N-glycosylation of fibroblast growth factor receptor 1 regulates ligand and heparan sulfate co-receptor binding.
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.
Duchesne
Laurence
L
School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom. lduchesn@liv.ac.uk
Tissot
Bérangère
B
Rudd
Timothy R
TR
Dell
Anne
A
Fernig
David G
DG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D006031:Glycosylation; D006497:Heparan Sulfate / Q000737:chemistry*; D008024:Ligands; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D006497:Heparan Sulfate; D008024:Ligands; D009841:Oligonucleotides; D051496:Receptor, Fibroblast Growth Factor, Type 1; D011994:Recombinant Proteins