Abstract

Stimulation of the β₂-adrenergic receptor (β₂AR) in human embryonic kidney (HEK) 293 cells causes a transient activation of Extracellular Signal-Regulated Kinase (ERK) 1/2. One of the mechanisms proposed for this activation is a PKA-mediated phosphorylation of the β₂AR that switches receptor coupling from G_s to G_i and triggers internalization of the receptor. To examine these phenomena, we characterized agonist activation of ERK1/2 in HEK293 cells by the endogenous β₂AR and in HEK293 cells stably overexpressing either the wild-type β₂AR or a substitution mutant β₂AR (PKA⁻) that lacks the cyclic AMP-dependent protein kinase (PKA) consensus phosphorylation sites (S261A, S262A and S345A, S346A). As the baseline, we established that epinephrine stimulation of the endogenous β₂AR in HEK293 cells (20–30 fmol/mg) caused a rapid and transient activation of ERK1/2 with an EC₅₀ of 5 to 6 nM. In contrast, the potency of epinephrine stimulation of ERK1/2 in cells stably overexpressing WTβ₂AR and PKA⁻ (2–4 pmol of β₂AR/mg) was increased by over 100-fold relative to HEK293 cells, the EC₅₀ values being 20 to 60 pM. The nearly identical 100-fold shift in EC₅₀ for ERK1/2 activation in the PKA⁻ and WTβ₂AR relative to that in the HEK293 showed that the PKA⁻ are fully capable of activating ERK1/2. We also found maximal activation of ERK1/2 in the overexpressing cell lines at concentrations of epinephrine that cause no internalization (i.e., the EC₅₀ for internalization was 75 nM). Pertussis toxin pretreatment caused only a weak inhibition of epinephrine activation of ERK1/2 in the HEK293 (7–16%) and no inhibition in the PKA⁻ cells. Finally we found that the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (10 μM) caused a >90% inhibition of epinephrine or forskolin activation of ERK1/2 in both cell lines. Our results indicate that the dominant mechanism of β₂AR activation of ERK1/2 does not require PKA phosphorylation of the β₂AR, receptor internalization or switching from activation of G_s to G_i but clearly requires activation of a Src family member that may be downstream of PKA.