Figure 1.

Protease-activated receptor-1 (PAR1) is essential for thrombin-induced ErbB transactivation in breast carcinoma cells. Serum-deprived MDA-MB-231 cells were incubated with 10 nM thrombin (a and b) for various times at 37 °C. Cells were lysed, immunoprecipitated with anti-PY99 antibody and immunoblotted with anti-epidermal growth factor (EGF) receptor (EGFR) or anti-ErbB2 antibody. Total cell lysates were immunoblotted for EGFR or ErbB2 as a control. The time-course of thrombin-induced EGFR and ErbB2 transactivation shown is from a representative experiment. The data (mean ± s.e.) are expressed as fold increase over basal from three independent experiments. In reciprocal experiments, serum-deprived MDA-MB-231 cells were incubated with 10 nM thrombin for 4 min at 37 °C, lysed and immunoprecipitated with anti-EGFR or anti-ErbB2 antibodies and then immunoblotted with anti-PY99 antibody. (c) Control, P1 and P2 shRNA-expressing MDA-MB-231 cells were incubated with anti-PAR1 antibody or preimmune serum, fixed and the amount of antibody bound to the cell surface was measured by enzyme-linked immunosorbent assay (ELISA). The data (mean ± s.d., n = 3) shown are from a representative experiment repeated at least three times. MDA-MB-231 control and P1 shRNA cells labeled with myo-[³H]inositol were incubated in the absence or presence of 10 nM thrombin, 100 µM TFLLRNPNDK or 100 µM SLIGKV for 60 min at 37 °C and the amounts of [³H]inositol phosphates generated were then measured. The data (mean ± s.d., n = 3) are expressed as fold increase over untreated control and are representative of three different experiments. (d) Control and PAR1-deficient P1 shRNA-expressing MDA-MB-231 cells were incubated in the absence or presence of 10 nM thrombin or 16 nM EGF for 4 min at 37 °C, lysed and immunoprecipitated with anti-PY99 antibody and immunoblotted with anti-EGFR or anti-ErbB2 antibody. Cell lysates were immunoblotted for EGFR or ErbB2 as a control. The data are expressed as fold increase over untreated control and are representative of three independent experiments.