Date of Award

Fall 2006

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Rick H Cote

Abstract

Rod photoreceptor phosphodiesterase (PDE6), the central enzyme of visual transduction in vertebrate photoreceptors, associates with the disk membranes of the rod outer segment (ROS) of the photoreceptor cell. This association insures the high efficiency of activation by the G-protein, transducin, and the precise control of its inactivation. In addition to binding to transducin during visual excitation, PDE6 is hypothesized to be regulated by other interacting proteins.

The first aim of this research was to isolate and identify the proteins that interact with PDE6 during various stages of the visual signaling pathway. We evaluated methods for solubilizing PDE6 and its binding partners from the ROS disk membrane, and analyzed the protein composition by immunoprecipitation or size exclusion chromatography. Our results suggested that additional, as-yet unidentified, proteins bind to PDE6 in its nonactivated and activated states. This work led to enhanced protocols for purifying rod and cone PDE6 from photoreceptor cells.

The second aim of the study was to evaluate the importance of the Glutamic Acid-Rich Protein-2 (GARP2) as a PDE6-interacting protein. First, we confirmed earlier observations that GARP2 is a high-affinity PDE6 interacting protein, and is present in amounts sufficient to stoichiometrically bind PDE6. Addition of purified GARP2 to ROS membranes containing PDE6 demonstrated that it is able to inhibit the basal activity of the nonactivated PDE6 holoenzyme. In contrast with a previous report, GARP2 does not alter the ability of transducin to activate PDE6. These results suggest a role for GARP2 in reducing the spontaneous activation of dark-adapted PDE6, thereby serving to increase the light sensitivity of rod photoreceptors as "single photon detectors.".

The final aim was to investigate the molecular mechanism by which GARP2 interacts with PDE6 and regulates its catalytic properties. We found that GARP2 exerted a destabilizing effect on the regulatory cGMP binding sites of the PDE6 catalytic dimer, as well as interacting directly with two distinct subdomains of the inhibitory subunit of PDE6. These latter interactions are believed to account for the observed ability of GARP2 to enhance the affinity of the inhibitory subunits for the catalytic dimer of PDE6.

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