Interaction sites of the C-terminal region of the 7 subunit of cgmp phosphodiesterase(P7) with the Q subunit of transducin (AT)

In photoreceptor cells, visual transduction occurs through at and P7 interaction. Trp70 has been implicated in PDE activation, at-P7 interaction and GAP activity. We have derivatized P7 with a reversible photoactivatable reagent-I25/-ACTP at Cys68. A light-dependent cross-linked complex of ot.GTP7S and m/-ACTP-derivatized P-, formed after photolysis. The specificity of complex formation between at and the P7 was demonstrated by specific protection by the C68A P7. The crossed-linked complex was treated with 3-mercaploethanol to transfer the 125/ photomoiety from P7 to at. Combined techniques involving SDS-PAGE, chemical and enzymatic cleavage, chemical and radiosequencing were used to identify three photoinsertion residues on the a3(His 244) and o4/56(Met308, Arg310) regions of ot.We conclude that CysGB of P7 is located at a position between the exposed face of the o3 and o4 helix of at. We propose that P7 interacts with at.GTP at multiple sites with the Cys68-Trp70 sequence interacting in the o3/u4//?6 region of at.GTP. The GDP form of at also formed a complex with P7 after photolysis, but at ~30% of that from at.GTP7S. Without photolysis, the GDP form of at disulfide exchanged the 12o/-ACTP from P7 to at. This disulfide exchange was protected by excess /37t arid C68A P7. It was shown that the disulfide exchange occurred at Cys250 and Cys210 of at. These data suggest that in the nonactivated state, a unique interaction may exist between the GTP and GDP bound form of G protein a subunits and effectors.(Supported by NIH grant GM3313S).

Duke Scholars

Author Vadim Y Arshavsky Ophthalmology, Vitreoretinal Diseases & Surgery