Functional modifications of transducin induced by cholera or pertussis‐toxin‐catalyzed ADP‐ribosylation
Authors: BORNANCIN Frédéric , FRANCO Michel , BIGAY Joëlle , CHABRE Marc 
Published: March 2005

Transducin (Tαβγ), 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 α 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 Tα by the resulting covalent modifications are studied such as: the binding of Tα to Tβγ to the membrane and to Rh*; the spontaneous or Rh*‐catalysed exchange of GDP for GTP or guanosine 5‐[γ‐thio]triphosphate (GTP[γS]), the conformational switch and activation undergone by transducin upon this exchange, the activation of TαGDP by fluoride complexes and the activation of the PDE by TαGTP.

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*‐Tαempty‐Tβγ and does not activate Tα. Subsequent to CTx‐catalyzed ADP‐ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive CTxTα (CTxTα, ADP‐ribosylated α subunit of transducin) which remains associated to Tβα; (b) CTxTαGDP‐Tβγ exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP[γS] in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as γ‐phosphate analogs, are kinetically modified, suggesting that the ADP‐ribose on Arg174 specifically perturbs binding of the γ‐phosphate in the nucleotide site; (c) CTxTαGDP‐Tβγ can still couple to Rh* and undergo fast nucleotide exchange; (d) CTxTβGTP[γS] and CTxTβGDP‐AlFx, (AlFx, 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) CTxTβGTP hydrolyses GTP more slowly than unmodified TβGTP, which entirely accounts for the prolonged action of CTxTβGTP on the PDE; (f) after GTP hydrolysis, CTxTβGDP reassociates to Tβγ and becomes inactive. Thus, CTx catalyzed ADP‐ribosylation only perturbs in Tβ the GTP‐binding domain, but not the conformational switch nor the domains of contact with the Tβγ subunit, with Rh* and with the PDE.

PTx is active on TβGDP in the absence of membrane and of ARF, but the cooperation of Tβγ is needed. Subsequent to PTx catalyzed ADP‐ribosylation (a)PTxTβGDP remains associated to Tβγ and is inactive, (b) PTxTβGDP‐Tβγ displays the usual slow kinetics of spontaneous exchange of GDP for GTP[γS] in the absence of Rh* and unmodified association and dissociation kinetics for fluoride complexes, suggesting that the nucleotide‐binding domain is not perturbed, (c) PTxTβGDP‐Tβγ does not bind to Rh* and thus does not undergo the fast‐receptor‐catalyzed nucleotide exchange, but (d) PTxTβGDP‐AlFx activates PDE with the same efficiency as its unmodified counterpart. Thus, PTx‐catalyzed ADP‐ribosylation only perturbs the receptor contact domain of Tβ but not the GTP binding domain, the conformational switch, or the effector and the Tβγ contact domains.

These two modifications are analogous to point mutations at Arg174 or Cys347, which would affect two structurally and functionally independent domains of Tβ. Sequential ADP‐ribosylations by CTx and PTx are, however, much hindered.