Epidermal Growth Factor (EGF) Receptor-dependent ERK Activation by G Protein-coupled Receptors

• Published in: The Journal of biological chemistry Vol. 276; no. 25; pp. 23155 - 23160
• Main Authors: Pierce, Kristen L., Tohgo, Akira, Ahn, Seungkirl, Field, Michael E., Luttrell, Louis M., Lefkowitz, Robert J.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 22.06.2001; American Society for Biochemistry and Molecular Biology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M101303200

“Transactivation” of epidermal growth factor receptors (EGFRs) in response to activation of many G protein-coupled receptors (GPCRs) involves autocrine/paracrine shedding of heparin-binding EGF (HB-EGF). HB-EGF shedding involves proteolytic cleavage of a membrane-anchored precursor by incompletely characterized matrix metalloproteases. In COS-7 cells, α2A-adrenergic 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. We have developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells. In this system, α2AAR expressing “donor” cells are co-cultured with “acceptor” cells lacking the α2AAR. Each population expresses a uniquely epitope-tagged ERK2 protein, allowing the selective measurement of ERK activation in the donor and acceptor cells. Stimulation with the α2AR selective agonist UK14304 rapidly increases 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 α2AAR-expressing donor cells. Experiments with various chemical inhibitors and dominant inhibitory mutants demonstrate that EGFR-dependent activation of the ERK cascade after α2AAR stimulation requires Gβγ subunits upstream and dynamin-dependent endocytosis downstream of HB-EGF shedding and EGFR activation, whereas Src kinase activity is required both for the release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.