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
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
"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.