The long-term goal of this research program is to elucidate the molecular mechanisms of the function and regulation of rod and cone cGMP-phosphodiesterases (PDE6). PDE6s serve as key effector enzymes in the vertebrate visual transduction cascade. Transducin activates PDE6 by relieving the inhibition imposed on the PDE6 catalytic dimer by two ?-subunits (P3). Rod PDE6 is unique among cyclic nucleotide PDEs in that it is a catalytic heterodimer composed of the PDE6A and PDE6B catalytic subunitsWe hypothesize that the heterodimerization of rod PDE6 plays an important role in rod phototransduction. The individual properties of PDE6A and PDE6B and the functional significance of rod PDE6 heterodimerization will be examined in aim 1. The goal is to bring a new level of understanding to the mechanism of rod PDE6 activation by transducin and advance the current models of the rod phototransduction cascade. Based on the existing evidence and our preliminary studies, we hypothesize that the interaction of P? with the PDE6 catalytic subunits is critical to the proper transport of the holoenzyme in photoreceptor cells.
Aim 2 is to test this hypothesis and probe the mechanisms of P? in expression and transport of PDE6.
Aims 1 and 2 will be achieved through extensive use of transgenic Xenopus laevis as a novel approach for the expression of mutant PDE6. Mutations in the human PDE6A and PDE6B genes each are responsible for 3-4% of cases of recessive retinitis pigmentosa (RP).
As aim 3 we will investigate the mechanism of the recessive mutation N605S in the mouse PDE6B subunit causing atypical retinal degeneration (atrd3) in mice. Based on the initial evidence, we hypothesize that atrd3 is triggered by functional changes in PDE6 and may reflect certain forms and mechanisms of human RP. The atrd3 mechanism will be examined using the mutant mouse model in comparison to a transgenic Xenopus laevis model of the cone PDE6C mutant counterpart. The molecular details of PDE6 inhibition by its ?-subunit will be elucidated by means of protein crystallography in aim 4. The structure of the chimeric PDE5/6 catalytic domain complexed with P?-63-87 and sildenafil (Viagra) will provide the first structural insights into the critical interaction between PDE6 and P? and should reveal the structural basis for the side effects of Viagra on vision. The structure of the chimeric atrd3 mutant will be investigated to uncover the molecular mechanism for retinal disease associated with this PDE6 mutation. Overall, these studies will advance our understanding of the regulation of PDE6 and help to elucidate the mechanisms of RP caused by mutations of PDE6 genes.
Rod and cone cGMP phosphodiesterases (PDE6 family) are the key effector enzymes in vision. Mutations in PDE6 lead to human retinal diseases such as retinitis pigmentosa, congenital stationary night blindness, and achromatopsia. This research would develop a new level of understanding of structure, function, and regulation of PDE6 necessary to uncover the mechanisms of PDE6 mutations in diseased retina.
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