Transducin (Talphabetagamma), the heterotrimeric GTP-binding protein that interacts with photoexcited rhodopsin (Rh*) and the cGMP-phosphodiesterase (PDE) in retinal rod cells, is sensitive to cholera (CTx) and pertussis toxins (PTx), which catalyze the binding of an ADP-ribose to the alpha subunit at Arg174 and Cys347, respectively. These two types of ADP-ribosylations are investigated with transducin in vitro or with reconstituted retinal rod outer-segment membranes. Several functional perturbations inflicted on Talpha by the resulting covalent modifications are studied such as: the binding of Talpha to Tbetagamma to the membrane and to Rh*; the spontaneous or Rh*-catalysed exchange of GDP for GTP or guanosine 5-[gamma-thio]triphosphate (GTP[gammaS]), the conformational switch and activation undergone by transducin upon this exchange, the activation of TalphaGDP by fluoride complexes and the activation of the PDE by TalphaGTP. ADP-ribosylation of transducin by CTx requires the GTP-dependent activation of ADP-ribosylation factors (ARF), takes place only on the high-affinity, nucleotide-free complex, Rh*-Talpha(empty)-Tbetagamma and does not activate Talpha. Subsequent to CTx-catalyzed ADP-ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive (CTx)TalphaGDP (CTx)Talpha, ADP-ribosylated alpha subunit of transducin) which remains associated to Tbetagamma; (b) ''TalphaGDP-Tbetagamma exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP[gammaS] in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as gamma-phosphate analogs, are kinetically modified, suggesting that the ADP-ribose on Arg174 specifically perturbs binding of the gamma-phosphate in the nucleotide site; (c) (CTx)TalphaGDP-Tbetagamma can still couple to Rh* and undergo fast nucleotide exchange; (d) (CTx)TalphaGTP[gammaS] and (CTx)TalphaGDP-AlF(x). (AlF(x), Aluminofluoride complex) activate retinal cGMP-phosphodiesterase (PDE) with the same efficiency as their unmodified counterparts, but the kinetics and affinities of fluoride activation are changed; (e) (CTx)TalphaGTP hydrolyses GTP more slowly than unmodified TalphaGTP, which entirely accounts for the prolonged action of (CTx)TalphaGTP on the PDE; (f) after GTP hydrolysis, (CTx)TalphaGDP reassociates to Tbetagamma and becomes inactive. Thus, CTx catalyzed ADP-ribosylation only perturbs in Talpha the GTP-binding domain, but not the conformational switch nor the domains of contact with the Tbetagamma subunit, with Rh* and with the PDE. PTx is active on TalphaGDP in the absence of membrane and of ARF, but the cooperation of Tbetagamma is needed. Subsequent to PTx catalyzed ADP-ribosylation (a) (PTx)TalphaGDP remains associated to Tbetagamma and is inactive, (b) (PTx)TalphaGDP-Tbetagamma displays the usual slow kinetics of spontaneous exchange of GDP for GTP[gammaS] in the absence of Rh* and unmodified association and dissociation kinetics for fluoride complexes, suggesting that the nucleotide-binding domain is not perturbed, (c) (PTx)TalphaGDP-Tbetagamma does not bind to Rh* and thus does not undergo the fast-receptor-catalyzed nucleotide exchange, but (d) (PTx)TalphaGDP-AIF(x) activates PDE with the same efficiency as its unmodified counterpart. Thus, PTx-catalyzed ADP-ribosylation only perturbs the receptor contact domain of Talpha but not the GTP binding domain, the conformational switch, or the effector and the Tbetagamma contact domains. These two modifications are analogous to point mutations at Arg174 or Cys347, which would affect two structurally and functionally independent domains of Talpha. Sequential ADP-ribosylations by CTx and PTx are, however, much hindered.
