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 alpha-subunit of transducin (alphat.GTP) and the cGMP phosphodiesterase inhibitory gamma-subunit (Pgamma) has been studied. It is widely accepted that the alphat.GTP is the active form of transducin and that the GDP-bound transducin alpha-subunit (alphat. GDP) is the inactive form. We have reported previously that the binding region of the C-terminal of Pgamma on alphat.GTP is in a region between the exposed face of the alpha3 and alpha4 helices of alphat.GTP [Liu, Arshavsky and Ruoho (1996) J. Biol. Chem. 271, 26900-26907]. We now report that N-[(3-[125I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) ]cysteine ([125I]ACTP)-derivatized Pgamma (at Cys-68) reversibly undergoes a unique disulphide exchange of the radioiodinated moiety N-(3-[125I]iodo-4-azidophenylpropionamido)cysteine ([125I]APC) from Cys-68 of Pgamma to alphat.GDP but not to the guanosine 5'-(gamma-thio)-triphosphate (GTP[S])-bound transducin alpha-subunit (alphat-GTP[S]). The specificity of the interaction was demonstrated by the fact that exchange was protected by the functionally active Cys-68-->Ala Pgamma mutant, and by pretreatment of the alphat.GDP with the betagamma-subunit of transducin. Chemical cleavage and amino acid sequencing demonstrated that the [125I]ACTP-derived Pgamma specifically transferred the [125I]APC group to Cys-250 and Cys-210 of alphat.GDP. These data indicate that the C-terminal region (especially Cys-68-Trp-70) of Pgamma interacts with alphat. GDP on the exposed interface between alpha2/beta4 and alpha3/beta5 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 alphat.GDP. The variation in the binding pattern between alphat.GDP and alphat.GTP with the C-terminal region of Pgamma may contribute to the functional difference between the GDP- and GTP-bound states.
Liu
Y
Y
Department of Pharmacology, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706-1532, USA.
Arshavsky
V Y
VY
Ruoho
A E
AE
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003488:Cyanogen Bromide; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000378:metabolism; D004789:Enzyme Activation; D006153:Guanosine Diphosphate / Q000378:metabolism*; D019811:Hydroxylamine; D007457:Iodine Radioisotopes; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D015293:Transducin / Q000378:metabolism*
D004220:Disulfides; D007457:Iodine Radioisotopes; D006153:Guanosine Diphosphate; D019811:Hydroxylamine; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D015293:Transducin; D003545:Cysteine; D003488:Cyanogen Bromide