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Vol. 62, Issue 5, 1094-1102, November 2002
2-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.
Department of Integrative Biology and Pharmacology, the
University of Texas Health Science Center at Houston Medical School,
Houston, Texas
Stimulation of the 
2-adrenergic receptor
(2AR) 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 2AR 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 2AR and in HEK293 cells stably overexpressing either the wild-type 2AR or a substitution mutant
2AR (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 2AR 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 WT2AR and PKA (2-4 pmol of
2AR/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 WT2AR 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 2AR activation of ERK1/2 does not
require PKA phosphorylation of the 2AR, 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.
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