Resonance energy transfer as a direct monitor of GTP-binding protein-effector interactions: activated alpha-transducin binding to the cGMP phosphodiesterase in the bovine phototransduction cascade.
Resonance energy-transfer approaches have been used to directly monitor the interactions of the GTP gamma S-bound alpha subunit of transducin (alpha T GTP gamma S) with the retinal cyclic GMP phosphodiesterase (PDE). The PDE was labeled with 5-(iodoacetamido) fluorescein (IAF-PDE) and served as the fluorescence donor in these experiments while the alpha T GTP gamma S was labeled with eosin-5-isothiocyanate (EITC-alpha T GTP gamma S) and served as the energy acceptor. The EITC-alpha T GTP gamma S species was able to quench a significant percentage of the IAF-PDE fluorescence (typically greater than or equal to 30%) due to resonance energy transfer between the IAF and EITC moieties. The quenching by the EITC-alpha T GTP gamma S species was dose-dependent, saturable (Kd = 21 nM), and specific for the GTP gamma S-bound form of the alpha T subunit. Limited trypsin treatment of the IAF-PDE, which selectively removes a fluorescein-labeled gamma subunit (gamma PDE), completely eliminates the quenching of the IAF fluorescence by the EITC-alpha T GTP gamma S complex. Although the EITC-alpha T GTP gamma S complex competes with the unlabeled alpha T GTP gamma S for a binding site on the IAF-PDE, as well as for a site on the native PDE, it is not able to stimulate PDE activity. Thus, the modification of a single EITC-reactive residue on the alpha T GTP gamma S complex prevents this subunit from eliciting a key activation event within the retinal effector enzyme.
Erickson
J W
JW
Department of Pharmacology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853-6401.
Cerione
R A
RA
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004735:Energy Transfer; D004789:Enzyme Activation; D004801:Eosine Yellowish-(YS) / Q000737:chemistry / Q000031:analogs & derivatives; D019204:GTP-Binding Proteins / Q000737:chemistry*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006868:Hydrolysis; D066298:In Vitro Techniques; D010777:Photochemistry; D012374:Rod Cell Outer Segment / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D015293:Transducin / Q000378:metabolism*; D014357:Trypsin
D016244:Guanosine 5'-O-(3-Thiotriphosphate); C012957:eosine-5-isothiocyanate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D014357:Trypsin; D019204:GTP-Binding Proteins; D015293:Transducin; D004801:Eosine Yellowish-(YS)