MEMBRANE STIMULATION OF CGMP PHOSPHODIESTERASE ACTIVATION BY TRANSDUCIN - COMPARISON OF PHOSPHOLIPID-BILAYERS TO ROD OUTER SEGMENT MEMBRANES

To clarify the role of phospholipids in G protein-effector interactions of vertebrate phototransduction, transducin activation of cGMP phosphodiesterase (PDE) has been reconstituted on the surface of well-defined phosphatidylcholine (PC) vesicles, using purified proteins from bovine rod outer segments (ROS). PC vesicles enhanced PDE stimulation by the GTP-gammaS-bound transducin alpha subunit (T(alpha)-GTPgammaS) as much as 17-fold over activation in the absence of membranes. In the presence of 3.5 muM accessible PC in the form of large (100 nm) unilamellar vesicles, 500 nM T(alpha)-GTPgammaS stimulated PDE activity to more than 70% of the maximum activity induced by trypsin. Activation required PC, PDE, and T(alpha)-GTPgammaS, but did not require prior incubation of any of the components, and occurred within 4 s of mixing. The PC vesicles were somewhat more efficient than urea-washed ROS membranes in enhancing PDE activation. Half-maximal activation occurred at accessible phospholipid concentrations of 3.8 muM for PC vesicles, and 13 muM for ROS membranes. Titrations of PDE with T(alpha)-GTPgammaS in the presence of membranes indicated a high-affinity (K(act) < 250 pM) activation of PDE by a small fraction (0.5-5%) of active T(alpha)-GTPgammaS, as did titrations of ROS with GTPgammaS. When activation by PC vesicles was compared to PDE binding to membranes, the results were consistent with activation enhancement resulting from formation of a T(alpha)-GTPgammaS-dependent PDE-membrane complex with half-maximal binding at phospholipid concentrations in the micromolar range. The value of the apparent dissociation constant, K(PL), associated with the activation enhancement was estimated to be in the range of 2.5 nM (assuming an upper limit value of 1600 phospholipids/site) to 80 nM (for a lower limit value of 50 phospholipids/site). Another component of membrane binding was more than 100-fold weaker and was not correlated with activation by T(alpha)-GTPgammaS. Low ionic strength disrupted the ability of ROS membranes, but not PC vesicles, to bind and activate PDE. Removal of PDE's membrane-binding domain by limited trypsin digestion eliminated both the binding of PDE to vesicles and the ability of PDE to be activated by T(alpha)-GTPgammaS and membranes. These results suggest that ROS membrane stimulation of PDE activation by T(alpha)-GTPgammaS is due almost exclusively to the phospholipids in the disk membrane.