The catalytic and GAF domains of the rod cGMP phosphodiesterase (PDE6) heterodimer are regulated by distinct regions of its inhibitory gamma subunit

The central effector of visual transduction in retinal rod photoreceptors, cGMP phosphodiesterase (PDE6), is a catalytic heterodimer (alpha beta) to w hich low molecular weight inhibitory gamma subunits bind to form the nonact ivated PDE holoenzyme (alpha beta gamma (2)). Although it is known that gam ma binds tightly to alpha beta, the binding affinity for each gamma subunit to alpha beta, the domains on gamma that interact with alpha beta, and the allosteric interactions between gamma and the regulatory and catalytic reg ions on alpha beta are not well understood. We show here that the gamma sub unit binds to two distinct sites on the catalytic alpha beta dimer (K-D1 < 1 pM, K-D2 = 3 pM) when the regulatory GAF domains of bovine rod PDE6 are o ccupied by cGMP. Binding heterogeneity of <gamma> to alpha beta is absent w hen cAMP occupies the noncatalytic sites. Two major domains on gamma can in teract independently with alpha beta with the N-terminal half of gamma bind ing with 50-fold greater affinity than its C-terminal, inhibitory region. T he N-terminal half of gamma is responsible for the positive cooperativity b etween gamma and cGMP binding sites on alpha beta but has no effect on cata lytic activity. Using synthetic peptides, we identified regions of the amin o acid sequence of gamma that bind to alpha beta, restore high affinity cGM P binding to low affinity noncatalytic sites, and retard cGMP exchange with both noncatalytic sites. Subunit heterogeneity, multiple sites of gamma in teraction with alpha beta, and positive cooperativity of gamma with the GAF domains are all likely to contribute to precisely controlling the activati on and inactivation kinetics of PDE6 during visual transduction in rod phot oreceptors.