Visualizing the Effects of Engineered Chimeric Growth Factors on Receptor Distribution
EMSL Project ID
44662
Abstract
Members of the epidermal growth factor receptor (EGFR/HER/ErbB) family - EGFR, HER2, HER3, and HER4 - regulate diverse cell behaviors including survival, proliferation, differentiation and migration. Overwhelming evidence supports the concept that HER family members must homo- or heterodimerize (or oligomerize) to initiate intracellular signaling via receptor tyrosine kinase domains, and that ligand binding to at least one dimer member is required under most physiological circumstances. However, mechanisms of dimerization and activation remain debated. Recent evidence also suggests that EGFR family members interaction with other cell surface receptors, including other tyrosine kinase receptors and mucins, regulates their activity. Three family members - - EGFR, HER3 and HER4 -- each have multiple known monovalent ligands, which are often produced in autocrine fashion to regulate cellular homeostasis or responses to stress. Although HER2 lacks known ligands, it heterodimerizes with other family members to form potent signaling complexes. HER2 acts in symbiotic fashion with HER3, which can bind ligand but has only weak kinase activity of its own; together, they each contribute to a robust signaling partnership. Although multivalency is not a recognized feature of HER family ligands, it drives receptor dimerization or oligomerization in other receptor systems, such as CD95-FasL. In this study, we use a panel of bivalent ligands for EGFR family members to probe the interactions between family members and other cell surface receptors, testing a hypothesis that bivalent ligands can bias receptor interactions away from those which occur naturally when cells are stimulated by monovalent ligands. Such biasing of EGFR family receptor interactions by these engineered bivalent ligands may have therapeutic value in regenerative medicine and in targeted cancer therapy. To accomplish a bias in receptor interactions, we create a bivalent ligand by linking together two neuregulin-1b (NRG) moieties (ligand for HER3 and HER4), two epidermal growth factor (EGF) moieties (ligand for EGFR), or an EGF and a NRG moiety, via a protease-resistant spacer of sufficient length to allow the two ligands to bind sites in adjacent (dimerized) receptors. Such bivalent ligands may drive particular dimerization events between monomeric receptors; stabilize pre-existing dimers or higher order oligomers; or disrupt pre-existing unoccupied receptor interactions and drive new ones. Initial phenotypic studies have demonstrated that bivalent EE, EN, and NN ligands alter the migration properties of human mesenchymal stem cells compared to cells that are either unstimulated or stimulated with WT monvalent ligands. Further, bivalent NN ligands inhibit invasive properties and induce apoptsis in certain cancer cells, hence have potential therapeutic value. Experimental work at EMSL using the multiphoton FLIM instrument to visualize receptor distributions in the presence and absence of either wild type monovalent or engineered bivalent ligands, combined with computational modeling of ligand-receptor interactions, will illuminate the ways EGFR family receptors interact on the cell surface in unperturbed cells and in cells exposed to environmental and inflammatory stimuli. Importantly, the dynamic structural information will provide a critical foundation for improving the design of engineered proteins that hold promise for therapeutic applications in cancer and regenerative medicine.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2011-10-01
End Date
2012-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members