Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

doi:10.1073/pnas.131200398

Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

^*Laboratory of Molecular Biology and Biochemistry, ^‡Howard Hughes Medical Institute, ^†The Rockefeller University, New York, NY 10021

Contributed by R. B. Merrifield

Abstract

We prepared a stable cell line expressing the glucagon receptor to characterize the effect of G_s-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation.

Footnotes

↵ § To whom reprint requests should be addressed at: Box 284, The Rockefeller University, 1230 York Avenue, New York, NY 10021. E-mail: sakmar{at}mail.rockefeller.edu.
Abbreviations:

AC,

adenylyl cyclase;

BAPTA/AM,

1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate/acetoxymethyl ester;

[Ca2+]i,

intracellular calcium concentration;

EGF,

epidermal growth factor;

ERK1/2,

extracellular signal-regulated protein kinase 1/2;

GPCR,

G protein-coupled receptor;

GR,

glucagon receptor;

GST,

glutathione S-transferase;

HEK,

human embryonic kidney;

MAP,

mitogen-activated protein;

MEK,

MAP kinase kinase/ERK kinase;

PKA,

cAMP-dependent protein kinase;

PKC,

protein kinase C;

PLC-β,

phospholipase C-β