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Studying the role of heparin in the formation of FGF1‐FGFR2 complexes using gel chromatography
Author(s) -
Robinson Christopher J.,
Harmer Nicholas J.,
Blundell Tom L.,
Gallagher John T.
Publication year - 2004
Publication title -
international journal of experimental pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.671
H-Index - 72
eISSN - 1365-2613
pISSN - 0959-9673
DOI - 10.1111/j.0959-9673.2004.390ak.x
Subject(s) - fibroblast growth factor , fast protein liquid chromatography , heparin , chemistry , heparan sulfate , fgf1 , biochemistry , size exclusion chromatography , hepes , receptor , fibroblast growth factor receptor , biophysics , chromatography , biology , high performance liquid chromatography , enzyme
The fibroblast growth factors (FGFs) are a family of at least 24 proteins which have diverse roles in regulating cell proliferation, migration and differentiation during embryogenesis, angiogenesis and tissue repair. Four FGF receptor tyrosine kinases (FGFRs) have been identified, each of which binds a specific subset of the FGF family. FGF binding induces dimerization of the FGFRs leading to their activation through transphosphorylation. Heparin or heparan sulfate (HS) is essential for the activity of the FGFs, because they form an integral part of the signalling complex on the cell surface. Our group has been using gel filtration FPLC to study the role that heparin plays in the formation of FGF1‐FGFR2 complexes. Methods A Superdex 200 HR 10/30 FPLC column was calibrated using protein standards and used to estimate the size of protein complexes which form in the presence of heparin. The column was run in 150 m m NaCl, 50 m m HEPES, pH 7.2 buffer and complexes could be visualized by protein UV absorbance at 280 nm. Results In agreement with published data (Pellegrini et al . 2000), we found that 2 : 2 : 1 complexes formed spontaneously in solution between FGF1, FGFR2IIIc and heparin decasaccharides (dp10). Although heparin dp6–8 fragments could bind FGF1 and FGFR2 simultaneously, only dp10 and larger fragments efficiently formed 2 : 2 : 1 complexes. These complexes appear to be relatively stable, as they remain associated following prolonged incubation with heparinase. FGF1 was able to bind heparin saccharides strongly even in the absence of receptor. In contrast, FGFR2 had very little affinity for either FGF1 or heparin dp12 separately, but bound efficiently to the FGF1‐heparin complex. Dimerization of FGF1 appears to be essential for subsequent dimerization of FGFR2. Selectively desulfated heparin saccharides could only bind FGF1 singly and formed 1 : 1 : 1 complexes with FGFR2, which did not then associate with one another. Discussion Our data supports the idea that heparin plays a dual role in the formation of FGF1 signalling complexes, firstly by mediating interactions between FGF1 and FGFR2 and secondly by acting as a template for the dimerization of FGF1‐FGFR2 subunits. This technique could be easily adapted to study other heparin/HS‐mediated ligand–receptor interactions and could provide new insights into the mechanisms of formation of multiprotein signalling systems on the cell surface.