Abstract
Abstract: :
The visual signaling pathway is initiated by photoactivation of the GPCR rhodopsin, which activates nucleotide exchange on the trimeric G–protein transducin (Gt). Previously we have shown by high–resolution NMR that the Gt–gamma subunit acts as a regulatory switch during rhodopsin–transducin interactions (Kisselev, O.G. and Downs, M.A. (2003) Structure, Cell Press 11, 367–373). The farnesylated C–terminal region of gamma (60–71), DKNPFKELKGGC, assumes an amphipathic helical conformation that interacts with Metarhodopsin II. Purpose: To evaluate the contribution of specific amino acids of Gt–gamma C–terminus involved in binding and release of transducin from native rhodopsin membranes. Methods: We have systematically substituted Alanine for each of the amino acids, including the highly conserved NPF region. Each of the eleven mutants was co–expressed with histidine labeled Gt–beta subunits in SF9 insect cells. The beta–His–gamma mutant protein was purified to homogeneity and assayed in the presence of Gt–alpha for the GTP–dependent interactions with light activated rhodopsin. Results: Several of the Alanine mutants, including N62A, P63A and F64A exhibited significant functional defects. Conclusions: These results indicate specific amino acids; including N62, P63 and F64 have a direct effect on transducin binding and release of rhodopsin membranes.
Keywords: signal transduction • protein structure/function • photoreceptors