BINDING OF TRANSDUCIN TO LIGHT-ACTIVATED RHODOPSIN PREVENTS TRANSDUCIN INTERACTION WITH THE ROD CGMP PHOSPHODIESTERASE GAMMA-SUBUNIT

In photoreceptor cells of vertebrates, the GTP-bound cr-subunit of rod G-protein, transducin (G(t alpha)), interacts with the cGMP phosphodi esterase inhibitory gamma-subunit (Py) to activate the effector enzyme . The GDP-bound Gr, can also bind the Py subunit, albeit with a lower affinity than G(t alpha)GTP. In this work, interactions between Gt,GDP and Py or Py-24-45Cys labeled with the fluorescent probe 3-(bromoacet yl)7-(diethylamino)coumarin (P gamma BC, P gamma-24-45BC) have been in vestigated. Addition of G(t alpha)GDP to P gamma BC produced approxima tely a 6-fold maximal increase in the probe fluorescence, while the fl uorescence of P alpha-24-45BC was enhanced by 2.3-fold. The K-d's for the G(t alpha)GDP binding to P gamma BC and P gamma-24-45BC were 75 +/ - 8 nM and 400 +/- 110 nM, respectively. The Gt(beta gamma) subunits h ad no notable effect on the binding of Gt(alpha)GDP to P gamma BC or P gamma-24-45BC, suggesting that P gamma and G(t beta gamma) bind to G( t alpha)CDP noncompetitively. The G(t alpha beta gamma) interaction wi th the fluorescently labeled Py was effectively blocked in the light-a ctivated rhodopsin (R)-G(t alpha beta gamma) complex. Furthermore, ad dition of excess P gamma or P gamma-24-45 prevented binding of Gt alph a beta gamma to R, indicating that the R* and Py binding surfaces on Gt alpha beta gamma may overlap. It is likely that R has a binding si te within the alpha 3 -beta 5 region of G(t alpha), which is a propose d site of G(t alpha)GDP binding to P-gamma-24-45. Alternatively, R ma y induce conformational changes of the Gt(alpha) alpha 3-beta 5 region such that the resulting structural changes alter the adjacent consens us sequence for the guanine ring binding of GDP/GTP(NKXD), and lead to a reduction in the affinity of G-protein for guanine nucleotides.