Abstract
Signaling pathways of many G protein-coupled receptors overlap with those of receptor tyrosine kinases. We have found previously that α1-adrenergic receptors stimulate DNA synthesis and cell proliferation in human vascular smooth muscle cells; these effects were attenuated by the tyrosine protein kinase (TPK) inhibitor genistein and the mitogen-activated protein kinase (MAPK) antagonist 2-aminopurine. Experiments were designed to determine if activation of α1 receptors directly stimulated TPKs and MAPKs in human vascular smooth muscle cells. Norepinephrine stimulated time- and concentration-dependent tyrosine phosphorylation of multiple proteins, including p52-, 75-, 85-, 120-, and 145-kDa proteins. Increased TPK activity was demonstrated in proteins precipitated by an antiphosphotyrosine antibody, both in autophosphorylation assays and with a peptide substrate. These effects of norepinephrine were completely blocked by α1 receptor antagonists. A membrane-permeable Ca2+ chelator [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester], completely blocked norepinephrine stimulation of phosphorylation of tyrosine proteins, suggesting that intracellular Ca2+ plays a critical role in α1 receptor stimulation phosphorylation of tyrosine proteins. Of the tyrosine-phosphorylated proteins, the results suggest that two of them are PLCγ1 and adapter protein Shc. Also, α1 receptor stimulation caused a time-dependent increase in MAPK activity due to increased phosphorylation of p42/44ERK1/2. The α1 receptor-mediated activation of MAPK was also attenuated by TPK inhibitors and intracellular Ca2+ chelator [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester]. These results suggest that phosphorylation of tyrosine proteins and intracellular Ca2+plays a critical role in α1 receptor-stimulated MAPK signaling pathways, potentially contributing to increased DNA synthesis and cell proliferation.
Footnotes
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Send reprint requests to: Dr. Zhuo-Wei Hu, M.D., Ph.D., Veterans Affairs Palo Alto Health Care System, GRECC 182B, 3801 Miranda Ave., Palo Alto, CA 94304. E-mail: huzhwei{at}leland.stanford.edu
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↵1 This work was supported in part by National Institutes of Health Grant HL41315 and a Grant-in-Aid from the American Heart Association, California Affiliate. R.Z.L. and J.C. were supported by a Pharmaceutical Research and Manufacturers of America Foundation Fellowship for Careers in Clinical Pharmacology during the course of this work.
- Abbreviations:
- 2-AP
- 2-aminopurine
- AR
- adrenergic receptor
- p125FAK
- focal adhesion kinase
- IGF-I
- insulin-like growth factor I
- IP3
- inositol-1,4,5-triphosphate
- MAPK
- mitogen-activated protein kinase
- MBP
- myelin basic protein
- PDBu
- 4β-phorbol 12,13-dibutyrate
- PDGF-BB
- platelet-derived growth factor BB
- PI-3 kinase
- phosphatidylinositol 3-kinase
- PKC
- protein kinase C
- PYK2
- proline-rich tyrosine kinase 2
- RTK
- receptor tyrosine kinase
- TPK
- tyrosine protein kinase
- VSMC
- vascular smooth muscle cell
- HVSMC
- human vascular smooth muscle cell
- PAGE
- polyacrylamide gel electrophoresis
- GPCR
- G protein-coupled receptor
- DMEM
- Dulbecco’s modified Eagle’s medium
- BAPTA-AM
- [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester]
- Received September 10, 1998.
- Accepted March 18, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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