This project will combine resources of three laboratories to study molecular details of the interaction of epidermal growth factor (EGF) and its receptor. Among the large class of receptors with intrinsic protein kinase activity, the EGF / EGF receptor system is one of the most intensively studied members, both because of its importance in regulating cellular proliferation and because it serves as a model for this growing class. Nonetheless, fundamental questions concerning the mechanisms of signal transduction triggered by EGF / EGF receptor interactions remain unresolved. The studies proposed will be directed toward developing an in-depth understanding of some of the earliest events that occur when EGF binds to its plasma membrane receptor. Site-directed mutagenesis of EGF will be employed to engineer unique reaction sites for spin-labels and optical probes at selected locations in the known three-dimensional structure of the hormone. These labelled EGF's will be employed in EPR and fluorescence/phosphore scence spectroscopic investigations of the rotational dynamics of the occupied receptor complex. These studies will address the hypothesis that the subpopulation of receptors which bind EGF with high affinity interacts with the cytoskeleton. New spin-labeled and phosphorescent affinity probes for the ATP binding site of the kinase domain of the receptor will be synthesized and employed in EPR and phosphorescence investigations, respectively, of the rotational species of the receptor which exist prior to EGF binding and at various stages of saturation. These studies will address the alternative hypothesis that high affinity receptors are a subpopulation that are dimeric in the absence of ligand. Fluorescence resonance energy transfer will be employed to address the fundamental question of the number of EGF's bound per activated receptor dimer. Stopped flow fluorescence anisotropy experiments on A431 cells of EGF association / dissociation will directly measure relative populations and on- and off-rates asocciated with high and low affinity classes of receptors. The kinetic effects of phorbol esters of EGF binding will be determined and correlated with rotational populations of receptors observed using EPR and phosphorescence anisotropy decay.
