Stabilization of an active dimeric form of the epidermal growth factor receptor by introduction of an inter-receptor disulfide bond.

Populations of the epidermal growth factor receptor (EGFR) with both high and low affinity for EGF are found on the surface of cells. It has been hypothesized that the high-affinity state of the EGFR represents the receptor dimer and that this is also the kinase-active form. We describe here studies aimed at addressing this question directly. To stabilize homodimers of EGFR, we have generated a mutated form of the receptor by inserting a cysteine residue in the extracellular juxtamembranous region, in order to cross-link the extracellular domains of two receptors via disulfide bond formation. The mutation resulted in ligand-induced appearance of covalently linked EGF receptor dimers and, in parallel, increased the number of high-affinity receptors present on the surface of cells expressing the mutated EGFR. Comparison of the tyrosine kinase activity of the covalently linked dimeric and the monomeric forms of the EGF receptor, separated by sucrose density gradient centrifugation, showed that the dimer was significantly more active than monomer in the phosphorylation of exogenous substrate. We conclude that the dimeric form of the EGF-receptor represents the active form, and that dimer formation is associated with the appearance of high-affinity binding EGF receptors on the cell surface.