Signaling Mechanisms and Mobility of the EGF Receptor

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9304393 Gross This project focuses on the regulation of binding of epidermal growth factor (EGF) by its receptor (EGF-R). The time course of binding of a fluorescent derivative of EGF (f-EGF) to the EGF-R will be studied at the level of single cells by the use of quantitative optical microscopy. Preliminary data indicates that the binding of f-EGF to cultured A431 cells proceeds over a biphasic time course that is not consistent with the commonly accepted model of EGF/EGF-R in which two EGF-R affinity classes are postulated to exist at fixed densities on the cell surface. A ternary complex model has been developed that is consistent with the individual cell binding data. It is proposed that actin/EGF-R interaction through specific binding domains modulates the affinity state of the receptor. Recent studies have shown that the EGF-R is an actin-binding protein. Microinjection of HL-33, a peptide that competes for the actin binding site with the EGF-R, will be used as a probe of this interaction. Preliminary f-EGF binding data to individual A431 cells also indicates that the kinetics of binding are variable between cells. The consequences of this variable timing and degree of primary signal input on subsequent individual cell responses will also be examined by quantitative, single-cell fluorescence imaging. A consequence of the predicted interaction of the EGF-R with actin is that some individual EGF-Rs will be immobilized in the plasma membrane, and that the immobile receptors will be preferentially of the high-affinity class. Measurement of the mobilities of individual EGF receptors on stimulated and unstimulated cells forms an important test of the actin-regulation model. Single receptors will be labeled by biotinylated anti- receptor Fab' fragments and fluorescent streptavidin-latex nanoparticles or streptavidin-colloidal gold particles as visualization probes. The lateral mobility of individual EGF-Rs will be followed before and during EGF challen ge, and will be examined in individual cells microinjected with the HL-33 competing peptide. Correlations between individual EGF receptor movements and the affinity state of the receptor will be examined. The lateral mobilities of individual EGF-Rs will be compared to those for individual LDL receptors. (The LDL receptor system is thus far the only one in which individual receptor movements have been described; in this system three classes of lateral movement were characterized including random, directed, and no mobility.) %%% The goal of this research is to better understand the molecular interactions between a cell surface protein that is known to regulate cell growth, the epidermal growth factor receptor, and the signal molecule that activates it, epidermal growth factor (EGF). The interaction of these two molecules on individual cultured cells will be studies using fluorescently labeled EGF and quantitative optical microscopy. The results of this study of the EGF receptor system will add much to knowledge about molecular details of the mechanism by which growth factors signal cells to divide. ***