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A systematic study using solid phase peptide synthesis has been undertaken to examine the role of the disulfide bonds in the structure and function of mEGF. A combination of one, two and three native disulfide pair analogues of an active truncated (4-48) form of mEGF have been synthesised by replacing specific cysteine residues with isosteric alpha-amino-n-butyric acid (Abu). Oxidation of the peptides was performed using either conventional aerobic oxidation at basic pH, in DMSO under acidic conditions or via selective disulfide formation using orthogonal protection of the cysteine pairs. The contribution of individual, or pairs of, disulfide bonds to EGF structure was evaluated by CD and H-1-NMR spectroscopy. The mitogenic activity of each analogue was determined using Balb/c 3T3 mouse fibroblasts. As we have reported previously (Barnham et al. 1998), the disulfide bond between residues 6 and 20 can be removed with significant retention of biological activity (EC50 20-50 nM). The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. We now show that removal of any other disulfide bond, either singly or in pairs, results in a major disruption of the tertiary structure, and a large loss of activity (EC50>900 nM). Remarkably, the linear analogue appears to have greater activity (EC50 580 nM) than most one and two disulfide bond analogues although it does not have a definable tertiary structure.

The epidermal growth factor receptor (EGFR) plays an important role in many types of human cancers. Receptor amplification, autocrine activation and/or deletion of exons 2-7 of EGFR gene have all been associated with tumor development. The traditional model of EGFR activation via ligand induced dimerization and consequential kinase activation does not provide full understanding of its tumorigenicity. The main function of the receptor extracellular domain (ECD) has been thought to be ligand recognition and binding. We report that the EGFR ECD, through its association also negatively regulates the activity of the intracellular kinase in the absence of ligand. Even in the absence of its ligands, the EGF receptor forms homodimers, however, the ECD prevents constitutive receptor kinase activation through its intrinsic ligand-independent interaction. The removal of this domain, either partial or total, results in constitutive activation of the receptor kinase as observed by its phosphorylation in intact cells. Furthermore, EGF receptors truncated in the ECD induce phosphorylation of the wild-type full-length receptor, indicating an inter-molecular inhibitory mechanism by the receptor ECD. The tumor associated delta2-7EGFR mutant also dimerizes with and phosphorylates the wild type EGFR in the absence of ligand. Thus, in addition to its role in ligand recognition, EGFR ECD interacts with each other, imposing an inhibitory effect on the activation of the intracellular kinase.