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

Abstract

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.

Dissociation of Heparan Sulfate and Receptor Binding Domains of Hepatocyte Growth Factor Reveals That Heparan Sulfate-c-Met Interaction Facilitates Signaling

“…A comparison of NK1 and c-Met-Fc retention by immobilized heparin-TSK using high performance liquid chromatography gave results consistent with those obtained using the biotin-HS ELISA and also revealed that a control Fc protein was not retained by immobilized heparin (data not shown). Thus, by all of the means used here to compare HS binding by ligand and receptor for the HGF/c-Met system, results were generally similar to those reported for other growth factor/ receptor systems, such as FGFs 1, 2, and 7 (23,35,37,38).…”

“…Ligand binding by that construct was not dependent on added heparin, whereas ligand binding and signaling by wildtype KGF receptor expressed on cell surfaces was significantly enhanced by HS (37). The explanation for this apparent discrepancy became clear after another, independent study of KGF receptor-HS interaction was published (38). Unlike the Ig-receptor chimera used by LaRochelle et al (37), which contained the Ig hinge region and was secreted as a disulfidelinked receptor dimer, Hsu et al (38) showed HS-dependent KGF binding by a soluble monomeric KGF receptor.…”

“…The explanation for this apparent discrepancy became clear after another, independent study of KGF receptor-HS interaction was published (38). Unlike the Ig-receptor chimera used by LaRochelle et al (37), which contained the Ig hinge region and was secreted as a disulfidelinked receptor dimer, Hsu et al (38) showed HS-dependent KGF binding by a soluble monomeric KGF receptor. The latter receptor construct formed a dimer in the presence of ligand and added heparin (38), suggesting that the normal HS dependence of KGF binding could be abrogated by the covalent linkage of two KGF receptors in the context of the Ig chimera.…”

“…Unlike the Ig-receptor chimera used by LaRochelle et al (37), which contained the Ig hinge region and was secreted as a disulfidelinked receptor dimer, Hsu et al (38) showed HS-dependent KGF binding by a soluble monomeric KGF receptor. The latter receptor construct formed a dimer in the presence of ligand and added heparin (38), suggesting that the normal HS dependence of KGF binding could be abrogated by the covalent linkage of two KGF receptors in the context of the Ig chimera. High affinity ligand binding to a soluble dimeric c-Met Ig fusion protein in the absence of heparin has been reported (25) and may also have been attributable to the existence of covalent receptor dimers.…”

Peptídeos de conformação restrita relacionados ao sítio 2 do fator de crescimento de fibroblastos ácido humano (hFGF-1): estudo sobre estrutura e atividade

“…Os modelos de Pantoliano e colaboradores e de Springer e colaboradores, embasados na estrutura do complexo receptor-ligante do hormônio do crescimento, propõem uma estequiometria de 1FGF:2FGFR:1HEP Springer et al, 1994). Já outros (Xu et al, 1992;Kan et al, 1993;Digabriele et al, 1998; Moy et al, 1997;Huhtala et al, 1999, Hsu et al, 1999 propõem uma estequiometria de 1FGF:1FGFR ligados por 1 ou 2 cadeias de HEP ou HS, formando complexos ternários com FGF-FGFR (Wang et al, 1999).…”