Fig. 3. mbGrk and secGrk bind Egfr, but only secGrk triggers signaling. (A-C) Aggregation of mbGrk-expressing cells with Egfr cells. Sf9 insect cells were independently infected with wild-type baculovirus, or virus encoding Egfr, mbGrk or Kek1. The two populations of cells were mixed and incubated together for 1 hour and then examined. (A) Cells expressing mbGrk strongly aggregate to cells expressing Egfr. (B) Cells expressing mbGrk and cells expressing Kek1 do not aggregate. (C) Cells expressing mbGrk and cells expressing Egfr, in presence of conditioned medium from cells expressing secGrk, no longer aggregate. (D) secGrk but not mbGrk activates Egfr in vitro. S2:Egfr cells (2x106) were incubated for 3 hours with 60 µM CuSO4 to induce moderate levels of Egfr expression (Schweitzer et al., 1995a). The cells were then stimulated for 30 minutes with serum free media (SFM), mbGrk, secGrk or secSpi. Egfr precipitates were prepared using rabbit anti-Egfr polyclonal sera. Top: the level of tyrosine phosphorylation of the Egfr was assessed by western blotting with mAb RC-20 (PY). Bottom: Egfr expression was then assessed by western blotting with rabbit anti-Egfr polyclonal sera. The lower amounts of Egfr in secGrk and secSpi lanes are most probably due to incomplete stripping of the RC-20 antibody.
