Prydz K et al. (2025), Ephrin-B1 regulates cell surface residency of h...

XB-ART-61352

Glycobiology 2025 Apr 23;356:. doi: 10.1093/glycob/cwaf020.

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Ephrin-B1 regulates cell surface residency of heparan sulfate proteoglycans (HSPGs) and complexes with the HSPG CD44V3-10 and fibroblast growth factor receptors.

Prydz K , Simm R , Davydova E , Aasheim HC .

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The ephrin family of membrane proteins mediate intracellular signalling as ligands of transmembrane Eph tyrosine kinase receptors during cell-cell interactions. Ephrin/Eph signalling regulates processes like cell migration and angiogenesis and is of particular importance during embryonic development. Ephrins-A3 and -B3 can also bind to cell surface-associated and soluble heparan sulfate proteoglycans (HSPGs) that also play important roles during early development. Here we show that ephrins-B1, -B2, and -B3 all can bind in cis to cell surface HSPGs, while only ephrin-B1 interacts with cell surface HSPGs in a way that retards HSPG endocytosis. Expressing ephrin-B1 in HEK293T cells, using polyethyleneimine (PEI) as transfection agent, increased cell surface levels of HSPGs which were detected by an anti-heparan sulfate (HS) antibody or by ephrin-B3-Fc binding. Ephrin-B1 in the plasma membrane seemed to retard PEI-induced HSPG internalisation and degradation. Binding of HSPGs by ephrin-B1 was observed for the human, mouse, xenopus, and zebrafish homologs, and did not require the cytoplasmic tail of ephrin-B1 that contains tyrosines shown to be involved in intracellular signalling. Furthermore, ephrin-B1 could bind the HSPG variant of CD44 (CD44V3-10), a complex that could further associate with fibroblast growth factor receptors (1 and 4) after co-expression with one of these receptors. In summary, our data indicate that ephrin-B1 can regulate cellular HSPG turnover and is able to form complexes of potential biological importance with CD44V3-10 and fibroblast growth factor receptors.

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Species referenced: Xenopus laevis

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	Fig. 1. Flow cytometry analysis of cell surface HS in HEK293T cells transiently transfected using PEI (unless otherwise indicated) to express ephrins-B1, -B2, or -B3. A) Cells expressing ephrin-B1 (black) or mock transfected (grey) incubated with ephrin-B3-fc and PE-conjugated anti-human IgG (PE-IgG) 2nd layer. Light grey is 2nd layer control. B) Cells expressing ephrin-B1 incubated with heparin (grey) or not (black) followed by ephrin-B3-fc binding and PE-IgG. C) Cells expressing ephrin-B1 (black) or mock transfected (grey) incubated with anti-HS antibody followed by FITC-conjugated anti mouse IgM (FITC-IgM). Light grey 2nd layer control. D) Cells expressing ephrin-B1 (black) or ephrin-B2 (grey) incubated with anti-HS antibody and FITC-IgM. E) Cells expressing ephrin-B1 (black) or ephrin-B3 (grey) incubated with anti-HS antibody and FITC-IgM. F) Cells expressing ephrin-B1 (black) or ephrin-B2 (grey) incubated with ephrin-B3-fc and PE-IgG. G) Cells expressing ephrin-B1 (black) or ephrin-B3 (grey) incubated with ephrin-B3-fc and PE-IgG. H) Cells mock transfected using PEI (grey) or Lipofectamine (black) incubated with anti-HS antibody and FITC-IgM. I) Cells transfected using PEI (grey) or Lipofectamine (black) expressing ephrin-B1 incubated with EphB2-fc and PE-IgG.
	Fig. 2. Co-precipitation of HSPGs and ephrin-B1. Ephrin-B1 is not an HSPG by itself. HEK293T cells PEI-transfected expressing CD44V3–10 (44), ephrin-B1 (B1) or mock (M), metabolically labelled with 35S-sulfate and lysed, incubated with anti-ephrin-B1 antibodies (A20, C18, or IV) and protein G beads before SDS-PAGE. (B) Mock (M) and ephrin-B1 (B1) expressing cells 35S-sulfate labelled and lysed. Lysates of cells expressing ephrin-B1 treated or not with Heparinase (hep) precipitated with anti-ephrin-B1 and protein G beads before SDS-PAGE. (C) Ephrin-B1-expressing cells 35S-sulfate labelled, lysed and precipitated with protein-G beads only (Ctr), anti-ephrin-B1 (α-B1), or EphB2-fc (EB2). (D) HT-29 cells 35S-sulfate labelled, lysed and precipitated with protein-G beads only (Ctr), anti-ephrin-B1 (α-B1), or EphB2-fc (EB2). (E) Primary sequence ephrin-B1 (extracellular part). Serine-glycines (SG) in bold. (F) Flow cytometry of HEK293T cells expressing ephrin-B1 (left panel) and ephrin-B1Q (middle panel) incubated with anti-HS and FITC-antibody 2nd layer (black). Grey: 2nd layer control. Right panel: Overlay of cells expressing ephrin-B1 (black) and ephrin-B1Q (grey) incubated with EphB2-fc and PE-antibody 2nd layer. (G) Cells mock transfected (M) or expressing ephrin-B1 (B1) or ephrin-B1Q (B1Q), 35S-sulfate labelled, washed, lysed, and precipitated with EphB2-fc bound to protein G beads.
	Fig. 3. Ephrin-B1 cytoplasmic tail tyrosines are not required for HSPG binding. A) Mock transfected (M) or HEK293T cells expressing ephrin-B1 (B1), ephrin-B1Q (B1Q), ephrin-B1S (B1S), or ephrin-B1QS, using PEI were 35S-sulfate labelled, lysed and precipitated with EphB2-fc. B) Flow cytometry of cells expressing ephrin-B1 (grey) or ephrin-B1S (black) incubated with EphB2-fc and PE-antibody. C) Flow cytometry of cells expressing ephrin-B1Q (grey) or ephrin-B1QS (black) after binding of EphB2-fc followed by PE. D) Western blot of lysates from mock (M) transfected and cells expressing human ephrin-B1 (B1), ephrin-B1S (B1S), ephrin-B1Q (B1Q), ephrin-B1QS (B1QS), or mouse ephrin-B1 (B1M). Upper panel detection with anti-ephrin-B1, lower panel with anti-actin for expression level control. Band regions were quantified and relative intensities compared to actin bands and related to lane 3 (insert).
	Fig. 4. Ephrins-B1, -B2, and -B3 all have the ability to bind HSPGs. A) HEK293T cells transfected to express ephrin-B1 (B1), ephrin-B2 (B2), or ephrin-B3 (B3) using PEI or Lipofectamine (Lipo) were 35S-sulfate labelled, lysed, and precipitated with EphB2-fc (EB2). Lanes were quantified by image quant and relative values calculated (insert). B) Cells mock (M) or transfected (Lipo) to express ephrins-B1 (B1), -B2, or -B3, 35S-sulfate labelled, lysed, and precipitated with either EB2, HPA (recognizing all ephrin-Bs) or an anti-ephrin-B3 antibody.
	Fig 5. Ephrin-B1 associates with the HSPG CD44V3–10. (A) Mock (M) transfected (Lipo) or HEK293T cells expressing ephrin-B1 (B1), CD44V3–10 (44), or both (B1 44), 35S-sulfate labelled, lysed, and precipitated with EphB2-fc. B) Cells co-expressing ephrin-B1 and CD44V3–10 (Lipo) 35S-sulfate labelled, lysed, and precipitated with EphB2-fc and protein-G beads and then boiled off the protein-G beads in PBS/0.5% SDS (1/10 of precipitate shown in lane denoted EB2). Remaining sample precipitated with ephrin-B1-fc (Ctr) or α-CD44. C). Lysates from cells transfected as in 5A, but expressing ephrin-B1S were precipitated with EphB2-fc (EB2). Lysates and precipitates were separated by SDS-PAGE and western blotted. Blots were probed with anti-CD44 (upper panel) or anti-ephrin-B1 (lower panel) D) lysates from cells transfected as in 5C precipitated with anti-CD44. Precipitates and lysates separated by SDS-PAGE and western blotted. Blots probed with anti-ephrin-B1 (upper) or anti-CD44 (lower panel).
	Fig 6. Ephrin-B1 can form a complex with CD44V3–10 and fibroblast growth factor receptors. A) HEK293T cells transfected (Lipo) to express combinations of ephrin-B1S (B1), FGFR1 (FR1), and CD44V3–10 (44). Cell lysates precipitated with anti-FGFR1 (left), anti-CD44 (mid), or EphB2-fc (EB2; right panel), separated by SDS-PAGE and western blotted with anti-ephrin-B1 (upper) or anti-FGFR1 (lower panels). B) Cells were transfected (Lipo) to express combinations of ephrin-B1S (B1), FGFR4 (FR4), and CD44V3–10 (44). Cell lysates precipitated with anti-FGFR4 (left), anti-CD44 (mid) or EphB2-fc (EB2; right panel), separated by SDS-PAGE and western blotted with anti-ephrin-B1 (upper) or anti-FGFR4 (lower panels). C) Cells transfected as in 6A (left) or 6B (right panels) were precipitated with anti-ephrin-B1, separated by SDS-PAGE and western blotted with anti-CD44 (upper), anti-FGFR1, anti-FGFR4 (mid), or anti-ephrin-B1 (lower panels).