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 gamma-subunit, Gtgamma(50-71)farnesyl, can replace the holoprotein in stabilizing rhodopsin's active intermediate metarhodopsin II (MII). However, the peptide cannot replace the Gtbeta gamma complex in direct activation assays. Competition by Gtgamma(50-71)farnesyl with Gt for the active receptor suggests a pivotal role for Gtbeta 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, Gtalpha(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 Gtalpha(340-350) and Gtgamma(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.
Kisselev
O G
OG
Institut für Medizinische Physik und Biophysik, Universitätsklinikum Charité, Humboldt Universität zu Berlin, D-10098 Berlin, Germany.
Meyer
C K
CK
Heck
M
M
Ernst
O P
OP
Hofmann
K P
KP
D000818:Animals; D001665:Binding Sites; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010786:Photoreceptor Cells / Q000378:metabolism*; D011485:Protein Binding; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction
D012243:Rhodopsin; D019204:GTP-Binding Proteins