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M.H. Elliott, A.J. Ghalayini, R.V. Rajala, K. Barkus, R.T. Dobrowsky, R.E. Anderson; Direct interaction with caveolin–1 regulates the activity of the alpha subunit of transducin. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1274.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have recently reported on an endogenous association between caveolin–1 (Cav–1) and the alpha subunit of transducin (Tα) (Biochemistry, 2003, 42: 7892–7903). Here, we show that Tα can interact directly with the Cav–1 scaffolding domain (amino acids 82–101) and that this interaction inhibits Tα GTPase activity. Methods: Full–length Cav–1 (residues 1–178) and various deletion mutants of Cav–1 (residues 1–140, 1–81, 61–101, 102–134, and 135–178) were expressed as glutathione S–transferase (GST) fusions and immobilized on glutathione–sepharose beads. For pull–down experiments, expressed Cav–GST fusion proteins were incubated with extracts of transducin purified from bovine rod outer segments (ROS). To assess the effects of the Cav–1/Tα interaction on transducin activity, dark–adapted bovine ROS were incubated with Cav–GST fusion proteins, exposed to light, and GTP hydrolysis was monitored by a multiple turnover GTPase assay. Results: In GST–fusion pull–down assays from purified holo–transducin, Tα was found to interact with Cav–1–GST–fusion proteins containing the scaffolding domain [full–length Cav–1 (1–178), 1–140, and 61–101], but not with other Cav–1–GST fusions (1–81 and 102–134) or GST alone. A weak interaction was detected for the C–terminal 135–178 fusion. Furthermore, as assessed by a multiple turnover GTPase assay, scaffolding domain–containing fusions (1–178, 1–140, 61–101) significantly decreased Tα–mediated GTP hydrolytic rate when compared with controls (untreated, 1–81, and GST alone). Conclusions: Our results imply functional consequences to Cav–1/Tα interactions. Based upon these results and our earlier observation of an endogenous Cav–1/Tα association, a model involving Cav–1–mediated sequestration of Tα will be presented.
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