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 binding fibroblast growth factor (FGF) family and is a paracrine mediator of proliferation and differentiation of a wide variety of epithelial cells. To examine the stoichiometry of complexes formed between KGF and its receptor, we have utilized a soluble variant of the extracellular region of the KGF receptor containing two tandem immunoglobulin-like loops, loops II and III (sKGFR). Ligand-receptor complexes were examined by size exclusion chromatography, light scattering, N-terminal protein sequencing, and sedimentation velocity. In the presence of low-molecular mass heparin ( approximately 3 kDa), we demonstrate the formation of complexes containing two molecules of sKGFR and 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 (Kd >/= 70 microM). Furthermore, using heparin fragments of defined size, we demonstrate that a heparin octamer or decamer can promote formation of a 2:1 complex, while a hexamer does not. Utilizing the highly purified proteins and defined conditions described in this study, we find that heparin is obligatory for formation of a KGF-sKGFR complex. Finally, 32D cells, which appear to lack low-affinity FGF binding sites, were transfected with a KGFR-erythropoeitin receptor chimera and were found to require heparin to achieve maximal KGF stimulation. Our data are consistent with the previously described concept that cell- or matrix-associated heparan sulfate proteoglycans (HSPGs) and FGF ligands participate in a concerted mechanism that facilitates FGFR dimerization and signal transduction in vivo.
Hsu
Y R
YR
Department of Protein Structure, Amgen Inc., Thousand Oaks, California 91320-1789, USA.
Nybo
R
R
Sullivan
J K
JK
Costigan
V
V
Spahr
C S
CS
Wong
C
C
Jones
M
M
Pentzer
A G
AG
Crouse
J A
JA
Pacifici
R E
RE
Lu
H S
HS
Morris
C F
CF
Philo
J S
JS
D000595:Amino Acid Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002850:Chromatography, Gel; D004247:DNA / Q000096:biosynthesis; D019281:Dimerization; D005110:Extracellular Space / Q000737:chemistry / Q000378:metabolism*; D051526:Fibroblast Growth Factor 10; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D006133:Growth Substances / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D006493:Heparin / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008027:Light; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor; D017978:Receptors, Growth Factor / Q000737:chemistry / Q000378:metabolism*; D012542:Scattering, Radiation; D014461:Ultracentrifugation
C496453:FGF7 protein, human; D051526:Fibroblast Growth Factor 10; D006133:Growth Substances; D046911:Macromolecular Substances; D010446:Peptide Fragments; D017468:Receptors, Fibroblast Growth Factor; D017978:Receptors, Growth Factor; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D006493:Heparin; D004247:DNA; D051497:Receptor, Fibroblast Growth Factor, Type 2; C065104:keratinocyte growth factor receptor