Complementary interactions of the rod PDE6 inhibitory subunit with the catalytic subunits and transducin

Abstract

Activation of the cyclic GMP phosphodiesterase (PDE6) by transducin is the central event of visual signal transduction. How the PDE6 inhibitory γ-subunit (Pγ) interacts with the catalytic subunits (Pαβ) and the transducin α-subunit (αt) in this process is not entirely clear. Here we have investigated this issue, taking advantage of site-specific label transfer from throughout the full-length Pγmolecule to both αt and Pαβ. The interaction profiling and pull-down experiments revealed that the Pγ C-terminal domain accounted for the major interaction with αt bound with guanosine 5′-3-O-(thio)triphosphate (αtGTPγS) in comparison with the central region, whereas an opposite pattern was observed for the Pγ-Pαβ interaction. This complementary feature was further exhibited when both αtGTPγS and Pαβ were present and competing for Pγ interaction, with the Pγ C-terminal domain favoring αt, whereas the central region demonstrated a preference for Pαβ. Furthermore, αtGTPγS co-immuno-precipitated with PDE6 and vice versa in a Pγ-dependent manner. Either Pαβ or αtGTPγS could be pulled down by the Btn-Pγmolecules on streptavidin beads that were saturated by the other partner, indicating simultaneous binding of these two partners to Pγ. These data together indicate that complementary Pγ interactions with its two targets facilitate the αt·PDE6 " transducisome" formation. Thus, our study provides new insights into the molecular mechanisms of PDE6 activation. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.