Figure 3.

UK14304, Isoproterenol- and EGF-stimulated tyrosine phosphorylation of the EGFR and the effect of the EGFR-specific tyrphostin, AG1478, on α_2A AR- and β₂ AR-mediated ERK 1/2 phosphorylation. (A) Serum-starved COS-7 cells transiently expressing the α_2A AR or β₂ AR or pCDNA3 were stimulated with 1 μM UK14304, 1 μM isoproterenol, or 10 ng/ml EGF for 2 min. Monolayers were lysed in glycerol lysis buffer, and endogenous EGFRs were immunoprecipitated by using a sheep anti-human EGFR polyclonal antiserum. Immunoprecipitates were resolved by SDS/PAGE, and EGFR tyrosine phosphorylation was determined by immunoblotting by using a horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antiserum as described in Materials and Methods. (B) Cells transiently overexpressing the α_2A AR-, the β₂ AR-, or vector-transfected cells were preincubated for 15 min with tyrphostin AG1478 (125 nM) before stimulation with isoproterenol (1 μM), UK14304 (1 μM), or EGF (10 ng/ml) for 5 min. ERK 1/2 phosphorylation was determined from whole-cell lysates as described in Materials and Methods. Data shown are the mean ± SEM of four independent experiments and are normalized to the level of ERK 1/2 phosphorylation in untreated cells.