Interaction sites of the C-terminal region of the cGMP phosphodiesterase inhibitory subunit with the GDP-bound transducin alpha-subunit

In the present report, the region of interaction between the GDP-bound alph a-subunit of transducin (alpha t.GTP) and the cGMP phosphodiesterase inhibi tory gamma-subunit (P gamma) has been studied. It is widely accepted that t he alpha t.GTP is the active form of transducin and that the GDP-bound tran sducin alpha-subunit (alpha t.GDP) is the inactive form. We have reported p reviously that the binding region of the C-terminal of P gamma on alpha t.G TP is in a region between the exposed face of the alpha 3 and alpha 4 helic es of alpha t.GTP [Liu, Arshavsky and Ruoho (1996) J. Biol. Chem. 271, 2690 0-26907].We now report that N-[(3-[<SUP>125</SUP>I]iodo-4-<INF></INF> azido phenylpropionamido-S-(2-thiopyridyl)]cysteine ([<SUP>125</SUP>I]ACTP)-deriv atized P gamma (at Cys-68) reversibly undergoes a unique disulphide exchang e of the radioiodinated moiety N-(3-[<SUP>125</SUP>I]iodo-4-azidophenylprop ionamido)cysteine ([<SUP>125</SUP>I]APC) from Cys-68 of P gamma to alpha t. GDP but not to the guanosine 5'-(gamma-thio)-triphosphate (GTP[S])-bound tr ansducin alpha-subunit (alpha t-GTP[S]). The specificity of the interaction was demonstrated by the fact that exchange was protected by the functional ly active Cys-68 --> Ala P gamma mutant, and by pretreatment of the alpha t .GDP with the beta gamma-subunit of transducin. Chemical cleavage and amino acid sequencing demonstrated that the [<SUP>125</SUP>I]ACTP-derived P gamm a specifically transferred the [<SUP>125</SUP>I]APC group to Cys-250 and Cy s-210 of alpha t.GDP. These data indicate that the C-terminal region (espec ially Cys-68-Trp-70) of P gamma interacts with alpha t.GDP on the exposed i nterface between alpha 2/beta 4 and alpha 3/beta 5 of the alpha-subunit of transducin. Disulphide exchange was also observed with the alpha-subunit of holotransducin but this was only approx. 60 % of that of pure alpha t.GDP. The variation in the binding pattern between alpha t.GDP and alpha t.GTP w ith the C-terminal region of P gamma may contribute to the functional diffe rence between the GDP- and GTP-bound states.