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Heparan Sulfate Regulates Ephrin-A3/Epha Receptor Signaling

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Heparan Sulfate Regulates Ephrin-A3/Epha Receptor Signaling Heparan sulfate regulates ephrin-A3/EphA receptor signaling Fumitoshi Irie*, Misako Okuno*, Kazu Matsumoto*, Elena B. Pasquale†, and Yu Yamaguchi*‡ *Sanford Children’s Health Research Center and †Cancer Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037 Communicated by Erkki Ruoslahti, Burnham Institute for Medical Research, Santa Barbara, CA, February 8, 2008 (received for review August 3, 2007) Increasing evidence indicates that many signaling pathways in- The function of many important morphogens and guidance volve not only ligands and receptors but also various types of molecules has been shown to involve accessory molecules as coreceptors and matrix components as additional layers of regu- coreceptors or as a mechanism to control the topological dis- lation. Signaling by Eph receptors and their ephrin ligands plays a tribution of the guidance molecules (7, 8, 12–14). Curiously, key role in a variety of biological processes, such as axon guidance there is little information on whether ephrin-Eph signaling is and topographic map formation, synaptic plasticity, angiogenesis, influenced by heparan sulfate or other accessory regulatory and cancer. Little is known about whether the ephrin-Eph receptor molecules. In this study, we examined whether ephrin-Eph signaling system is subject to such additional layers of regulation. signaling is modulated by heparan sulfate. Our results surpris- Here, we show that ephrin-A3 binds to heparan sulfate, and that ingly reveal that ephrin-A3 uniquely binds heparan sulfate. the presence of cell surface heparan sulfate is required for the full Ephrin-A3-mediated EphA receptor activation and biological biological activity of ephrin-A3. Among the ephrins tested, includ- activities are attenuated in cells lacking heparan sulfate. Cortical ing ephrin-A1, -A2, -A5, -B1, and -B2, only ephrin-A3 binds heparin neurons deficient in heparan sulfate synthesis, isolated from or heparan sulfate. Ephrin-A3-dependent EphA receptor activation conditional Ext1 knockout mice (7), exhibit greatly impaired is reduced in mutant cells that are defective in heparan sulfate growth cone collapse in response to ephrin-A3, as do wild-type synthesis, in wild-type cells from which cell surface heparan sulfate cortical neurons from which cell surface heparan sulfate has has been removed, and in the hippocampus of conditional knock- been removed by heparitinase digestion. These results provide out mice defective in heparan sulfate synthesis. Ephrin-A3-depen- evidence for an involvement of heparan sulfate in ephrin/Eph dent cell rounding is impaired in CHO cells lacking heparan sulfate, receptor signaling and suggest that heparan sulfate modulates and cortical neurons lacking heparan sulfate exhibit impaired ephrin-A3-dependent biological processes. growth cone collapse. In contrast, cell rounding and growth cone collapse in response to ephrin-A5, which does not bind heparan Results sulfate, are not affected by the absence of heparan sulfate. These Binding of Ephrin-A3 to Heparin and Heparan Sulfate. To examine the results show that heparan sulfate modulates ephrin/Eph signaling binding of various ephrins to heparan sulfate, we generated a and suggest a physiological role for heparan sulfate proteoglycans library of the recombinant ectodomains of most A and B ephrins. in the regulation of ephrin-A3-dependent biological processes. These recombinant ephrins all included the entire extracellular domain, up to the last amino acid before the stretch of hydro- proteoglycan ͉ cell adhesion ͉ growth cone collapse phobic amino acids that serve as either the signal for GPI-anchor attachment (in the case of the A ephrins) or the transmembrane eparan sulfate is a class of sulfated glycosaminoglycans. It domain (in the case of the B ephrins), followed by the FLAG Hoccurs as heparan sulfate proteoglycans, in which one or epitope. Culture supernatants from transfected 293T cells con- more heparan sulfate chains are covalently attached to a variety taining the secreted ephrin ectodomains were applied to hepa- of core proteins (1). Heparin is a specialized form of heparan rin-Sepharose and eluted by a stepwise increase in NaCl con- sulfate synthesized exclusively by connective tissue mast cells, centration. Only ephrin-A3 bound to heparin (Fig. 1A). None of whereas heparan sulfate is expressed in many cell types. The the other ephrins examined (ephrin-A1, -A2, -A5, -B1, -B1, or -B2) nor the ectodomains of several Eph receptors (EphA2, -A3, negatively charged sulfate groups of heparan sulfate mediate -A4, or -B2) exhibited detectable binding to heparin. Elution of interactions with a variety of proteins. Increasing evidence ephrin-A3 from heparin-Sepharose required 0.4–0.8 M NaCl. indicates that heparan sulfate acts as an integral component of This suggests that ephrin-A3 binds with an affinity similar to a number of morphogen and growth factor signaling pathways by those of several known heparin-binding factors, such as Sonic interacting with these molecules. For example, fibroblast growth hedgehog, Wnt1, the Ig-like domain-containing isoform of neu- factors, Wnts, Sonic hedgehog, bone morphogenetic proteins, regulin 1, and various chemokines (15–18). and neuregulins bind to heparan sulfate and are functionally To further confirm the significance of the above results, we modulated by it (2, 3). Molecules involved in axon pathfinding, performed two additional binding experiments. First, we exam- such as netrin-1, Slit, and semaphorins, are other major targets ined binding of full-length untagged ephrins to heparin. Cell of regulation by heparan sulfate (4). We have used genetic lysates from 293T cells that had been transfected with full-length ablation of the Ext1 gene, which encodes a glycosyltransferase untagged ephrin-A3 or -A5 were applied to heparin-Sepharose, essential for heparan sulfate synthesis (5, 6), to demonstrate the and eluted ephrin-A3 and -A5 were detected with the respective physiological significance of the interactions of some of these antibodies. Consistent with the results obtained with FLAG- molecules with heparan sulfate (7–9). CELL BIOLOGY The Eph receptors form the largest family of receptor tyrosine kinases. Together with their membrane-bound ligands, the Author contributions: F.I., E.B.P. and Y.Y. designed research; F.I. and M.O. performed ephrins, they are involved in bidirectional signaling between two research; E.B.P. contributed new reagents/analytic tools; K.M. analyzed data; and F.I. and interacting cells (10, 11). Signals generated by the engagement of Y.Y. wrote the paper. ephrin ligands to Eph receptors generally result in repulsive The authors declare no conflict of interest. responses, such as retraction of the cell periphery and cell ‡To whom correspondence should be addressed. E-mail: [email protected]. rounding, and growth cone collapse in neurons. These repulsive This article contains supporting information online at www.pnas.org/cgi/content/full/ responses are thought to be the basis for the function of the 0801302105/DCSupplemental. ephrin-Eph system in axon guidance. © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801302105 PNAS ͉ August 26, 2008 ͉ vol. 105 ͉ no. 34 ͉ 12307–12312 Downloaded by guest on September 25, 2021 Fig. 1. Ephrin-A3 binds heparin. (A) Binding of soluble ephrin and Eph proteins to heparin-Sepharose. FLAG-tagged ectodomains of the indicated ephrins and Eph receptors were expressed in 293T cells and the culture supernatants (Sample) were incubated with heparin-Sepharose. Unbound materials were collected (Pass) and bound materials were eluted with indi- cated final concentrations of NaCl. Each fraction was analyzed by immuno- blotting with anti-FLAG antibody. (B) Binding of full-length, native ephrin-A3 and ephrin-A5 to heparin-Sepharose. Lysates of 293T cells transfected with full-length ephrin-A3 or ephrin-A5 were incubated with heparin-Sepharose. After washing with 0.2 M NaCl, bound materials were eluted with 1.2 M NaCl (Bound). Each fraction was analyzed by immunoblotting with anti-ephrin-A3 or anti-ephrin-A5 antibody. tagged ectodomains, the native form of ephrin-A3 bound to heparin, whereas that of ephrin-A5 did not (Fig. 1B). Second, we examined whether ephrin-A3 binds not only to heparin but also to heparan sulfate. Heparin is synthesized exclusively in con- nective tissue mast cells (19) and, therefore, binding to heparin does not necessarily imply the biological significance of the Fig. 3. Ephrin-A3-dependent EphA signaling is impaired in cells defective in interaction in other cell type. Binding of soluble forms of ephrins heparan sulfate synthesis and in cells treated with heparitinase. Levels of EphA receptor autophosphorylation were analyzed in cells lacking heparan sulfate and Eph receptors to heparan sulfate was examined in an expression. (A) Experiments using CHO cells. (Left) Comparison between ELISA-type assay. As shown in Fig. 2, ephrin-A3 displayed wild-type (WT) and pgsD-677 mutant (pgsD-677) cells. pgsD-677 cells show reduced EphA2 phosphorylation in response to ephrin-A3 (compare lane 3 and lane 4). (Right) Removal of cell surface heparan sulfate by heparitinase treatment reduces EphA2 autophophorylation in wild-type CHO cells (com- pare lane 9 and lane 10). (H’ase) heparitinase treated cells; (Cont) mock treated cells. Note that phosphorylation in response to ephrin-A5 was not affected in either pgsD-677 cells or wild-type cells treated with heparitinase (lanes 5, 6, 11, and 12). (B) Experiments using cortical neurons. (Left) Compar- ison between wild-type (WT) and Ext1 null (Ext1-KO) cortical neurons. Ext1 null neurons show reduced EphA4 phosphorylation in response to ephrin-A3 (compare lanes 3 and 4). (Right) Removal of cell surface heparan sulfate by heparitinase treatment also reduces EphA4 autophophorylation in wild-type cortical neurons (compare lane 9 and lane 10). (H’ase) heparitinase treated cells; (Cont) mock treated cells. Note that the response to ephrin-A5 was not affected in either Ext1 null neurons or wild-type neurons treated with hep- aritinase (lanes 5, 6, 11, and 12).
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