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A. S. Shavkunov, S. A. Baker, M. Kosloff, K. A. Martemyanov, V. Y. Arshavsky; Molecular Mechanism of RGS9 Regulation by R9AP. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4664.
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Proper timing of the photoresponse requires rapid termination of transducin signaling. R9AP is an important member of the GTPase activating protein complex, also containing RGS9 and Gß5, which inactivates transducin by increasing the rate of its intrinsic GTPase activity. R9AP was previously shown to potentiate the activity of the entire complex. Our goal was to elucidate the underlying molecular mechanism.
His-tagged R9AP (wild type and mutant), RGS9 and Gß5 were cloned in baculovirus vectors and expressed in an Sf9 insect cell line. Purified recombinant proteins were reconstituted with rod outer segment disc membranes and transducin from dark-adapted bovine retinas. Transducin GTPase activity was measured in single turnover and multiple turnover assays. Protein-protein interactions were assayed by pull-down experiments.
R9AP causes a major enhancement of the catalytic efficiency of RGS9·Gß5 but has little effect on the affinity between the complex and the GTP-bound transducin. Earlier studies showed that R9AP lacking its transmembrane domain can bind RGS9·Gß5 but can not potentiate its activity. We replaced this transmembrane with either another transmembrane domain from syntaxin 1A or with a prenylation site CIIL. Both mutants were able to restore RGS9·Gß5 activation, which indicates the importance of membrane attachment of the GTPase activation complex for its activity.
Our data indicate that the potentiation of RGS9·Gß5 may result from the secondary protein-lipid interactions following the membrane anchoring of RGS9·Gß5 by R9AP. We are evaluating this hypothesis in the current round of mutagenesis.
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