New Connections Between Integrins and Axon Guidance Dispatch

Neurobiology: New Connections Dispatch between Integrins and Axon Guidance

Tetsuya Nakamoto, Kristin H. Kain and Mark H. domain V1–3, and a carboxy-terminal basic domain. The Ginsberg γ VI and V1–3 regions of Netrin-1, 2, 3 are similar to the chain of laminin and these regions in Netrin-4 resemble the β chain of laminin. In the carboxy-terminal domain, Axon guidance molecules such as netrins, sema- netrins have an RGD integrin-binding motif. Netrins phorins, Slits and ephrins provide the cues required have two families of known receptors, the DCC (deleted for accurate patterning of axonal projections in the in colorectal cancer) family, including neogenin, and the nervous system. Recent reports have described mul- UNC5H family. DCC/neogenin receptors mediate tiple paradigms by which these molecules interact attraction to netrins, while UNC5H family members bind with integrin adhesion receptors in and outside neu- DCC receptors to induce repulsion. ronal tissues. Netrins attract axons ventrally toward the midline of the central nervous system. However, Netrin-1 is also expressed in several epithelial tissues such as the Four major families of axon guidance molecules — inner ear, lung and mammary gland [4]. In the netrins, semaphorins, Slits and ephrins — guide axons mammary gland, Netrin-1 is expressed by prelumenal to specific sites by providing attractive or repulsive cells where it acts as a short-range attractant for the cues during the formation of neural networks. Axonal adjacent neogenin-expressing cap cell layer, which is guidance requires spatiotemporal control of cell adhe- necessary to maintain proper mammary gland mor- sion and assembly of the actin and microtubule phology [4]. The mechanisms responsible for netrin- cytoskeletons. Similar processes control the migration mediated regulation of cell migration are currently of all animal cells and integrin adhesion receptors often unclear, but the recent report by Yebra et al. [3] sheds play pivotal roles in each of them [1]. Integrins are new light on the interplay between integrins, netrins transmembrane, heterodimeric receptors for compo- and the ECM in epithelial cell motility. nents of the extracellular matrix (ECM) and for cellular Yebra et al. show that Netrin-1 localizes to a discrete counter-receptors. In humans, combinations of 18 α population of epithelial cells within the fetal pancreatic subunits and 8 β subunits form at least 25 integrins, ductal epithelium and to the basal membrane of ducts, each of which binds specific ECM components or possibly by binding to collagen IV. Using primary fetal ligands on the surfaces of other cells [2]. Integrin sig- pancreatic epithelial cells and pancreatic adenocarci- naling regulates cytokeletal dynamics, adhesion, and noma-derived cells, they show that Netrin-1 induces migration events, through associated proteins such as cell migration by serving as an adhesive ligand that talin, vinculin, integrin-linked kinase (ILK), focal adhe- associates with integrins. A 25 amino acid sequence, sion kinase (FAK), paxillin, p130Cas, Abl kinase and lacking the RGD motif, from the carboxy-terminal basic many other signaling or cytoskeletal proteins. domain mediates the netrin–integrin interaction. Block- Recently published work [3] showing that Netrin-1 ing antibodies showed that this adhesion is mediated regulates epithelial cell motility through the engage- by α6β4 integrin and not by DCC/neogenin or by other ment of integrins highlights the conserved function of integrins. However, a peptide column of the carboxy- axonal guidance molecules in regulating migration in terminal 25 amino acids of Netrin-1 bound α3 and β1 many tissue types and the importance of integrin integrins together with α6 and β4 integrins suggesting function in this process. Indeed, a burgeoning litera- a role for α3β1. The authors found that Netrin-1–α3β1 ture has underscored the connections between inte- association was necessary for the induction of hapto- grins and axonal guidance cues. Here, we summarize taxis on Netrin-1 by hepatocyte growth factor/scatter four families of neuronal guidance molecules that are factor. Both integrins colocalize with Netrin-1 in the fetal now known to show a conservation of function in non- pancreatic epithelial duct, suggesting a role in the neuronal cells and connect with integrins through migration of epithelial precursors and in the morpho- three paradigms: firstly, integrins act as receptors for genesis of the pancreas. the guidance cues; secondly, binding of the axonal Both α6β4 and α3β1 integrins are laminin receptors guidance molecules to their receptors alters the func- expressed in epithelial cells. Binding of these integrins tional states of integrins; thirdly, signals from axonal to laminins is mediated by basic residue-rich domains guidance receptors and integrins converge to coordi- of laminins that are similar to the Netrin-1 carboxy-ter- nate cellular behaviors (Figure 1). minal basic sequence. This sequence is different from the DCC/neogenin-binding site in Netrin-1. In the Netrins nervous system, netrins act as diffusible chemotropic Netrins are structurally composed of domain VI, three factors [5]. In epithelial cells, however, soluble Netrin-1 consecutive epidermal growth factor (EGF)-like repeats, promotes migration via direct association with integrins in pancreatic epithelial cells. Thus, this work identifies a Department of Cell Biology, VB-2, The Scripps Research new mechanism of Netrin-1 action in regulation of Institute, 10550 N. Torrey Pines Road, La Jolla, California motility events in non-neuronal cells. Furthermore it 92037, USA. E-mail: [email protected] provides an example of a paradigm in which integrins Dispatch R122

Figure 1. Three paradigms of the connec- 2. Signals from AG 3. Signals from AG 1. Integrins are tion between integrins and axon guidance receptor regulate receptors and receptors for AG: molecules. integrin activation: integrins converge: Netrin-1, Sema7A Paradigm 1: integrins serve as receptors Sema3, Eph/Ephrin Sema3, Slit, Eph/Ephrin for axon guidance molecules (AG). Para- digm 2: the signaling from axon guidance Integrin receptors alters the functional states of ligands AG integrins. Paradigm 3: signals from axon guidance receptors and integrins con- AG AG verge. Integrin Integrin Integrin

Activation or suppression

Convergence Current Biology act as receptors for axonal guidance molecules (para- In contrast, Class 3 semaphorins — secreted sema- digm 1 in Figure 1). phorins that bind to neuropilins in a complex with plexin A — have no RGD sequence. Neuropilins are also Semaphorins receptors for vascular endothelial growth factor and are The semaphorins are secreted and membrane-associ- expressed in vascular endothelial cells. Studies in ated proteins defined by the presence of an approxi- knockout mice suggest that Class 3 semaphorins are mately 500 amino acid extracellular domain termed as involved in vascular formation. Serini et al. [7] showed the Sema domain. There are more than 20 sema- Sema3A inhibits attachment and migration of endothe- phorins, which are divided into 8 classes. The common lial cells and this inhibition is reversed by adding the β1 receptors for semaphorins are the plexins, except for integrin activating antibody. In contrast, a dominant- class 3 semaphorins, which bind to neuropilins in a negative form of neuropilin-1 and plexin A1 enhances complex with plexins. Initial research revealed that the attachment and migration of endothelial cells through a semaphorins are chemorepellent molecules for axons, process requiring α5β1 or αvβ3 integrins. Thus, sema- but semaphorins can also work as chemoattractants. phorins can regulate integrin function via paradigm 2 Semaphorins are expressed in many organs and regu- (Figure 1). Although the molecular connection between late motility of many migratory cell types, such as Class 3 semaphorin–neuropilin–plexin complexes and immune, epithelial and endothelial cells. Semaphorins integrin signaling is unclear, one possible link is con- can interact with integrins through all three of the par- vergence at the signaling molecule MICAL, which binds adigms outlined in Figure 1. both plexins and CasL, a key intermediary in integrin Semaphorin 7A (Sema7A) is a glycosylphosphatidyl signaling [8,9] (paradigm 3, Figure 1). inositol (GPI)-anchored semaphorin that binds to plexin C1 in vitro and has a role in the immune system. Slits Pasterkamp et al. [6] detected expression of Sema7A The three Slit family members (Slit1, Slit2 and Slit3) in the nervous system where it promotes axon growth are secreted ligands that bind the extracellular from olfactory bulb neurons. In Sema7A-deficient mice, domain of the transmembrane family of Roundabout the lateral olfactory tract is reduced, although this phe- (Robo) receptors (Robo1, Robo2, Robo3, and Robo4) notype was not observed in plexin C1 deficient mice and mediate axonal guidance at the midline. Slit is a and the response of olfactory bulb neurons to Sema7A chemorepellant molecule, promoting commissural was not disrupted, suggesting a plexin C independent axonal growth on the contralateral side of the midline mechanism for Sema7A-directed migration. Sema7A and preventing ipsilateral recrossing. Recent studies also contains an RGD integrin-binding motif. By using have established that Robo and Slit family members a short RGD-containing peptide from Sema7A, these are expressed at extra-neuronal sites where they reg- authors found that a β1 integrin is a receptor for ulate cell migration (for review see [10]). Sema7A. Furthermore, stimulation of olfactory bulb Robo signals through four conserved sequences in neurons by binding to Sema7A induces FAK and ERK its cytoplasmic domain (CC0, CC1, CC2, CC3). The phosphorylation, which are downstream events in inte- CC3 domain of Robo interacts with a family of Rho grin activation. Several other semaphorins contain the GTPase-activating proteins (GAPs) called Slit–Robo RGD sequence, raising the possibility that they may GAPs (srGAPs) [11], which can mediate chemorepul- also serve as integrin ligands. sion by inactivating Cdc42. In addition, Robo can Current Biology R123

signal through molecules such as Ena/VASP [12] and generated by axonal guidance receptors to coordinate Abl kinase [12], which are implicated in integrin-medi- cellular behaviors (Sema3, Slit, Eph/Ephrin). The many ated functions [13,14]. Furthermore, studies in connections between axonal patterning and integrin- Drosophila indicate that integrin-mediated adhesion mediated cell migration suggest that workers in these regulates the responsiveness of axons to Slit [15]. The hitherto divergent fields will look to each other for shared effectors downstream of integrins and Robo novel guidance cues! and the potential regulation of Robo signaling by integrins strongly suggest that integrin signaling and Robo References 1. Horwitz, A.R., and Parsons, J.T. (1999). Cell migration–movin’ on. signaling are convergent (paradigm 3 in Figure 1). Science 286, 1102–1103. 2. Hynes, R.O. (2002). Integrins: bidirectional, allosteric signaling Ephrins machines. Cell 110, 673–687. Ephrins are the ligands of Eph family receptor tyrosine 3. Yebra, M., Montgomery, A.M., Diaferia, G.R., Kaido, T., Silletti, S., Perez, B., Just, M.L., Hildbrand, S., Hurford, R., Florkiewicz, E., et al. kinases. EphrinA ligands are GPI-anchored type ligands (2003). Recognition of the neural chemoattractant Netrin-1 by inte- and bind EphA receptors. EphrinB ligands are trans- grins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion membrane proteins that principally bind EphB recep- and migration. Dev. Cell 5, 695–707. 4. Srinivasan, K., Strickland, P., Valdes, A., Shin, G.C., and Hinck, L. tors. The Ephrin–Eph system is known to regulate (2003). Netrin-1/neogenin interaction stabilizes multipotent progeni- retinal topography, axonal patterning in the midline and tor cap cells during mammary gland morphogenesis. Dev. Cell 4, arterial–venous differentiation. Ephs and Ephrins have 371–382. 5. Kennedy, T.E., and Serafini, T. (1994). Netrins are diffusible been found to regulate integrin functions in a wide chemotropic factors for commissural axons in the embryonic spinal variety of settings (paradigms 2 and 3). cord. Cell 78, 425–435. The regulation of integrins by Ephrin–Eph signaling is 6. Pasterkamp, R.J., Peschon, J.J., Spriggs, M.K., and Kolodkin, A.L. (2003). Semaphorin 7A promotes axon outgrowth through integrins complex and the reports are sometimes contradictory. and MAPKs. Nature 424, 398–405. Stimulation of EphB1 by EphrinB1 and expression of 7. Serini, G., Valdembri, D., Zanivan, S., Morterra, G., Burkhardt, C., Cac- EphA8 activates integrins [16], while stimulation of cavari, F., Zammataro, L., Primo, L., Tamagnone, L., Logan, M., et al. EphB2 by EphrinB1 has been reported to suppress (2003). Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function. Nature 424, 391–397. integrins by way of R-Ras [16]. Stimulation of EphA2 by 8. Suzuki, T., Nakamoto, T., Ogawa, S., Seo, S., Matsumura, T., EphrinA1 has also been shown to suppress integrin- Tachibana, K., Morimoto, C., and Hirai, H. (2002). MICAL, a novel mediated cell adhesion and to promote dephosphory- CasL interacting molecule, associates with vimentin. J. Biol. Chem. 277, 14933–14941. lation of FAK and paxillin by recruiting the SHP2 9. Terman, J.R., Mao, T., Pasterkamp, R.J., Yu, H.H., and Kolodkin, A.L. phosphatase [16]. In another report [17], however, stim- (2002). MICALs, a family of conserved flavoprotein oxidoreductases, ulation of EphA2 by EphrinA1 induces FAK and p130Cas function in plexin-mediated axonal repulsion. Cell 109, 887–900. phosphorylation leading to cell adhesion and spread- 10. Wong, K., Park, H.T., Wu, J.Y., and Rao, Y. (2002). Slit proteins: molecular guidance cues for cells ranging from neurons to leukocytes. ing. These contradictory results suggest that the regu- Curr. Opin. Genet. Dev. 12, 583–591. lation of integrins by Eph–Ephrin signaling might 11. Wong, K., Ren, X.R., Huang, Y.Z., Xie, Y., Liu, G., Saito, H., Tang, H., depend on various contexts, such as the expression Wen, L., Brady-Kalnay, S.M., Mei, L., et al. (2001). Signal transduction in neuronal migration: roles of GTPase activating proteins and the level of each protein. small GTPase Cdc42 in the Slit-Robo pathway. Cell 107, 209–221. Both Ephs and Ephrins are membrane proteins and 12. Bashaw, G.J., Kidd, T., Murray, D., Pawson, T., and Goodman, C.S. Eph/Ephrin signaling is bidirectional. In reverse signal- (2000). Repulsive axon guidance: Abelson and Enabled play oppos- ing roles downstream of the roundabout receptor. Cell 101, 703–715. ing, stimulation of EphrinA5 or EphrinA2 by EphA3 and 13. Lewis, J.M., Baskaran, R., Taagepera, S., Schwartz, M.A., and Wang, stimulation of EphrinB1 by EphB1 enhance integrin- J.Y. (1996). Integrin regulation of c-Abl tyrosine kinase activity and mediated cell adhesion in the Ephrin-containing cells cytoplasmic-nuclear transport. Proc. Natl. Acad. Sci. USA 93, [18–20]. As previously mentioned, EphrinA ligands are 15174–15179. 14. Reinhard, M., Jarchau, T., and Walter, U. (2001). Actin-based motility: GPI-anchored proteins and they do not have a cyto- stop and go with Ena/VASP proteins. Trends Biochem. Sci. 26, plasmic domain but localise to lipid rafts of the plasma 243–249. membrane where they can recruit various proteins 15. Stevens, A., and Jacobs, J.R. (2002). Integrins regulate responsiveness to slit repellent signals. J. Neurosci. 22, 4448–4455. including Src family protein kinases. Given that both 16. Kullander, K., and Klein, R. (2002). Mechanisms and functions of Eph integrins and Eph/Ephrins are involved in cell migration and ephrin signalling. Nat. Rev. Mol. Cell Biol. 3, 475–486. and axon guidance, it is possible that Eph/Ephrin sig- 17. Carter, N., Nakamoto, T., Hirai, H., and Hunter, T. (2002). EphrinA1- naling operates through integrins in the formation of induced cytoskeletal re-organization requires FAK and p130(cas). Nat. Cell Biol. 4, 565–573. the neuronal and vascular networks and in epithelial 18. Davy, A., and Robbins, S.M. (2000). Ephrin-A5 modulates cell adhe- morphogenesis. sion and morphology in an integrin-dependent manner. EMBO J. 19, 5396–5405. Conclusions 19. Huai, J., and Drescher, U. (2001). An ephrin-A-dependent signaling pathway controls integrin function and is linked to the tyrosine phos- Accumulating evidence shows that axonal guidance phorylation of a 120-kDa protein. J. Biol. Chem. 276, 6689–6694. molecules operate outside the nervous system. 20. Huynh-Do, U., Vindis, C., Liu, H., Cerretti, D.P., McGrew, J.T., Axonal guidance molecules can bind directly to inte- Enriquez, M., Chen, J., and Daniel, T.O. (2002). Ephrin-B1 transduces signals to activate integrin-mediated migration, attachment and grins (Netrin-1 and Sema7A) to provide direct adhe- angiogenesis. J. Cell Sci. 115, 3073–3081. sive signals that control cell migration. In other cases (Sema3, Eph/Ephrin), the engagement of axonal guidance receptors can regulate the adhesive function of integrins, and thus indirectly provide adhesive cues. In addition, integrins are signaling receptors in their own right and integrin signals can converge with those