REAL-TIME MEASUREMENTS OF KINETICS OF EGF BINDING TO SOLUBLE EGF RECEPTOR MONOMERS AND DIMERS SUPPORT THE DIMERIZATION MODEL FOR RECEPTOR ACTIVATION

We have tested one aspect of the allosteric dimerization model for the activation of EGF receptor (EGFR) by EGF: whether EGF binding favors dimerization of the receptor. For this to be true, EGF molecules must bind with higher affinity to dimeric receptors than to monomeric recep tors. We have tested this directly in a defined system using the solub le, extracellular ligand binding domain of EGFR monomers (sEGFR) and s EGFR dimers stabilized by treatment with a covalent cross-linking agen t. We describe real-time kinetic measurements of EGF binding to recept or monomers and dimers employing the method of total internal reflecti on (surface plasmon resonance). Our data show that sEGFR dimers bound EGF with 30-40-fold higher affinity [K(D) = (2-3) X 10(-8) M] than did sEGFR monomers. The enhanced binding affinity of sEGFR dimers resulte d mainly from a reduced off-rate with k(off) = 0.001 s-1 for sEGFR dim ers as compared to k(off) = 0.06 s-1 for sEGFR monomers. These measure ments indicate that dimerization of sEGFR increases its affinity for E GF by prolonging the amount of time that EGF remains bound to the rece ptor. This provides evidence that EGF binding stabilizes receptor dime rization and provides further support for the allosteric dimerization model as a mechanism for ligand induced receptor activation.