Figure 5. Dominant negative Rit strongly interacts with Par6 independent of its PDZ domain.

(A) COS cells were transfected with GST or GST-Par6C and Flag-tagged Rit^WT, Rit^L79 or Rit^N35. GST precipitations were performed as in Figure 4B. Note that Rit^N35 displays stronger association with Par6 than either Rit^WT or Rit^L79. Blots are representative of three independent trials. (B,C) Biotinylated Par6C was bound to streptavidin-coated plates and incubated with the indicated concentrations of recombinant GST, GST-PCKι, or GST-Rit pre-loaded with non-hydrolyzable GTPγS (GTP) or GDP. Binding isotherms for PKCι (B) and GTPγS-Rit and GDP-Rit (C) to Par6 indicate that PKCι binds Par6 with much higher apparent K_D than Rit and that GTP- and GDP-Rit bind to Par6 with similar affinities. (D) COS cells were co-transfected with GST, GST-Par6C full length, GST-Par6CΔPDZ, or GST-Par6CΔN and Flag-Rit^N35and whole cell lysates were generated and subjected to GST pull-down analysis as described under “Materials and Methods”. Blots are representative of three independent trials.