Mechanism of transducin activation of frog rod photoreceptor phosphodiesterase - Allosteric interactions between the inhibitory gamma subunit and thenoncatalytic cGMP-binding sites

The rod photoreceptor phosphodiesterase (PDE) is unique among all known ver tebrate PDE families for several reasons. It is a catalytic heterodimer (al pha beta); it is directly activated by a G-protein, transducin; and its act ive sites are regulated by inhibitory gamma subunits, Rod PDE binds cGMP at two noncatalytic sites on the ap dimer, but their function is unclear. We show that transducin activation of frog rod PDE introduces functional heter ogeneity to both the noncatalytic and catalytic sites. Upon PDE activation, one noncatalytic site is converted from a high affinity to low affinity st ate, whereas the second binding site undergoes modest decreases in binding. Addition of gamma to transducin-activated PDE can restore high affinity bi nding as well as reducing cGMP exchange kinetics at both sites. A strong co rrelation exists between cGMP binding and gamma binding to activated PDE; d issociation of bound cGMP accompanies gamma dissociation from PDE, whereas addition of either cGMP or gamma to clip dimers can restore high affinity b inding of the other molecule, At the active site, transducin can activate P DE to about one-half the turnover number for catalytic cup dimers completel y lacking bound gamma subunit, These results suggest a mechanism in which t ransducin interacts primarily with one PDE catalytic subunit, releasing its full catalytic activity as well as inducing rapid cGMP dissociation from o ne noncatalytic site, The state of occupancy of the noncatalytic sites on P DE determines whether gamma remains bound to activated PDE or dissociates f rom the holoenzyme, and may be relevant to light adaptation in photorecepto r cells.