Enhanced Tumorigenic Behavior of Glioblastoma Cells Expressing a Truncated Epidermal Growth Factor Receptor Is Mediated through the Ras-Shc-Grb2 Pathway

doi:10.1074/jbc.271.41.25639

Enhanced Tumorigenic Behavior of Glioblastoma Cells Expressing a Truncated Epidermal Growth Factor Receptor Is Mediated through the Ras-Shc-Grb2 Pathway*

From the ^¶ Ludwig Institute for Cancer Research, the
^‴ Department of Medicine, and
^∥ Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0660, the
^‡ University of California, San Diego Cancer Center, and
^″ Departments of Medicine and Pharmacology, University of California, San Diego, La Jolla, California 92093-0684, and the
^∥ Department of Medicine, University of California, San Diego, La Jolla, California 92093-0652

^# To whom correspondence should be addressed. Tel.: 619-534-7814; Fax: 619-534-7750; E-mail: hhuang{at}ucsd.edu

Abstract

A mutant epidermal growth factor receptor (ΔEGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG.ΔEGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated ΔEGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTP-bound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.ΔEGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of ΔEGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins.

Footnotes

↵^§ Recipient of a NATO postdoctoral fellowship.
↵* This work was supported in part by National Institutes of Health Grant GM49360 (to G. R. B.) and the Tobacco related Disease Research Program (to J. R. F.). The microinjection core is supported by grants from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

BrdUrd

5-bromodeoxyuridine

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine

PTB

phosphotyrosine-binding domain

GST

glutathione S-transferase.
↵² E. Stockert, L. J. Old, W. K. Cavenee, and H.-J. S. Huang, unpublished data.
↵³ H.-J. Su Huang, M. Nagane, C. K. Klingbeil, H. Lin, R. Nishikawa, X.-D. Ji, C.-M. Haung, G.-N. Gill, H. S. Wiley, and W. K. Cavenee, unpublished data.
- Received May 15, 1996.
- Revision received July 23, 1996.