Epidermal Growth Factor Receptor Transactivation Mediates Substance P-induced Mitogenic Responses in U-373 MG Cells

Ignazio Castagliuolo,Leyla Valenick,Jennifer Liu,Charalabos Pothoulakis

doi:10.1074/jbc.m003990200

Ignazio Castagliuolo, Leyla Valenick + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m003990200

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Abstract

Ligand-induced activation of G protein-coupled receptors is emerging as an important pathway leading to the activation of certain receptors with intrinsic tyrosine kinase activity, such as the epidermal growth factor receptor (EGFR). Substance P (SP) exerts many effects via activation of its G protein-coupled receptor (neurokinin-1, NK-1). SP participates in acute inflammation and activates key proteins involved in mitogenic pathways, such mitogen-activated protein kinases (MAPKs), stimulating DNA synthesis. We tested the hypothesis that SP-induced MAPK activation and DNA synthesis require activation of the EGFR. In U-373 MG cells, which express functional NK-1, SP induced tyrosine phosphorylation of several proteins including EGFR. SP induced formation of an activated EGFR complex containing the adapter proteins SHC and Grb2, but not c-Src. SP activated the MAPK pathway as shown by increased Erk2 kinase activity. SP induced Erk2 activation, and DNA synthesis was inhibited in cells transfected with a dominant negative EGFR plasmid lacking kinase activity, as well as in cells treated with a specific EGFR inhibitor. In addition, pertussis toxin, an inhibitor of Galpha(iota) protein subunits, prevented SP-induced EGFR transactivation and subsequent DNA synthesis. Our results implicate EGFR as an essential regulator in SP/NK-1-induced activation of the MAPK pathway and cell proliferation in U-373 MG cells, and these events are mediated by a pertussis toxin-sensitive Galpha protein. We suggest that this mechanism by which SP controls cell proliferation is an important pathway in tissue restoration and healing.

Highlights

Substance P (SP),1 an 11-amino acid peptide member of the tachykinin family, is distributed widely in the central and peripheral nervous systems [1]
SP participates in acute inflammation and activates key proteins involved in mitogenic pathways, such mitogen-activated protein kinases (MAPKs), stimulating DNA synthesis
We demonstrate that SPinduced MAPK activation and cell proliferation require the presence of a functional epidermal growth factor receptor (EGFR) kinase domain and involve a pertussis toxin (PTX)-sensitive G protein

Summary

Introduction

Substance P (SP), an 11-amino acid peptide member of the tachykinin family, is distributed widely in the central and peripheral nervous systems [1]. SP binding to SPR and consequent activation of G protein subunits results in the activation of a variety of second messengers, including inositol3-phosphate kinase resulting in hydrolysis of phosphoinositides, cAMP formation [24], and arachidonic acid release [25]. Mitogenic pathways are tightly regulated by a variety of molecular events, most notably the phosphorylation/dephosphorylation of tyrosine residues on several key effector proteins Growth factors, such EGF and platelet-derived growth factor, bind to membrane receptors with intrinsic tyrosine kinase activity, resulting in autophosphorylation of the receptor. Once activated, such receptor tyrosine kinases (RTKs) recruit adapter proteins to the cell plasma membrane, forming an activated complex that initiates a signaling cascade leading to nuclear translocation of transcription factors and cell proliferation [26] Mitogenic mediators such as lysophosphatidic acid [27], thrombin [27], angiotensin II [28], and SP. Angiotensin II [30] and thrombin [31] stimulate phosphorylation of different growth factor RTKs, suggesting that transactivation of distinct RTKs may contribute to GPCR-mediated mitogenic signaling

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