Abstract 888: β-Arrestin mediated β1-Adrenergic Receptor Transactivation of the Epidermal Growth Factor Receptor Requires GRK 5 and 6
Recent evidence suggests that stimulation of β adrenergic receptors (βARs) can induce mitogenic and cell survival signaling that is independent of G protein activation. However, the precise molecular mechanism by which β1ARs activate these signaling pathways, and whether it involves transactivation of epidermal growth factor receptor (EGFR) is unknown. To address this issue, we used small interfering RNA to knock down expression of β-arrestin and G protein-coupled receptor kinase (GRK) isoforms and studied β1AR-mediated EGFR transactivation. HEK 293 cells were cotransfected with wild-type β1AR or a mutant β1AR that lacked phosphorylation sites for protein kinase A (PKA-β1AR), along with siRNA targeting β-arrestin 1, 2, or both. Stimulation of β1AR with dobutamine (Dob) in the presence of the β2AR antagonist ICI 118,551 (ICI) resulted in internalization and tyrosine phosphorylation of the EGFR and robust activation of the extracellular regulated kinase (ERK) cascade. However, siRNA targeting of β-arrestin 1, 2, or both abolished EGFR transactivation and ERK activation upon Dob/ICI stimulation. Moreover, in experiments using siRNA to knock down the expression of the ubiquitously expressed GRK isoforms 2, 3, 5, and 6, only siRNA targeting GRK 5 or 6 resulted in inhibition of EGFR transactivation and attenuated ERK activation, demonstrating the preferential requirement of GRK 5 and 6 for EGFR transactivation. To determine in vivo whether transactivation of EGFR by β1ARs requires β-arrestin and GRK5 and/or 6, we administered Dob following ICI pretreatment to knockout (KO) mice lacking either the β-arrestin 2, GRK5, or GRK6 gene. EGFR immunoprecipitated from myocardial lysates was immunoblotted for phosphotyrosine and western blots were carried out for ERK activation. Consistently, Dob-mediated EGFR phosphorylation and ERK activation were completely blocked in β-arrestin 2, GRK5, and GRK6 KO mice. In conclusion, these findings show the essential requirement for both β-arrestin and GRK5/6 in β1AR-mediated transactivation of the EGFR. This newly defined β1AR signaling pathway is likely important for the activation of growth and cell survival cascades in the heart.