Branching Tubulogenesis but Not Scatter of Madin-Darby Canine Kidney Cells Requires a Functional Grb2 Binding Site in the Met Receptor Tyrosine Kinase

doi:10.1074/jbc.271.36.22211

Branching Tubulogenesis but Not Scatter of Madin-Darby Canine Kidney Cells Requires a Functional Grb2 Binding Site in the Met Receptor Tyrosine Kinase*

From the Molecular Oncology Group, Royal Victoria Hospital, Departments of ^¶ Medicine,
** Oncology, and
^‡ Biochemistry, McGill University, Montreal, Quebec H3A 1A1, Canada

Abstract

Hepatocyte growth factor is a multifunctional cytokine that induces mitogenesis, motility, invasion, and branching tubulogenesis of several epithelial and endothelial cell lines in culture. The receptor for hepatocyte growth factor has been identified as the Met tyrosine kinase. To investigate the signaling pathways that are involved in these events, we have generated chimeric receptors containing the colony stimulating factor-1 receptor fused to the transmembrane and intracellular domains of the Met receptor. Madin-Darby canine kidney epithelial cells expressing the Met chimera dissociate scatter and form branching tubules in response to colony stimulating factor-1. From structure-function analyses, tyrosine residue 1356 within the carboxyl terminus of the Met receptor is critical for these events. The amino acid sequence downstream from tyrosine 1356 represents a consensus binding site for the Grb2 adaptor protein and forms a multisubstrate binding site for the p85 subunit of phosphatidylinositol 3-kinase, phospholipase Cγ, and the Shc adaptor protein. To distinguish which of these signaling pathways are required, we generated a mutant receptor that selectively fails to associate with the Grb2 adaptor protein. Cells expressing this mutant receptor scattered but were unable to form branching tubules, indicating that a Grb2 binding site in the Met receptor is critical for morphogenic responses.

Footnotes

↵^§ Recipient of a Royal Victoria Hospital Research Institute Studentship.
↵^∥ Recipient of a Steve Fonyo Research Studentship from the National Cancer Institute of Canada.
↵^‡‡ Senior scholar of National Cancer Institute of Canada. To whom reprint requests should be addressed. Tel.: 514-842-1231 (ext. 5834); Fax: 514-843-1478.
↵* This research was supported by operating grants from the National Cancer Institute of Canada and the Medical Research Council of Canada (to M. P.). 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:

HGF

hepatocyte growth factor

SF

scatter factor

CSF-1

colony stimulating factor-1

SH2

Src homology domain 2

PTB

phosphotyrosine binding domain

PLCγ

phospholipase Cγ

PI 3-kinase

phosphatidylinositol 3-kinase

MDCK

Madin-Darby canine kidney

PAGE

polyacrylamide gel electrophoresis

GST

glutathione S-transferase.
- Received March 22, 1996.
- Revision received June 10, 1996.