Abstract
The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, also known as ErbB or HER, plays crucial roles in the development of multicellular organisms. Mutations and over-expression of the ErbB receptors have been implicated in a variety of human cancers. It is widely thought that the ErbB receptors are located in the plasma membrane, and that ligand binding to the monomeric form of the receptors induces its dimeric form for activation. However, it still remains controversial whether prior to ligand binding the receptors exist as monomers or dimers on the cell surface. Using bimolecular fluorescence complementation (BiFC) assays in the present study, we demonstrate that in the absence of bound ligand, all the ErbB family members have preformed, yet inactive, homo- and heterodimers on the cell surface, except for ErbB3 homodimers and heterodimers with cleavable ErbB4, which exist primarily in the nucleus. BiFC assays of the dimerization have also suggested that the ligand-independent dimerization of the ErbB receptors occurs in the endoplasmic reticulum (ER) before newly synthesized receptor molecules reach the cell surface. Based on BiFC and mammalian two-hybrid assays, it is apparent that the intracellular domains of the receptors are responsible for the spontaneous dimer formation. These provide new insights into an understanding of transmembrane signal transduction mediated by the ErbB family members, and are relevant to the development of anti-cancer drugs.
Highlights
Receptor tyrosine kinases (RTKs) mediate a variety of cellular responses in normal biological processes and in pathological states (Hunter, 2000; Schlessinger, 2000)
When plasmid constructs encoding fluorescent protein (FP)-fused receptors were transfected into Chinese hamster ovary (CHO) cells, epidermal growth factor receptor (EGFR)-CFP, ErbB2-YFP and ErbB4-JMa-GFP were expressed on the cell surface as expected
Co-expression of FP-fused receptors in a pair-wise manner in CHO cells revealed that all the receptors except for coexpressed ErbB3-CFP and ErbB4-JMa-YFP were produced on the cell surface (Fig. 1B; supplementary material Fig. S2)
Summary
Receptor tyrosine kinases (RTKs) mediate a variety of cellular responses in normal biological processes and in pathological states (Hunter, 2000; Schlessinger, 2000). The epidermal growth factor receptor family belongs to subclass I RTKs and consists of four members; EGFR ( known as ErbB1 or HER1), ErbB2 (Neu or HER2), ErbB3 (HER3) and ErbB4 (HER4) (Yarden and Sliwkowski, 2001; Olayioye et al, 2000). The ErbB receptor contains an extracellular ligand-binding domain, a single-pass transmembrane α-helix and an intracellular domain that encodes a tyrosine kinase followed by a regulatory region (Ullrich et al, 1984; Warren and Landgraf, 2006). The four ErbB family members recognize at least 11 different but structurally related growth factors including epidermal growth factor (EGF), transforming growth factor α (TGF-α) and neuregulins (NRGs) (Citri and Yarden, 2006; Linggi and Carpenter, 2006a). ErbB4 consists of two pairs of naturally occurring isoforms differing in their juxtamembrane domain (JMa or JMb) and C termini (cyt or cyt). An isoform pair is characterized by alternative splicing of exons located in the extracellular juxtamembrane region, conferring the JMa isoforms with susceptibility to proteolytic cleavage (Junttila et al, 2000; Gambarotta et al, 2004)
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