Figure 1. β₁AR-mediated transactivation of EGFR requires GRK phosphorylation sites.

HEK293 cells stably expressing WT β₁AR (A), PKA^– β₁AR (B), GRK^– β₁AR (C), or PKA^– GRK^– β₁AR (D) and transfected with FLAG-EGFR were treated with ICI and Dob (as described in Methods) and compared with cells with no stimulation (NS) or EGF stimulation. WT β₁AR (A) and PKA^– β₁AR (B) induced increases in phospho-EGFR and phospho-ERK1/2 after 5 minutes in response to treatment with Dob, while GRK^– β₁AR (C) and PKA^–/GRK^– β₁AR (D) lacked this effect; *P < 0.05 versus NS. EGFR internalization following Dob or EGF stimulation for 30 minutes was visualized using confocal microscopic analysis of HEK293 cells stably expressing the above β₁AR mutants and transfected with EGFR-GFP. In the absence of agonist, EGFR-GFP was visualized on the membrane in each stable cell line (A–D, i, arrows), while EGF stimulation resulted in redistribution of EGFR-GFP into cellular aggregates (A–D, iii). Treatment of either WT β₁AR or PKA^– β₁AR cells with Dob resulted in a similar redistribution of EGFR into cellular aggregates (A and B, ii, arrowheads), an effect that was absent in GRK^– β₁AR and PKA^–/GRK^– β₁AR cells, where EGFR-GFP remained on the membrane (C and D, ii, arrows). Original magnification, ×100.