FIGURE 3.

A CD151 mutant with impaired α₃β₁ integrin association fails to recruit Rac1 to the membrane. A, CD151^VR mutant cDNA that encodes CD151 with a large extracellular loop substituted with that of TM4SF2, as illustrated, was generated by PCR and subcloned into a pcDNA3 vector. The wild-type (WT) and mutant (MT^VR) CD151 expression vectors were transiently transfected into 293 cells, and their expression levels were examined by immunoblotting analysis using mAbs H-80 and N-20, which recognize large and small extracellular loops of CD151, respectively. B, following transfection with the wild-type and mutant CD151 expression constructs, Brij 97 detergent lysates were subjected to immunoprecipitation (IP) with anti-CD151 mAb (N20) followed by immunoblotting analysis with Abs against integrin α₃, α₆, or β₁. C, following sucrose density gradient ultracentrifugation, 12 fractions were examined for the presence of β₁ integrin, CD151, and Rac1 by immunoblotting analysis.