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 phosphodiesterase 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-(bromoacetyl)7-(diethylamino)coumarin (P gamma BC, P gamma-24-45BC) have been investigated. Addition of G(t alpha)GDP to P gamma BC produced approximately a 6-fold maximal increase in the probe fluorescence, while the fluorescence 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 had 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 with the fluorescently labeled Py was effectively blocked in the light-activated rhodopsin (R*)-G(t alpha beta gamma) complex. Furthermore, addition of excess P gamma or P gamma-24-45 prevented binding of Gt alpha 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 site within the alpha 3 -beta 5 region of G(t alpha), which is a proposed site of G(t alpha)GDP binding to P-gamma-24-45. Alternatively, R* may induce conformational changes of the Gt(alpha) alpha 3-beta 5 region such that the resulting structural changes alter the adjacent consensus sequence for the guanine ring binding of GDP/GTP(NKXD), and lead to a reduction in the affinity of G-protein for guanine nucleotides.