Regulation of Retinal cGMP Phosphodiesterases

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DESCRIPTION (provided by applicant): The long-term goal of this research program as to elucidate molecular mechanisms of function and regulation of rod and cone cGMP-phosphodiesterases (PDE6) Rod and cone PDE6 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 gamma-subunits (Pgamma). Activated PDE6 hydrolyzes cGMP with a uniquely high catalytic rate. To study the structure-and-function relationships of PDE6 we have developed a system for expression of PDE6alpha/PDE5 chimeras in insect cells. A chimeric PDE6/PDE5 enzyme maximally equivalent to PDE6 will be generated through a progressive incorporation of PDE6 sequence into existing functional PDE6/PDE5 chimeras. Mutational analysis of the PDE6-like chimeric enzymes guided by the model of PDE6 catalytic domain will be carried out to identify the structural elements of PDE6 critical for the efficient hydrolysis of cGMP and the enzyme sensitivity to selective competitive inhibitors. PDE6 catalytic subunits contain two N-terminal GAF domains, GAFa and GAFb, implicated in noncatalytic binding of cGMP. We have found that the polycationic region of Pgamma binds to the GAFa domains of PDE6. We hypothesize that a direct stabilization of the cGMP-binding pocket by Pgamma is the mechanism for known positive cooperativity between Pgamma and noncatalytic cGMP binding. To test this hypothesis. Pgamma contact residues within the GAFa domain of PDE6 will be identified by mutagenesis. The GAF domains and residues involved in the noncatalytic cGMP-binding will be identified by substituting selected residues within potential cGMP-pockets of PDE6. Models of PDE6 GAF domains will be utilized to elucidate the nature of the reciprocal relationship between the cGMP and Pgamma-binding sites. Rod PDE6 catalytic alpha and beta subunits form indissociable alphabeta heterodimers, whereas cone PDE6 alpha' subunit forms alpha' alpha' homodimers. Although the GAF domains of PDE6 are thought to be involved in the dimerization, the PDE6 intersubunit interface has not been investigated. Sites and residues responsible for the specific dimerization of PDE6 will be identified through the analysis of dimer formation between chimeric and mutant PDEs. The role of specific PDE6 residues will be established by inducing homodimerization between mutant GAF domains of PDE6 alpha and beta. The map of PDEalpha/beta-Pgamma interactions indicates two possible types of assembly of PDE6. A Pgamma molecule may bind to GAFa and the catalytic site from the same or different subunits of the catalytic dimer. The assembly of PDE6 subunits will be tested using a cross-linking approach. These studies will advance our understanding of the structure, function, and regulation of PDE6, and help to elucidate the mechanisms of retinal degeneration caused by mutations of PDE6 genes.