Date of Award
Program or Major
Doctor of Philosophy
Rick H Cote
The photoreceptor phosphodiesterase (PDE6) must be precisely regulated to control the sensitivity, amplitude, and kinetics of the photoresponse during excitation, termination and adaptation to light stimulation in rod and cone photoreceptors. The central hypothesis of this thesis is that one PDE6 binding partner, the glutamic acid-rich protein (GARP2), may regulate PDE6 to reduce its "dark noise", enhance its sensitivity and conserve metabolic energy during rod photoresponse saturation.
The first aim of this research is to better understand the unique biochemical and biophysical characteristics of GARP2 in order to reveal its functional attributes for regulating PDE6 during phototransduction in rod photoreceptors. We first improved immunological methods to better characterize GARP2. We then developed approaches to purify PDE6 free of GARP2; we also generated recombinant GARP2 to permit study of its biochemical and biophysical properties, and discovered that recombinant GARP2 suppresses the basal activity of PDE6, and behaves as a natively unfolded protein in solution.
The second aim is to identify the interacting sites between GARP2 and PDE6 and determine the regulatory mechanism of GARP2 on PDE6 in visual phototransduction. We demonstrated that the N-terminal half of the inhibitory gamma subunit of PDE6 (Pgamma), interacts with high affinity for GARP2. The C-terminal portion of GARP2 is most effective in suppressing the basal activity of PDE6, whereas the central region of GARP2 reduces cGMP binding to PDE6 GAF domains. This suggests that GARP2 alters both the allosteric and catalytic properties of PDE6 to regulate PDE6 activity and lifetime through multiple interacting sites with the PDE6 holoenzyme. GARP2 may play an important role in lowering the level of dark noise, and reducing cGMP metabolic flux during rod photoresponse saturation under bright light.
The final aim of this research is to investigate the effects of zinc on the catalytic mechanism and structural stability of PDE6. Several different zinc-chelator systems were used to make solutions with defined concentrations of free zinc. We demonstrate that low concentrations of zinc are able to activate PDE6 catalytic activity, while high concentrations cause the loss of PDE6 activity.
Yao, Wei, "Regulation of the catalytic and allosteric properties of photoreceptor phosphodiesterase (PDE6) by the glutamic acid-rich protein-2 (GARP2)" (2013). Doctoral Dissertations. 747.