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.

Stimulation of the beta(2)-adrenergic receptor (beta(2)AR) 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 beta(2)AR 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 beta(2)AR and in HEK293 cells stably overexpressing either the wild-type beta(2)AR or a substitution mutant beta(2)AR (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 beta(2)AR 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 WTbeta(2)AR and PKA(-) (2-4 pmol of beta(2)AR/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 WTbeta(2)AR 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 muM) caused a >90% inhibition of epinephrine or forskolin activation of ERK1/2 in both cell lines. Our results indicate that the dominant mechanism of beta(2)AR activation of ERK1/2 does not require PKA phosphorylation of the beta(2)AR, 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.