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.
M Dean
MD
Chamberlain
Deborah H
DH
Anderson
D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D020696:rab5 GTP-Binding Proteins