Interaction between the retinal cyclic GMP phosphodiesterase inhibitor and transducin. Kinetics and affinity studies.
In the retinal cyclic GMP phosphodiesterase (PDE), catalysis by the alpha beta-heterodimer is inhibited in the dark by two identical gamma-subunits and stimulated in the light by the GTP-bearing alpha-subunit of the heterotrimeric G-protein transducin (T beta gamma-T alpha GDP). Two T alpha GTP molecules, dissociated from T beta gamma, bind to and displace the PDE gamma subunits from their inhibitory sites on PDE alpha beta. With GTP gamma S in lieu of GTP, this association becomes persistent. Under physiological conditions, the PDE alpha beta (gamma T alpha)2 active complex stays on the membrane. But in low-salt buffers, it becomes soluble and dissociates into a partially active PDE alpha beta catalytic moiety and two PDE gamma-T alpha GTP gamma S complexes. This indicates that T alpha binds preferentially to PDE gamma. We have studied the interaction of recombinant bovine PDE gamma with purified T alpha in solution or with retinal rod outer segments (ROS) containing both T beta gamma-T alpha GDP and PDE alpha beta gamma 2. When added to dark ROS, recombinant PDE gamma did not bind to inactive PDE alpha beta gamma 2 but extracted T alpha GDP from membrane-bound holo-transducin to form a soluble PDE gamma-T alpha GDP complex. PDE gamma also bound to purified T alpha GDP in solution. The kinetics and affinity of the interaction between PDE gamma and T alpha GDP or T alpha GTP gamma S were determined by monitoring changes in the proteins' tryptophan fluorescence. The Kd's for the binding of recombinant PDE gamma to soluble T alpha GTP gamma S and T alpha GDP are < or = 0.1 and 3 nM, respectively. PDE gamma-T alpha GDP falls apart in 3 s. This slow dissociation means that, in situ, T alpha-PDE gamma cannot physically leave the active PDE alpha beta, since after GTP hydrolysis, an isolated T alpha-PDE gamma complex would dissociate too slowly to allow a fast PDE reinhibition by the liberated PDE gamma. When recombinant PDE gamma was added to PDE that had been persistently activated by T alpha GTP gamma S, reinhibition occurred and T alpha GTP gamma S, complexed to the native PDE gamma, was released, indicating that both had hitherto stayed bound to PDE alpha beta. The mutation W70F does not prevent recombinant PDE gamma from inhibiting PDE alpha beta but diminishes its affinity for T alpha GTP and T alpha GDP 100-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
Otto-Bruc
A
A
CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.
Antonny
B
B
Vuong
T M
TM
Chardin
P
P
Chabre
M
M
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002417:Cattle; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012160:Retina / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D015293:Transducin / Q000378:metabolism*; D014364:Tryptophan
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014364:Tryptophan; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin