Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors - A co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding

Citation
Kl. Pierce et al., Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors - A co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding, J BIOL CHEM, 276(25), 2001, pp. 23155-23160
Citations number
25
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
0021-9258 → ACNP
Volume
276
Issue
25
Year of publication
2001
Pages
23155 - 23160
Database
ISI
SICI code
0021-9258(20010622)276:25<23155:EGF(RE>2.0.ZU;2-H
Abstract
"Transactivation" of epidermal growth factor receptors (EGFRs) in response to activation of many G protein-coupled receptors (GPCRs) involves autocrin e/ paracrine shedding of heparin binding EGF (HB-EGF). HB-EGF shedding invo lves proteolytic cleavage of a membrane-anchored precursor by incompletely characterized matrix metalloproteases. In COS-7 cells, alpha (2A)-adrenergi c receptors (ARs) stimulate ERK phosphorylation via two distinct pathways, a transactivation pathway that involves the release of HB EGF and the EGFR and an alternate pathway that is independent of both HB-EGF and the EGFR. W e have developed a mixed culture system to study the mechanism of GPCR-medi ated HB-EGF shedding in COS-7 cells. In this system, alpha (2A)AR expressin g "donor" cells are co-cultured with "acceptor" cells lacking the alpha (2A )AR. Each population expresses a uniquely epitope-tagged ERK2 protein, allo wing the selective measurement of ERK activation in the donor and acceptor cells. Stimulation with the alpha (2)AR selective agonist UK14304 rapidly i ncreases ERK2 phosphorylation in both the donor and the acceptor cells. The acceptor cell response is sensitive to inhibitors of both the EGFR and HB- EGF, indicating that it results from the release of HB EGF from the alpha ( 2A)AR-expressing donor cells. Experiments with various chemical inhibitors and dominant inhibitory mutants demonstrate that EGFR-dependent activation of the ERK cascade after alpha (2A)AR stimulation requires G beta gamma sub units upstream and dynamin dependent endocytosis downstream of HB-EGF shedd ing and EGFR activation, whereas Src kinase activity is required both for t he release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.