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-gamma S-bound transducin alpha subunit (T alpha-GTP gamma S) as much as 17-fold over activation in the absence of membranes. In the presence of 3.5 microM accessible PC in the form of large (100 nm) unilamellar vesicles, 500 nM T alpha-GTP gamma S stimulated PDE activity to more than 70% of the maximum activity induced by trypsin. Activation required PC, PDE, and T alpha-GTP gamma S, 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 microM for PC vesicles, and 13 microM for ROS membranes. Titrations of PDE with T alpha-GTP gamma S in the presence of membranes indicated a high-affinity (Kact less than 250 pM) activation of PDE by a small fraction (0.5-5%) of active T alpha-GTP gamma S, as did titrations of ROS with GTP gamma S. 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-GTP gamma S-dependent PDE-membrane complex with half-maximal binding at phospholipid concentrations in the micromolar range. The value of the apparent dissociation constant, KPL, 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-GTP gamma S. 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-GTP gamma S and membranes. These results suggest that ROS membrane stimulation of PDE activation by T alpha-GTP gamma S is due almost exclusively to the phospholipids in the disk membrane.
Malinski
J A
JA
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Wensel
T G
TG
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D008051:Lipid Bilayers; D009994:Osmolar Concentration; D010713:Phosphatidylcholines; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D015398:Signal Transduction; D015293:Transducin / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015106:3',5'-Cyclic-GMP Phosphodiesterases; D014357:Trypsin; D015293:Transducin