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. 2015 Mar 24;35(8):1401–1413. doi: 10.1128/MCB.00112-15

FIG 3.

FIG 3

β8 integrin binds preferentially to RhoGDI1pY156 to regulate Rac1 and Cdc42 activation at the leading edge. (A, B) Myc-tagged RhoGDI1 protein binds to β8 integrin in wild-type astrocytes but not β8−/− cells, as revealed by coimmunoprecipitation (A) or pulldown assays using a recombinant protein consisting of GST fused to the cytoplasmic domain of β8 integrin (B). (C) Astrocytes were transfected with myc-tagged RhoGDI1 or Y156 point mutant constructs, and interactions with β8 integrin were analyzed by coimmunoprecipitation. Note that the mutant with the Y156E mutation showed enhanced binding to β8 integrin. A darker exposure would reveal relatively weak interactions with wild-type (WT) RhoGDI1. (D) Schematic showing constructs comprised of V5-tagged β8 integrin (100 kDa), myc-tagged full-length RhoGDI1 (21 kDa), or the 13-kDa myc-tagged RhoGDI1 CTD, which contains Y156 (asterisks). (E) V5-tagged β8 integrin interacts with full-length RhoGDI1 or the RhoGDI1 CTD, as revealed by coimmunoprecipitation. (F) In comparison to full-length RhoGDI1, the CTD of RhoGDI1 is hyperphosphorylated on tyrosine and binds weakly to Rac1. FL, Flag tag.