β2-Adrenergic Receptor Lacking the Cyclic AMP-Dependent Protein Kinase Consensus Sites Fully Activates Extracellular Signal-Regulated Kinase 1/2 in Human Embryonic Kidney 293 Cells: Lack of Evidence for Gs/Gi Switching.

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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 Gs to Gi 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 Gs to Gi but clearly requires activation of a Src family member that may be downstream of PKA.