HER4 Receptor Activation and Phosphorylation of Shc Proteins by Recombinant Heregulin-Fc Fusion Proteins

doi:10.1074/jbc.270.21.12857

HER4 Receptor Activation and Phosphorylation of Shc Proteins by Recombinant Heregulin-Fc Fusion Proteins (*)

From the (1) Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121

^§ To whom correspondence should be addressed:
Bristol-Myers Squibb Pharmaceutical Research Inst., 3005 First Ave., Seattle, WA 98121.
Tel.: 206-727-3641; Fax: 206-727-3602.

Abstract

Heregulins (HRGs) are mosaic glycoproteins that bind to and induce the tyrosine phosphorylation of the HER4/p180 receptor. This work was aimed at studying the biological effects induced by recombinant epidermal growth factor (EGF)-like domains of HRGs as well as identifying intracellular molecules involved in HER4 signaling. To this end, we cloned the EGF-like domains of HRG-α, -β2, and -β3 into a eukaryotic expression vector in frame with sequences encoding a thrombin cleavage site followed by the Fc portion of a human IgG1. These chimeric genes directed the expression of recombinant fusion proteins, rHRGs-T-Fc, which specifically stimulated the phosphorylation of HER4/p180. We also show that rHRG-α-T-Fc bound to human breast cancer cells that express HER4 receptors and induced the expression of intercellular adhesion molecule-1. After thrombin protease cleavage of rHRGs-T-Fc, their EGF-like domains were purified and shown to stimulate protein phosphorylation in HER4-expressing cells. Moreover, the rHRG-β2 EGF-like domain markedly induced the phosphorylation of Shc proteins on tyrosine, suggesting a role for these adaptor molecules in HRG-mediated signaling.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

HRGs

heregulins (the prefix “r” stands for recombinant)

reHRGs

EGF-like domains of recombinant HRGs

NDF

neu differentiation factor

EGF

epidermal growth factor

EGFR

EGF receptor

HER

human EGF receptor

ICAM-1

intercellular adhesion molecule-1

SH2

Src homology 2

CHO

Chinese hamster ovary

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

Bes

N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid.
↵²A. Aruffo and D. Hollenbaugh, unpublished data.
↵³J.-M. Culouscou, unpublished data.