INTERACTION BETWEEN THE RETINAL CYCLIC-GMP PHOSPHODIESTERASE INHIBITOR AND TRANSDUCIN - KINETICS AND AFFINITY STUDIES

In the retinal cyclic GMP phosphodiesterase (PDE), catalysis by the al phabeta-heterodimer is inhibited in the dark by two identical gamma-su bunits and stimulated in the light by the GTP-bearing alpha-subunit of the heterotrimeric G-protein transducin (Tbetagamma-TalphaGDP). Two T alphaGTP molecules, dissociated from Tbetagamma,bind to and displace t he PDEgamma subunits from their inhibitory sites on PDEalphabeta. With GTPgammaS in lieu of GTP, this association becomes persistent. Under physiological conditions, the PDEalphabeta(gammaTalpha)2 active comple x stays on the membrane. But in low-salt buffers, it becomes soluble a nd dissociates into a partially active PDEalphabeta catalytic moiety a nd two PDEgamma-TalphaGTPgammaS complexes. This indicates that Talpha binds preferentially to PDEgamma. We have studied the interaction of r ecombinant bovine PDEgamma with purified Talpha in solution or with re tinal rod outer segments (ROS) containing both Tbetagamma-TalphaGDP an d PDEalphabetagamma2. When added to dark ROS, recombinant PDEgamma did not bind to inactive PDEalphabetagamma2 but extracted TalphaGDP from membrane-bound holo-transducin to form a soluble PDEgamma-TalphaGDP co mplex. PDEgamma also bound to purified TalphaGDP in solution. The kine tics and affinity of the interaction between PDEgamma and TalphaGDP or TalphaGTPgammaS were determined by monitoring changes in the proteins ' tryptophan fluorescence. The K(d)'s for the binding of recombinant P DEgamma to soluble TalphaGTPgammaS and TalphaGDP are less-than-or-equa l-to 0.1 and 3 nM, respectively. PDEgamma-TalphaGDP falls apart in 3 s . This slow dissociation means that, in situ, Talpha-PDEgamma cannot p hysically leave the active PDEalphabeta, since after GTP hydrolysis, a n isolated Talpha-PDEgamma complex would dissociate too slowly to allo w a fast PDE reinhibition by the liberated PDEgamma. When recombinant PDEgamma was added to PDE that had been persistently activated by Talp haGTPgammaS, reinhibition occurred and TalphaGTPgammaS, complexed to t he native PDEgamma, was released, indicating that both had hitherto st ayed bound to PDEalphabeta. The mutation W70F does not prevent recombi nant PDEgamma from inhibiting PDEalphabeta but diminishes its affinity for TalphaGTP and TalphaGDP 100-fold. Thus, W70 of PDEgamma must take part in its binding to TalphaGTP and TalphaGDP. When reconstituted wi th [W70F]PDEgamma, PDE was fully inhibited but could no longer be acti vated by TalphaGTP or TalphaGTP-gammaS.