Functional analysis of the ligand binding site of EGF-receptor utilizing chimeric chicken/human receptor molecules.

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Abstract
The epidermal growth factor (EGF)‐receptor is composed of an extracellular ligand‐binding region connected by a single transmembrane region to the cytoplasmic kinase domain. In spite of its importance for understanding signal transduction, the ligand‐binding domain of the EGF‐receptor is not yet defined. We describe the identification of a major ligand‐binding domain of the EGF‐receptor by utilizing chimeras between the human EGF‐receptor and the chicken EGF‐receptor. This approach is based on the fact that murine EGF binds to the chicken EGF‐receptor with 100‐fold lower affinity as compared to the human EGF‐receptor. Hence, the substitution of various domains of the chicken EGF‐receptor by domains of the human EGF‐receptor may restore the higher binding affinity towards EGF, characteristic of the human receptor. We show that chimeric chicken/human EGF‐receptor, which contains domain III of the extracellular region of the human receptor, behaves like the human EGF‐receptor with respect to EGF binding affinity and biological responsiveness. However, a chimeric chicken/human EGF‐receptor containing domains I and II of the human receptor behaves like the chicken rather than the human EGF‐receptor. Moreover, two different monoclonal antibodies which compete for the binding of EGF to EGF‐receptor recognize specifically domain III of the human EGF‐receptor. It is concluded that domain III which is flanked by the two cysteine‐rich domains is a major ligand‐binding domain of the EGF‐receptor.