PROJECT SUMMARY/ABSTRACT Photoreceptor phosphodiesterase (PDE6) is the central enzyme of the visual signaling pathway. Precise regulation of its activation and deactivation is essential for the speed, sensitivity, and recovery of rod and cone photoreceptors to illumination. Inherited mutations in rod and cone PDE6 genes have been linked in a variety of retinal diseases, including retinitis pigmentosa, congenital stationary night blindness, and cone dystrophy. Next-generation sequencing is identifying a growing number of mutations in PDE6 genes, the large majority of which remain of uncertain clinical significance. Even less is known about the molecular etiology of retinal disease-causing mutations. Rod PDE6 consists of two catalytic subunits whose activity is inhibited in the dark- adapted state by binding of two identical γ-subunits (Pγ). Upon light-induced activation of the visual signaling pathway, PDE6 activity is stimulated by binding of the heterotrimeric G-protein, transducin. The lifetime of light- activated PDE6 is precisely controlled by the rate at which the transducin hydrolyzes its bound GTP, a process controlled by RGS9-1 (Regulator of G-protein Signaling9-1). While the proteins involved in regulation of PDE6 during phototransduction have been identified, the molecular sequence of events in which PDE6 dynamically interacts with its binding partners--as well as its allosteric regulation--during PDE6 activation and deactivation remain poorly understood. Until we understand the mechanistic basis of PDE6 regulation during normal phototransduction, we will be hampered in developing therapeutic interventions for those diseases arising from defects in PDE6 or its binding partners that result in retinal degenerative diseases and visual disorders. The overall objective of this application is to understand the sequence of events accompanying PDE6 activation by transducin and its subsequent inactivation by RGS9-1 and other proteins during recovery of PDE6 to its dark-adapted state. Our experimental plan is based on the hypothesis that the inhibitory Pγ subunit of PDE6 is the “master regulator” responsible for mediating multiple allosteric interactions that occur within the PDE6 catalytic dimer, as well as with the transducin α-subunit and RGS9-1. We propose two specific aims that will (1) delineate the sequence of binding interactions between transducin α-subunits and PDE6 catalytic and inhibitory Pγ subunits to provide a comprehensive model of rod PDE6 activation, and (2) determine the molecular architecture of the PDE6 inactivation complex upon RGS9-1 binding and the structural rearrangements of the Pγ subunit that accelerate termination of activated PDE6. The outcomes of this research advance the goals of the Retinal Diseases Program at the National Eye Institute by enhancing our ability to predict the pathogenicity of mutations in phototransduction proteins, thereby enabling development of personalized therapeutic interventions for retinal...