Project Summary Growth factor (GF)-based therapies hold great promise for tissue engineering, cancer treatment, and regenera- tive medicine but controlling their activity and selectivity can be challenging. GFs act as ligands for membrane- receptors controlling signaling cascades that drive gene expression and cellular functions, such as proliferation and differentiation. Tools that can selectively activate or suppress GF-mediated signaling activity in cells are needed to achieve control over the activity of these molecules and improve their therapeutic properties. Heparan sulfate (HS) proteoglycans (PGs), while often overlooked, are uniquely suited for this purpose, as they often serve as coreceptors for GFs at the cell surface. By promoting the formation of complexes between GFs and their receptors, they balance competing signaling pathways and regulate cellular responses. While the structure and activity of HS on cells can be controlled, to some extent, through genetic engineering of their biosynthesis, chemical tools for remodeling cell-sulfate HS to attain GF-binding specificity would be much mor general and better suited for therapeutic applications. This project establishes such tools, termed neoPG chimeras, that will be able to selectively activate or inhibit GF signaling activity in cells. This will be achieved by taking advantage of the GF-binding selectivities of recombinant HS polysaccharides produced through systematic mutation of HS biosynthetic enzymes in laboratory cell lines. The recombinant HS polysaccharides will be harvested, character- ized for GF binding specificity and merged with functional elements for targeting to the cells. Membrane targeting neoPG chimeras will be developed to promote GF association with receptors at the cell surface and promote GF signaling activity (Aim 1). Lysosome-targeting neoPG chimeras will be used to drive extracellular GFs into the cells for degradation, thus inhibiting signaling activity (Aim 2). The focus of this study will be on establishing and validating neoPG chimeras as actuators of signaling by members of the Fibroblast Growth Factor family of pro- teins in the context of cellular proliferation and differentiation. However, many other classes of GFs require cell surface HS for function and the new tools are expected to find broad application in many different aspects of biomedical research and GF-based therapies.