Figure 5.
Endogenous ligand activation of EphB1 requires cell–cell contact and promotes LMW–PTP recruitment. Equal numbers of HRMEC (5 × 105 cells) were plated on Matrigel-coated 35 (p35)-, 60-, (p60)-,100-, (p100)-, or 150 (p150)-mm-diam. dishes, representing 1×, 3.1×, 8.6×, or 19.5× unit surface areas, in medium supplemented with 1% bovine albumin. Cells were incubated for 2 hr at 37°C, then stimulated for 15 min with either no addition (NA), control Fc fusion (ORF/Fc, 250 ng/ml), preclustered ORF/Fc (250 ng/ml) plus anti-Fc (25 ng/ml), ephrin-B1/Fc (250 ng/ml), preclustered ephrin-B1/Fc (250 ng/ml) plus anti-Fc (25 ng/ml), or phorbol myristate acetate (PMA, 2 ng/ml). Immunoprecipitated EphB1 receptor complexes were analyzed for receptor activation (anti-pTyr, 130-kD band) and recruitment of the 18-kD LMW–PTP (anti-pTyr and anti-LMW–PTP). The 25-kD band is immunoglobulin light chain from the immunoprecipitation. Notably, PMA-stimulated cell density-dependent activation of EphB1 and recruitment of LMW–PTP to receptor complexes.