TY - JOUR
T1 - Intracellular Transactivation of Epidermal Growth Factor Receptor by alpha(1A)-Adrenoceptor Is Mediated by Phosphatidylinositol 3-Kinase Independently of Activation of Extracellular Signal Regulated Kinases 1/2 and Serine-Threonine Kinases in Chinese Hamster Ovary Cells
AU - Ulu, Nadir
AU - Henning, Robert H.
AU - Guner, Sahika
AU - Zoto, Teuta
AU - Duman-Dalkilic, Basak
AU - Duin, Marry
AU - Gurdal, Hakan
PY - 2013/10
Y1 - 2013/10
N2 - Transactivation of epidermal growth factor receptor (EGFR) by alpha(1)-adrenoceptor (alpha(1)-AR) is implicated in contraction and hypertrophy of vascular smooth muscle (VSM). We examine whether all alpha(1)-AR subtypes transactivate EGFR and explore the mechanism of transactivation. Chinese hamster ovary (CHO) cells stably expressing one subtype of alpha(1)-AR were transiently transfected with EGFR. The transactivation mechanism was examined both by coexpression of a chimeric erythropoietin (EPO)-EGFR with an extracellular EPO and intracellular EGFR domain, and by pharmacologic inhibition of external and internal signaling routes. All three alpha(1)-AR subtypes transactivated EGFR, which was dependent on the increase in intracellular calcium. The EGFR kinase inhibitor AG1478 [4-(3'-chloroanilino)-6,7-dimethoxyquinazoline] abrogated alpha(1A)-AR and alpha(1D)-AR induced phosphorylation of EGFR, but both the inhibition of matrix metalloproteinases by GM6001 [(R)-N4-hydroxy-N-1-[(S)-2-(1H-indol-3-yl)-1-methylcarbamoyl-ethyl]-2-isobutyl-succinamide] or blockade of EGFR by cetuximab did not. Stimulation of alpha(1A)-AR and alpha(1D)-AR also induced phosphorylation of EPO-EGFR chimeric receptors. Moreover, alpha(1A)-AR stimulation enhanced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 and serine-threonine kinases (Akt), which were both unaffected by AG1478, indicating that ERK1/2 and Akt phosphorylation is independent of EGFR transactivation. Accordingly, inhibitors of ERK1/2 or Akt did not influence the alpha(1A)-AR-mediated EGFR transactivation. Inhibition of calcium/calmodulin-dependent kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K), and Src, however, did block EGFR transactivation by alpha(1A)-AR and alpha(1D)-AR. These findings demonstrate that all alpha(1)-AR subtypes transactivate EGFR, which is dependent on an intracellular signaling route involving an increase in calcium and activation of CaMKII, PI3K, and Src, but not the of ERK1/2 and Akt pathways.
AB - Transactivation of epidermal growth factor receptor (EGFR) by alpha(1)-adrenoceptor (alpha(1)-AR) is implicated in contraction and hypertrophy of vascular smooth muscle (VSM). We examine whether all alpha(1)-AR subtypes transactivate EGFR and explore the mechanism of transactivation. Chinese hamster ovary (CHO) cells stably expressing one subtype of alpha(1)-AR were transiently transfected with EGFR. The transactivation mechanism was examined both by coexpression of a chimeric erythropoietin (EPO)-EGFR with an extracellular EPO and intracellular EGFR domain, and by pharmacologic inhibition of external and internal signaling routes. All three alpha(1)-AR subtypes transactivated EGFR, which was dependent on the increase in intracellular calcium. The EGFR kinase inhibitor AG1478 [4-(3'-chloroanilino)-6,7-dimethoxyquinazoline] abrogated alpha(1A)-AR and alpha(1D)-AR induced phosphorylation of EGFR, but both the inhibition of matrix metalloproteinases by GM6001 [(R)-N4-hydroxy-N-1-[(S)-2-(1H-indol-3-yl)-1-methylcarbamoyl-ethyl]-2-isobutyl-succinamide] or blockade of EGFR by cetuximab did not. Stimulation of alpha(1A)-AR and alpha(1D)-AR also induced phosphorylation of EPO-EGFR chimeric receptors. Moreover, alpha(1A)-AR stimulation enhanced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 and serine-threonine kinases (Akt), which were both unaffected by AG1478, indicating that ERK1/2 and Akt phosphorylation is independent of EGFR transactivation. Accordingly, inhibitors of ERK1/2 or Akt did not influence the alpha(1A)-AR-mediated EGFR transactivation. Inhibition of calcium/calmodulin-dependent kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K), and Src, however, did block EGFR transactivation by alpha(1A)-AR and alpha(1D)-AR. These findings demonstrate that all alpha(1)-AR subtypes transactivate EGFR, which is dependent on an intracellular signaling route involving an increase in calcium and activation of CaMKII, PI3K, and Src, but not the of ERK1/2 and Akt pathways.
KW - VASCULAR SMOOTH-MUSCLE
KW - PROTEIN-COUPLED RECEPTORS
KW - CARDIAC-HYPERTROPHY
KW - CALMODULIN
KW - EGFR
KW - SRC
KW - CALCIUM/CALMODULIN
KW - PHARMACOLOGY
KW - ASSOCIATION
KW - PATHWAYS
U2 - 10.1124/jpet.113.206243
DO - 10.1124/jpet.113.206243
M3 - Article
SN - 0022-3565
VL - 347
SP - 47
EP - 56
JO - Journal of Pharmacology and Experimental Therapeutics
JF - Journal of Pharmacology and Experimental Therapeutics
IS - 1
ER -