Signal transfer from rhodopsin to the G-protein: Evidence for a two-site sequential fit mechanism

Photoactivation of the retinal photoreceptor rhodopsin proceeds through a cascade of intermediates, resulting in protein–protein interactions catalyzing the activation of the G-protein transducin (Gt). Using stabilization and photoregeneration of the receptor’s signaling state and Gt activation assays, we provide evidence for a two-site sequential fit mechanism of Gt activation. We show that the C-terminal peptide from the Gt γ-subunit, Gtγ(50–71)farnesyl, can replace the holoprotein in stabilizing rhodopsin’s active intermediate metarhodopsin II (MII). However, the peptide cannot replace the Gtβγ complex in direct activation assays. Competition by Gtγ(50–71)farnesyl with Gt for the active receptor suggests a pivotal role for Gtβγ in signal transfer from MII to Gt. MII stabilization and competition is also found for the C-terminal peptide from the Gt α-subunit, Gtα(340–350), but the capacity of this peptide to interfere in MII-Gt interactions is paradoxically low compared with its activity to stabilize MII. Besides this disparity, the pH profiles of competition with Gt are characteristically different for the two peptides. We propose a two-site sequential fit model for signal transfer from the activated receptor, R*, to the G-protein. In the center of the model is specific recognition of conformationally distinct sites of R* by Gtα(340–350) and Gtγ(50–71)farnesyl. One matching pair of domains on the proteins would, on binding, lead to a conformational change in the G-protein and/or receptor, with subsequent binding of the second pair of domains. This process could be the structural basis for GDP release and the formation of a stable empty site complex that is ready to receive the activating cofactor, GTP.