Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition

Abstract

FGF signaling uses receptor tyrosine kinases that form high-affinity complexes with FGFs and heparan sulfate (HS) proteoglycans at the cell surface. It is hypothesized that assembly of these complexes requires simultaneous recognition of distinct sulfation patterns within the HS chain by FGF and the FGF receptor (FR), suggesting that tissue-specific HS synthesis may regulate FGF signaling. To address this, FGF-2 and FGF-4, and extracellular domain constructs of FR1-IIIc (FR1c) and FR2-IIIc (FR2c), were used to probe for tissue-specific HS in embryonic day 18 mouse embryos. Whereas FGF-2 binds HS ubiquitously, FGF-4 exhibits a restricted pattern, failing to bind HS in the heart and blood vessels and failing to activate signaling in mouse aortic endothelial cells. This suggests that FGF-4 seeks a specific HS sulfation pattern, distinct from that of FGF-2, which is not expressed in most vascular tissues. Additionally, whereas FR2c binds all FGF-4–HS complexes, FR1c fails to bind FGF-4–HS in most tissues, as well as in Raji-S1 cells expressing syndecan-1. Proliferation assays using BaF3 cells expressing either FR1c or FR2c support these results. This suggests that FGF and FR recognition of specific HS sulfation patterns is critical for the activation of FGF signaling, and that synthesis of these patterns is regulated during embryonic development.

FRS2 via Fibroblast Growth Factor Receptor 1 Is Required for Platelet-derived Growth Factor Receptor β-mediated Regulation of Vascular Smooth Muscle Marker Gene Expression

“…To study the role of FGFR1 signaling in VSMC and to avoid the confounding effects of heparan sulfate proteoglycans on FGF receptor signaling, we developed a constitutively activated FGFR1 with mutations that abolish signaling through specific pathways. These mutants eliminate the need for FGF2 stimulation, which will abrogate FGF-mediated signaling through syndecans (32,33) and allows dissection of signaling via FGFR1. These constitutively active FGFR1 mutants reveal that the majority of ERK signaling occurs through FGFR1 interaction with FRS2, although low level ERK signaling occurred with FGFR1 K562E: ϪFRS2.…”

HSulf-1 and HSulf-2 Are Potent Inhibitors of Myeloma Tumor Growth in Vivo

“…FR1cAP is a soluble FGF receptor 1c fusion protein that consists of the three extracellular Ig loop domains of the IIIc splice variant of FR1c fused to the NH 2 terminus of human placental alkaline phosphatase (18). The FR1cAP fusion protein was purified as described previously (19) and quantified based on its AP activity (20,21). Following incubation with FGF-2 or FGF-2 and FR1cAP, sections were washed three times in PBS containing Ca 2ϩ and Mg 2ϩ .…”

QSulf1, a heparan sulfate 6- O -endosulfatase, inhibits fibroblast growth factor signaling in mesoderm induction and angiogenesis

“…Heparin-mediated FGF2-FGFR1 binding was assayed by using a soluble FGFR1c containing the extracellular domain of FGFR1 fused to an AP tag (31). FGF2 and FGFR1c were incubated with increasing concentrations of QSulf1-or QSulf1(C-A)-treated heparin (0-1,000 ng͞ml), and FGF2-heparin-FGFR ternary complexes were then immunoprecipitated with anti-AP antibody for Western blot analysis.…”