Receptor tyrosine kinases (RTKs) from the epidermal growth factor (EGF) receptor, or ErbB, family have been implicated in a number of different human cancers. The EGF receptor itself (EGFR) and its relative ErbB2/HER2/Neu are now well-validated targets of approved therapies used in breast, lung, colorectal, and other cancers. Although these agents represent a significant advance in the fight against cancer, their modest efficacy, plus problems with resistance, indicate that they are not optimized. Consequently, there is significant interest in the development of additional ErbB receptor-targeted therapeutic agents that can be used alongside (or instead of) the existing drugs. For this reason, a detailed understanding of the regulation of the EGF receptor family is biomedically significant. Structural studies of isolated ErbB receptor extracellular and intracellular regions have yielded quite a sophisticated view of elements of the ErbB receptor activation process. However, the resulting models fail to explain several key, and long-established, properties of the cell surface receptors in their native membranes. Current structural models cannot explain the apparent existence of two affinity classes of EGF receptors on the cell surface, or negative cooperativity in EGF binding. Moreover, these models give no insight into the nature of the pre-formed dimers of EGFR and other ErbB receptors that have been observed using a wide variety of methods. In comparative studies of EGFR extracellular regions from different organisms, we have recently obtained insights into the likely structural nature of pre-formed EGF receptors. We have also seen evidence for communication between EGF binding sites in asymmetric receptor dimers of the sort that would be expected in a negatively cooperative system. To understand these processes, we propose to combine experiments on intact receptors at the cell surface with studies of the corresponding isolated extracellular regions, and structural analyses. By comparing ligand binding and dimerization properties in solution and at cell membranes, our goal is to bridge the current gulf between the structural information about EGFR family members and our understanding of their binding and signaling properties in cells. Our Specific Aims are: A To test the hypothesis that pre-formed dimers of untethered EGFR molecules are required for negative cooperativity in ligand binding B To test the hypothesis that `low-affinity' EGFR ligands occupy only one class of receptor binding site, and that this defines their signaling characteristics C To test the hypothesis that ErbB2-containing heterodimers resemble an asymmetric high-affinity EGF/EGFR complex. PUBLIC HEALTH RELEVANCE: Cell-surface growth factor receptors from the EGF receptor (or ErbB) family are involved in many human diseases from cancers to heart disease to schizophrenia, and were the first molecules to be specifically targeted with current-generation cancer therapeutics (e.g. Herceptin, Erbitux and Iressa). Many profound gaps still exist in our knowledge of how these receptors function: for example, it is not clear how a single receptor from this family is capable of eliciting different cellular responses upon stimulation by its several distinct ligands. Our studies suggest detailed explanations for this phenomenon, which should be exploitable for specifically blocking certain responses when they go awry in particular diseases (such as cancers and schizophrenia). We anticipate that the mechanistic insight provided by our proposed research will stimulate work towards a new generation of more sophisticated ErbB receptor-targeted therapeutics.