N-Glycosylation of Fibroblast Growth Factor Receptor 1 Regulates Ligand and Heparan Sulfate Co-receptor Binding

Abstract

The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble-and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi-and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs.The fibroblast growth factors (FGFs) 5 and their receptors co-evolved with metazoans (1, 2), a reflection of their central role in mediating cell-cell communication. FGFs regulate the specification of stem cell fate, organogenesis, skeletal growth (3), tissue repair (4) and phosphate metabolism. FGFs are involved in numerous pathologies such as genetic skeletal defects in children (5) and carcinomas in adults (6, 7).In humans and rodents there are 22 genes encoding more than 30 different FGF proteins and the fgfr 1-4 genes encode over 48 major isoforms of the cognate receptor tyrosine kinase (FGFR) (1, 2, 8). FGFs possess a glycosaminoglycan co-receptor, usually heparan sulfate (HS). The interaction of FGFs with HS is required for the stimulation of cell proliferation, which is initiated by the formation of a complex of FGF ligand, HS co-receptor and FGFR (9 -11). Not surprisingly for a family of key regulatory ligands, the activation of FGF signaling is subjected to multiple regulatory inputs, some of which operate at the level of the assembly of the receptor-ligand complex, whereas others operate inside the cell, on the active receptor complex. Regulatory inputs that operate extracellularly include partial selectivity in the choice of ligand by certain receptor isoforms. FGF-7 is the most specific FGF ligand, beca...

Initial FGF23-Mediated Signaling Occurs in the Distal Convoluted Tubule

“…It has been shown that both FGFR1 and KL are glycosylated within the TGN before residence in the cell membrane, consistent with the fact that a portion of the FGFR1 and KL staining also appears as intracellular. 10,17 In agreement with the above results from vehicle-injected animals, there was no p-ERK1/2 and FGFR1 colocalization in this experimental group (not shown).…”

NEU1 Sialidase Regulates the Sialylation State of CD31 and Disrupts CD31-driven Capillary-like Tube Formation in Human Lung Microvascular Endothelia

“…NEU1 Impairs EC Capillary-like Tube Formation-Because a number of EC sialoproteins participate in the angiogenic response (17)(18)(19)(20)(21)(22)(23) and the angiogenic phenotype is associated with altered sialylation patterns (3-6), we asked whether NEU1 might influence EC capillary-like tube formation in a Matrigel system as a measure of in vitro angiogenesis (Fig. 1).…”

“…Multiple galectins, endogenous lectins that bind galactose residues and regulate angiogenesis (16), were also up-regulated. Finally, a number of EC sialoproteins directly participate in the angiogenic process, including vascular endothelial cadherin (17), selectins and other adhesion molecules (18,19), CD31 (20), CD44 (21), fibroblast growth factor receptor (22), and ␣ v ␤ 3 integrin (23). Taken together, these combined studies establish a central role for glycan structures (and more specifically, sialylation) as intrinsic to the angiogenic process.…”

Blocking the FGF/FGFR system as a ⿿two-compartment⿿ antiangiogenic/antitumor approach in cancer therapy

“…FGFRs are internalized upon receptor activation [17], inducing receptor degradation or recycling. Relevant to this point, N-glycosylation of the receptor affects the assembly of the FGF/FGFR1/HSPG complex [18] and internalization of FGFR2 [19]. At intracellular level, MAPK signalling may phosphorylate threonine residues on FRS2, inhibiting the recruitment of GRB2 [20].…”