Heparin is essential for a single keratinocyte growth factor molecule to bind and form a complex with two molecules of the extracellular domain of its receptor

Keratinocyte growth factor (KGF or FGF-7) is a member of the heparin bindin g fibroblast growth factor (FGF) family and is a paracrine mediator of prol iferation and differentiation of a wide variety of epithelial cells. To exa mine the stoichiometry of complexes formed between KGF and its receptor, we have utilized a soluble variant of the extracellular region of the KGF rec eptor containing two tandem immunoglobulin-like loops, loops II and III (sK GFR). Ligand-receptor complexes were examined by size exclusion chromatogra phy, light scattering, N-terminal protein sequencing, and sedimentation vel ocity. In the presence of low-molecular mass heparin (similar to 3 kDa), we demonstrate the formation of complexes containing two molecules of sKGFR a nd one molecule of KGF. In the absence of heparin, we were unable to detect any KGF-sKGFR complexes using the above techniques, and additional studies in which sedimentation equilibrium was used show that the binding is very weak (K-d greater than or equal to 70 mu M). Furthermore, using heparin fra gments of defined size, we demonstrate that a heparin octamer or decamer ca n promote formation of a 2:1 complex, while a hexamer does not. Utilizing t he highly purified proteins and defined conditions described in this study, we find that heparin is obligatory for formation of a KGF-sKGFR complex. F inally, 32D cells, which appear to lack low-affinity FGF binding sites, wer e transfected with a KGFR-erythropoeitin receptor chimera and were found to require heparin to achieve maximal KGF stimulation. Our data are consisten t with the previously described concept that cell- or matrix-associated hep aran sulfate proteoglycans (HSPGs) and FGF ligands participate in a concert ed mechanism that facilitates FGFR dimerization and signal transduction in vivo.