EGF Receptor Activation and Interaction with ErbB Family Receptors

Washington University, Saint Louis MO

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Abstract

The EGF receptor is the first member of the ErbB family of growth factor receptor tyrosine kinases that[unreadable] also includes ErbB2, ErbB3 and ErbB4. Binding of EGF to its receptor induces the formation of EGF receptor[unreadable] homodimers, resulting in autophosphorylation of the EGF receptor. The EGF receptor is able to heterodimerize[unreadable] with other members of the ErbB family, with ErbB2 being the preferred heterodimerization partner. Recent[unreadable] data suggest that ErbB receptors may form heterotetramers and that activated oligomers may dissociate into[unreadable] monomers that re-dimerize with inactive monomers in a process referred to as secondary dimer formation.[unreadable] EGF shows heterogeneity in affinity when binding to its receptor. This observation has been attributed to[unreadable] the existence of two independent classes of sites. How these arise from the expression of a single EGF[unreadable] receptor protein has never been explained. Recently, we have shown that EGF binding can be described by[unreadable] a model that involves negative cooperativity in an aggregating system. In this model, the affinity of EGF for a[unreadable] monomer and an unoccupied dimer is similar. However, binding to the second site on an EGF receptor dimer[unreadable] is negatively cooperative. This model predicts that the negative cooperativity will lead to EGF-induced dissociation[unreadable] of the dimer at high concentrations of EGF, a prediction that we have confirmed using fluorescence[unreadable] correlation spectroscopy and enzyme complementation to examine EGF receptor oligomerization.[unreadable] Our long-term goal is to understand how ErbB receptors are activated and to develop a model for[unreadable] predicting the interactions among ErbB family members. Using our new model as the starting point for further[unreadable] investigations, the specific aims of this proposal are to: 1) Quantify the interaction of the EGF receptor and[unreadable] ErbB2 through ligand binding studies; 2) Determine whether EGF receptor-mediated activation of ErbB2[unreadable] occurs via the formation of a hetero-tetrameric complex and/or involves dissociation of dimeric receptor[unreadable] complexes; and, 3) Determine whether different EGF receptor ligands induce characteristic patterns of EGF[unreadable] receptor oligomers and whether this can be related to differences in downstream signaling.[unreadable] EGF equilibrium binding studies will be used to quantitatively characterize the interaction of the EGF[unreadable] receptor with ErbB2. Fluorescence correlation spectroscopy and brightness analysis along with enzyme[unreadable] complementation will be employed to assess the oligomerization state of EGFR/ErbB2 heteromeric complexes[unreadable] while chemically-induced dimer formation will be used to probe the activation mechanism of ErbB2 within these[unreadable] complexes. A combination of these methods will be applied to assess oligomerization of the EGF receptor[unreadable] elicited by three different agonists. Signaling stimulated by the three agonists will be assessed by measuring[unreadable] the activation of downstream pathways. Together, these data will characterize the interactions of the EGF[unreadable] receptor and ErbB2 allowing the development of a quantitative model for the activation of ErbB2 via the EGF[unreadable] receptor and provide insight into the role of ErbB family oligomers in the process of cell signaling.