Kinetic Model for FGF, FGFR, and Proteoglycan Signal Transduction Complex Assembly

Abstract

The current working model for fibroblast growth factor receptor (FGFR) dimerization and activation requires the assembly of a ternary complex of fibroblast growth factor (FGF), FGFR, and heparin or heparan sulfate proteoglycan (HSPG) on the plasma membrane. The recent FGF2-FGFR1-heparin crystal structure provides a detailed but static view of the FGF-FGFR-heparin complex. However, the kinetics of ternary complex assembly has yet to be investigated. Here, we characterize FGF2, FGFR1, and heparin interactions using surface plasmon resonance (SPR). Binding constants for binary FGF2/ FGFR1 (K D ) 62 nM), FGF2/heparin (K D ) 39 nM), and FGFR1/heparin (K D ) 3.2 µM) interactions correlate to the magnitude of binding interface observed in the FGF2-FGFR1-heparin crystal structure. Interestingly, comparison of sensorgrams of sequential injections of FGF2 and FGFR1 and equimolar FGF2-FGFR1 injections onto a heparin neoproteoglycan surface demonstrates that FGF2 dramatically enhances the association of FGFR1 with heparin and leads us to propose a model for the stepwise assembly of a ternary FGF-FGFR-HSPG complex. The weak binding affinity of the FGFR1-heparin interaction suggests that in this model, FGFR and HSPG are unbound in the absence of FGF ligand. The availability of FGF results in formation of initial FGF-HSPG complexes, which promotes the rapid binding of FGFR and creates a ternary complex capable of undergoing dimerization and subsequent FGFR activation. In contrast, alternative models for the kinetic assembly of a ternary complex in which binary FGF-FGFR or FGFR-HSPG complexes are intermediates do not conform well with the experimental data.The fibroblast growth factor (FGF) family consists of 22 structurally related proteins with pleiotropic signaling activities (1). Each FGF consists of a core region of homology of 100-120 residues known as a -trefoil core, in addition to variable N-and C-terminal regions. The biological responses to the FGF ligand are mediated by one of four receptor tyrosine kinases, FGFR1-4. Each FGFR is composed of an extracellular ligand-binding region comprised of three immunoglobulin (Ig)-like domains (D1-D3), a single transmembrane helix, and a cytoplasmic segment with tyrosine kinase activity. The minimal portion of the extracellular domain necessary for ligand binding and specificity is D2, D3, and the interconnecting linker. Alternative splicing of the second half of D3 in FGFR1-3 extends the number of functional FGFRs to seven and plays a central role in determining ligand-binding specificity (2-5).Receptor dimerization is a central step in FGFR activation and requires heparin or heparan sulfate proteoglycans (HSPGs), in addition to ligand binding (4,(6)(7)(8). The recent crystal structure of a dimeric 2:2:2 FGF2-FGFR1-heparin ternary complex has provided a mechanistic view by which FGF, FGFR, and HSPG cooperate to promote FGFR dimerization (9). In this two-end model, two 1:1:1 FGF-FGFRheparin ternary complexes associate to form a symmetrical dimer. Within the dimer, each...

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Nanomechanics on FGF-2 and Heparin Reveal Slip Bond Characteristics with pH Dependency

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Immobilization of Bioactive Fibroblast Growth Factor-2 into Cubic Proteinous Microcrystals (Bombyx mori Cypovirus Polyhedra) That Are Insoluble in a Physiological Cellular Environment

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