This project will take advantage of a unique combination of resources in biological spectroscopy available at Vanderbilt to probe structural and kinetic questions related to the mechanism of transmembrane signaling by the EGF-EGF receptor complex. An array of spectroscopic approaches including linear and saturation transfer electron paramagnetic resonance (EPR) spectroscopy, fluorescence energy-transfer and fluorescence and phosphorescence anisotropy decay will be employed in experiments utilizing wild type murine EGF and site-directed mutants of mEGF derivitized with appropriate spectroscopic probes. For this study, specific site-directed mutants of mEGF designed for spectroscopic studies will be prepared and characterized, and new spectroscopic probes will be synthesized. These molecular tools will be applied to three distinguishable areas of study: 1) Fluorescence energy transfer will be used to measure the distance between EGF molecules bound in the occupied receptor dimer. By employing site-directed mutants of mEGF derivitized at different points in the molecule, the spatial relationship of the two molecules in adjacent subunits of the receptor dimer will be mapped. 2) Kinetics of dissociation of EGF from the receptor, of ligand exchange, and of exchange of occupied receptor monomers in the occupied receptor dimer complex will be investigated using EPR and fluorescence methods. EPR will be used to extend our kinetic studies of EGF dissociation from the occupied receptor in vitro to investigate the effects of dissociation kinetics of receptor autophosphorylation, of phosphorylation of the receptor by protein kinase C, and of the interaction of the receptor with phospholipase Cgamma or domains thereof. EPR methods will also be applied to investigate whether EGF is released from the receptor following endocytosis. Fluorescence methods will be applied to investigation of the kinetics of the exchange of free EGF with EGF bound to the occupied receptor dimer and the kinetics of exchange of occupied receptor monomers between occupied receptor dimers. 3) The rotational diffusion of the occupied EGF receptor in membrane preparations and in intact cells will be investigated using saturation transfer EPR and/or phosphorescence anisotropy decay measurements.
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