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

Abstract

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.

Structural basis of function in heterotrimeric G proteins

“…Additional studies are needed to dissect the role of the N-terminus and its fatty-acid modifications in the modulation of effector interactions. In addition to the effector-binding site illustrated by the crystal structures, several studies indicate that the a4/b6 loop of Ga is important for PDEc binding to Ga t (Rarick et al 1992 ;Liu et al 1999) and the activation of AC by Ga s (Berlot & Bourne, 1992).…”

The Retinal cGMP Phosphodiesterase γ-Subunit — A Chameleon

“…The interaction site has been mapped to the α3-β5 region [30] (or probably also α4-β6) [107] on Gα t , which does not undergo conformational change upon GTP binding [36–38]. Moreover, PDEγ has been observed to interact also with GDP-bound Gα t [30, 106–108], albeit with much lower affinity (880 nM) than with the GTP-bound form (12 nM) [30]. Taken together, it is likely that the interaction of the PDEγ central domain with the Gα t α3-β5 region constitutes an important part of the PDEγ:Gα t GDP association, and remains in the PDEγ:Gα t GTP complex.…”