The Catalytic and GAF Domains of the Rod cGMP Phosphodiesterase (PDE6) Heterodimer Are Regulated by Distinct Regions of Its Inhibitory γ Subunit

Abstract

The central effector of visual transduction in retinal rod photoreceptors, cGMP phosphodiesterase (PDE6), is a catalytic heterodimer (αβ) to which low molecular weight inhibitory γ subunits bind to form the nonactivated PDE holoenzyme (αβγ2). Although it is known that γ binds tightly to αβ, the binding affinity for each γ subunit to αβ, the domains on γ that interact with αβ, and the allosteric interactions between γ and the regulatory and catalytic regions on αβ are not well understood. We show here that the γ subunit binds to two distinct sites on the catalytic αβ dimer (KD1 < 1 pM, KD2 = 3 pM) when the regulatory GAF domains of bovine rod PDE6 are occupied by cGMP. Binding heterogeneity of γ to αβ is absent when cAMP occupies the noncatalytic sites. Two major domains on γ can interact independently with αβ with the N-terminal half of γ binding with 50-fold greater affinity than its C-terminal, inhibitory region. The N-terminal half of γ is responsible for the positive cooperativity between γ and cGMP binding sites on αβ but has no effect on catalytic activity. Using synthetic peptides, we identified regions of the amino acid sequence of γ that bind to αβ, restore high affinity cGMP binding to low affinity noncatalytic sites, and retard cGMP exchange with both noncatalytic sites. Subunit heterogeneity, multiple sites of γ interaction with αβ, and positive cooperativity of γ with the GAF domains are all likely to contribute to precisely controlling the activation and inactivation kinetics of PDE6 during visual transduction in rod photoreceptors.