GPCR-mediated Rap1 activation and chemokine stimulation of VLA-4 avidity at prolonged contact are defective in LAD EBV cells. (A) A time course of SDF-1 stimulation (100 nM) of ERK1/2 phosphorylation in control or LAD EBV cells. Immunoblotting with anti-phosphospecific ERK1/2 (top row) and anti-ERK (bottom row) is depicted. SDF-1-induced ERK activation was blocked by pertussis toxin pretreatment of stimulated EBV cells (not shown). (B) Impaired SDF-1-induced Rap1 activation in LAD EBV cells. SDF-1-induced (100 nM, 30 seconds) stimulation of Rap1 activation in control and LAD cells. At this time point, maximal Rap1 activation was achieved in control cells. No Rap1 activation was observed in LAD cells at any SDF-1 dose between 0.5 to 5 minutes. (C) Similar PMA-induced Rap1 activation in control and LAD cells. Control and LAD cells were untreated (-) or treated (+) with 100 ng/mL PMA for 5 minutes, when maximal Rap1 activation was observed in both cell types. Rap1-GTP was detected with pull-down assays using GST-RalGDS-RBD (B-C, top panels). Total Rap1 is also shown (B-C, bottom panels). (D-E) Defective stimulation of VLA-4 avidity by surface-bound SDF-1 in LAD cells. Resistance to detachment by incremented shear forces by control or LAD cells settled for one minute at stasis on VCAM-1 (panel D, coated at 2 μg/mL) or on the α4-specific mAb HP1/2 (panel E, coated at 0.02 μg/mL), each coimmobilized with either inactivated SDF-1 (-) or intact SDF-1 (+), at 2 μg/mL. (E, inset) Shear resistance of control and LAD cells settled for 1 minute at stasis on high-density HP1/2 (coated at 0.2 μg/mL). Results are mean ± range of determinations in 2 fields and experiments are each representative of 3.