenerative diseases and visual disorders that result from dysfunction of the PDE6 signaling complex in photoreceptor cells.
The proposed research is relevant to public health because a greater understanding of the proper functioning and regulation of the photoreceptor phosphodiesterase enzyme (PDE6) and the proteins with which it interacts during visual signaling will enhance our ability to predict and develop therapeutic interventions for retinal diseases (e.g., retinitis pigmentosa, congenital stationary night blindness). The proposed research is relevant to the mission of the National Eye Institute because it will contribute to attaining several objectives/goals of the strategic plan established by the NEI Retinal Diseases Program, including: understanding the mechanisms underlying light adaptation and recovery in photoreceptors, increasing knowledge of phototransduction in cones, and, ultimately, determining the pathophysiological mechanisms underlying retinal degenerative disease gene mutations.
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