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 c ascade of intermediates, resulting in protein-protein interactions catalyzi ng the activation of the G-protein transducin (Gt), Using stabilization and photoregeneration of the receptor's signaling state and Gt activation assa ys, we provide evidence for a two-site sequential fit mechanism of Gt activ ation. We show that the C-terminal peptide from the Gt gamma-subunit, Gt ga mma(50-71)farnesyl, can replace the holoprotein in stabilizing rhodopsin's active intermediate metarhodopsin II (MII). However, the peptide cannot rep lace the Gt beta gamma complex in direct activation assays. Competition by Gt gamma(50-71)farnesyl with Gt for the active receptor suggests a pivotal role for Gt beta gamma in signal transfer from MII to Gt. MII stabilization and competition is also found for the C-terminal peptide from the Gt alpha -subunit, Gt alpha(340-350), but the capacity of this peptide to interfere in MII-Gt interactions is paradoxically low compared with its activity to s tabilize MII. Besides this disparity, the pH profiles of competition with G t are characteristically different for the two peptides, We propose a two-s ite 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 Gt alpha(340-350) and Gt gamma(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 su bsequent binding of the second pair of domains. This process could be the s tructural basis for GDP release and the formation of a stable empty site co mplex that is ready to receive the activating cofactor, GTP.