Measurement of the interaction of the p85alpha subunit of phosphatidylinositol 3-kinase with Rab5.
During endocytosis of the activated platelet-derived growth factor (PDGF) receptor, phosphatidylinositol 3-kinase ( PI3K) remains associated with the receptor. We found that the p85 alpha subunit of PI3 kinase binds directly to Rab5 and possesses GTPase activating protein (GAP) activity toward Rab5. Rab5 is a small monomeric GTPase involved in regulating vesicle fusion events during receptor-mediated endocytosis. We used two methods to characterize the direct binding between Rab5 in various nucleotide- bound states and the p85 protein. In the first assay, the ability of p85 to bind to Rab5 is measured using an enzyme-linked immunosorbent assay (ELISA). The second assay is a glutathione S-transferase (GST) pull-down approach in which GST-Rab5 proteins in various nucleotide-bound states are allowed to bind p85. In both instances, bound p85 is detected using anti-p85 antibodies.[1]References
- Measurement of the interaction of the p85alpha subunit of phosphatidylinositol 3-kinase with Rab5. Dean Chamberlain, M., Anderson, D.H. Meth. Enzymol. (2005) [Pubmed]
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