Oncogene (2000) 19, 5614 ± 5619 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Eph receptors and ephrin ligands: embryogenesis to tumorigenesis

Vincent C Dodelet1 and Elena B Pasquale*,1

1The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California, CA 92037, USA

Protein tyrosine kinase genes are the largest family of The Eph receptors become phosphorylated at speci®c oncogenes. This is not surprising since tyrosine kinases tyrosine residues in the cytoplasmic domain following are important components of signal transduction path- ligand binding (Figure 1). Phosphorylated motifs serve ways that control cell shape, proliferation, di€erentia- as sites of interaction with certain cytoplasmic signaling tion, and migration. At 14 distinct members, the Eph proteins such as non-receptor tyrosine kinases of the kinases constitute the largest family of receptor tyrosine Src and Abl families and the non-receptor phosphotyr- kinases. Although they have been most intensively osine phosphatase LMW ± PTP (low molecular weight studied for their roles in embryonic development, phosphotyrosine phosphatase); the enzymes phospholi- increasing evidence also implicates Eph family proteins pase C g, phosphatidylinositol 3-kinase, and Ras in cancer. This review will address the recent progress in GTPase activating protein; the adaptors SLAP, Grb2, understanding the function of Eph receptors in normal Grb10, and Nck; and SHEP1, a R-Ras- and Rap-1A- development and how disregulation of these functions binding protein (reviewed by Bruckner and Klein, 1998; could promote tumorigenesis. Oncogene (2000) 19, Dodelet et al., 1999; Flanagan and Vanderhaeghen, 5614 ± 5619. 1998; Kalo and Pasquale, 1999). Furthermore, through their C terminus the Eph receptors associate with PDZ Keywords: receptor tyrosine kinase; tissue patterning; (postsynaptic density protein, disc large, zona occlu- axon guidance; cell segregation; angiogenesis dens) domain-containing proteins such as AF6, a candidate e€ector for Rap1A (Linnemann et al., 1999), Pick1, a protein kinase C-interacting protein, Introduction Syntenin, a syndecan-interacting protein, and Grip1 and Grip2, two glutamate receptor interacting proteins The Eph receptors have been divided into two groups (Buchert et al., 1999; Hock et al., 1998; Torres et al., based on sequence homology: EphA and EphB 1998). Many of these proteins, which are candidates for receptors (Eph-Nomenclature-Committee, 1997; Pas- mediating the activities of the Eph receptors, have been quale, 1997). The ligands for the Eph receptors are the implicated in regulating cell morphology, attachment ephrins, which share the distinctive property of being and motility. membrane-bound. The ®ve ephrins of the A subclass The transmembrane ephrin-B ligands also become are linked to the membrane through a GPI linkage, phosphorylated on tyrosine residues following receptor while the three ephrins of the B subclass are binding (Bruckner et al., 1997; Holland et al., 1996). transmembrane proteins. Although the ephrins bind The identity of the ephrin-B phosphorylation sites and promiscuously to Eph receptors, some speci®city has their functional signi®cance are unknown. However, been demonstrated (Gale et al., 1996). The ligands of such phosphorylation suggests ephrin-B association the ephrin-A subclass bind preferentially to the EphA with a tyrosine kinase. This association may explain receptors whereas the ligands of the B subclass bind how the ephrin-B ligands, which lack enzymatic preferentially to the B receptors. activity, are able to transduce signals. Ephrin-B ligands The Eph receptors have a ligand-binding domain at have also been shown to interact through their C the amino terminus (Labrador et al., 1997). This terminus with several PDZ domain-containing proteins globular domain is followed by a central cysteine-rich such as Syntenin, Grip, Pick1, Phip and the phospho- region, and two ®bronectin type III repeats near the tyrosine phosphatase FAP-1 (Lin et al., 1999; Torres et membrane-spanning segment. The cytoplasmic region al., 1998) (Figure 1). These proteins may also of the Eph receptors contains a conserved kinase contribute to ephrin-B signaling pathways. Finally, domain ¯anked by less conserved juxtamembrane recent evidence suggests that the ephrin-A ligands can region and C-terminal tail (Pasquale, 1997) (Figure 1). also signal, even though they lack a cytoplasmic region (Davy et al., 1999). Although some of the proteins involved in Eph Bidirectional signaling receptor and ephrin signal transduction have been identi®ed, the exact mechanisms at play are still not An intriguing feature of the Eph receptors is their well understood. involvement in bidirectional signaling (Bruckner et al., 1997; Holland et al., 1996). Recent evidence has shown that signaling pathways can be propagated not only Functions of Eph receptors and ephrin ligands downstream of the Eph receptors but also downstream of the ephrin ligands. Activation of Eph receptors does not appear to have a major e€ect on cell proliferation. Rather, Eph receptors in conjunction with their ephrin ligands have *Correspondence: EB Pasquale been shown to control the directional movement of Eph receptors and ephrin ligands VC Dodelet and EB Pasquale 5615 of cells from adjacent segments (rhombomeres) and from adjacent streams of neural crest cells (Smith et al., 1997; Xu et al., 1995, 1999). Furthermore, the lamina- speci®c and area-speci®c patterns of expression of EphA proteins in the embryonic primate cerebral cortex suggest a role in early speci®cation of pre- sumptive functional domains (Donoghue and Rakic, 1999). A notable example of cell segregation mediated by Eph proteins outside the nervous system is that of EphB4, a receptor expressed in endothelial cells of embryonic veins, and ephrin-B2, a ligand for EphB4 that is expressed in endothelial cells of arteries (Adams et al., 1999; Gerety et al., 1999; Wang et al., 1998).

Axon guidance A classical example of Eph receptor-mediated axon guidance is the speci®cation of retinotectal topography. Temporal retinal axons, which have high EphA3 expression, grow to the anterior tectum, where expression of its ligands, ephrin-A2 and ephrin-A5, is low (Cheng and Flanagan, 1994; Drescher et al., 1995). In vitro experiments have con®rmed that both of these ligands behave as repulsive guidance molecules for axons that express their receptors (Cheng and Flanagan, 1994; Drescher et al., 1995; Monschau et al., 1997). Furthermore, changing the distribution of ephrin-A2 in vivo in the chicken optic tectum by Figure 1 Interactions and signal transduction of Eph receptors retroviral expression caused temporal axons to follow and ephrins. Upon cell-cell contact Eph receptors and ephrins aberrant trajectories that avoided ectopic areas of high engage in a class speci®c manner, with GPI-linked ephrin-A ligand expression (Nakamoto et al., 1996). Gene knock ligands binding to EphA receptors and transmembrane ephrin-B ligands binding to EphB receptors. Binding of ephrins leads to out experiments con®rmed that the repulsive activities receptor clustering, which in turn leads to receptor activation and of the ephrin-A2 and ephrin-A5 ligands are required in subsequent autophosphorylation of multiple tyrosine residues vivo for the proper guidance and mapping of retinal (shown for EphB). These phosphorylated tyrosine residues axons in the mammalian midbrain (Feldheim et al., provide docking sites for SH2 domain-containing signaling proteins. Upon receptor binding, ephrin-B ligands also become 1998; Frisen et al., 1998). Examples of Eph receptor- phosphorylated on tyrosine, presumably via an unidenti®ed mediated axon guidance, however, are not con®ned to associated tyrosine kinase. It is unknown at present whether the retinotectal/retinocollicular projection. Disruption SH2 domain-containing proteins also dock to phosphorylated of the ephrin-A5 gene, for example, caused a distortion ephrin-B ligands. Signaling proteins containing PDZ domains of the body map in the neocortical somatosensory area dock to the carboxy terminus of the Eph receptors (shown for EphA) and the ephrin-B ligands (Vanderhaeghen et al., 2000) and loss of topographic precision in a sensory projection to the forebrain (Feldheim et al., 1998). Furthermore, inactivation of the EphA4 gene caused major path®nding defects in the corticospinal tract (Dottori et al., 1998), inactiva- cells and neuronal growth cones and the establishment tion of the EphB3 receptor gene a€ected the path®nd- of the embryonic body plan, likely by regulating ing of the axons in the corpus callosum (Orioli et al., cytoskeletal organization and cell adhesion (Bruckner 1996), and inactivation of the EphB2 gene caused et al., 1997; Drescher, 1997; Flanagan and Vanderhae- inappropriate pathway selection at the midline in ghen, 1998; Pasquale, 1997). Eph activities are commissural axons that innervate the ear (Cowan et important in neurons and other types of cells, such al., 2000). EphB receptors may also play a role in as neural crest cells and endothelial cells. The functions path®nding of longitudinally projecting spinal cord and expression patterns of Eph family proteins suggest commissural axons (Imondi et al., 2000). that they also play a role in cancer progression and Gene knock out experiments suggest that ephrin-B metastasis. ligands can also transduce signals in the axons in which they are expressed (Birgbauer et al., 2000; Henkemeyer et al., 1996). For example, signals transduced by Cell sorting ephrin-B ligands upon interaction with EphB receptor In many developing tissues, areas where Eph receptors ectodomains appear to guide the growth of axons are expressed con®ne with areas where ephrin ligands forming the brain anterior commissure and, possibly, are expressed (Flenniken et al., 1996; Gale et al., 1996). retinal ganglion cell axons. In fact, these axons project Consistent with these expression patterns, the Eph abnormally in mice lacking certain EphB genes, but the receptor-ligand system restricts intermingling between guidance errors are ameliorated by expression of a adjacent populations of cells (Mellitzer et al., 1999). truncated form of EphB2 lacking the kinase domain Eph family proteins contribute to segmental patterning and containing the ectodomain. Thus, the intrinsic of the hindbrain, because they prevent the intermixing signaling function of EphB2 appears to be dispensable

Oncogene Eph receptors and ephrin ligands VC Dodelet and EB Pasquale 5616 for guidance of these axons while the ectodomain plays de®cient in EphB2 and EphB3, exhibit severe defects in an essential role, presumably by activating ephrin-B the remodeling of the embryonic vascular system. ligand signaling pathways.

Eph receptor and ephrin ligand expression in cancer Functions in synapses Eph family proteins may be involved not only in The ®rst receptor of this gene family, EphA1, was guiding axons to their targets, but also in the originally isolated from an erythropoietin-producing development of initial neuronal contacts into synapses hepatoma cell line (Hirai et al., 1987). Carcinomas of and in synapse maintenance. The recently described breast, liver, lung, and colon showed elevated expres- localization of Eph receptors and ephrins in neuronal sion of this receptor, despite the absence of gene synapses (Buchert et al., 1999; Torres et al., 1998) ampli®cation or rearrangement (Hirai et al., 1987; suggests previously unexpected roles for these proteins. Maru et al., 1988). Additionally, overexpression of They may activate bidirectional signaling pathways in EphA1 in NIH3T3 cells resulted in the formation of synapses, and contribute to synaptic plasticity by foci in soft agar and tumors in nude mice (Maru et al., destabilizing synapses. Alternatively, the interaction 1990). between the EphB2 and ephrin-B1 ectodomains may The expression levels of several other receptors of play adhesive roles. Synaptic localization of EphB2 and both the A and B class were also observed to be ephrin-B1 likely depends on their binding to proteins elevated in various tumor types. EphA2 was found to containing PDZ domains that are concentrated at be overexpressed in nearly all melanoma cell lines neuronal synapses (Buchert et al., 1999; Craven and tested, whereas it was not detected in normal Bredt, 1998; Torres et al., 1998). melanocytes (Easty et al., 1995). Interestingly, EphA2 expression levels were signi®cantly higher in cell lines from distant metastases than in primary melanomas. Angiogenesis EphA3 was identi®ed as an antigen present on the Recent evidence suggests that the Eph receptors are surface of a pre-B cell leukemic cell line and was also one of the families of receptor tyrosine kinases that, in found to be overexpressed in the absence of gene conjunction with their ligands, control blood vessel ampli®cation or rearrangements in some hemopoietic formation. Ephrin-A1, the ®rst ligand for an Eph tumors and several lymphoid tumor cell lines (Wicks et receptor to be identi®ed, was cloned from human al., 1992). EphB2 was found to be overexpressed in umbilical vein endothelial cells (HUVECs) as a gene about one third of 31 human tumor cell lines induced by tumor necrosis factor a (TNFa) (Holzman examined, in 75% of the gastric tumors examined, et al., 1990). Ephrin-A1 transcripts were also detected and in some esophageal and colon cancers (Kiyokawa in embryonic endothelial cells during early organogen- et al., 1994). EphB3 was detected in tumor cell lines of esis but not in adult tissues (Flenniken et al., 1996; breast and epidermoid origins (Bohme et al., 1993). McBride and Ruiz, 1998; Takahashi and Ikeda, 1995), Analysis of human breast carcinoma tissue by in situ suggesting a physiological role for this ligand in hybridization showed that expression of EphB4 is vascular morphogenesis. An angiogenic activity for elevated in primary in®ltrating ductal breast carcino- ephrin-A1 was demonstrated in vivo, in a rat corneal mas with a high grade of malignancy (Berclaz et al., angiogenesis assay (Pandey et al., 1995b). In this assay, 1996). Particularly strong expression was observed in ephrin-A1 speci®cally mediated the angiogenic e€ects grade III carcinomas, in the outer layer of the tumor of TNFa, but not FGF, suggesting that induction of cell mass, which are the layers most likely to release ephrin-A1 and subsequent activation of its receptor, tumor cells that may give rise to metastases. In EphA2, could be responsible for the angiogenic e€ects transgenic mouse models of mammary carcinogenesis, of TNFa. Ephrin-A1 does not cause endothelial cell EphB4 and EphA2 were detected in the undi€eren- proliferation, but acts as a chemoattractant for tiated and invasive mammary tumors of mice expres- endothelial cells. sing the H-Ras oncogene, but not in the well- Three other Eph receptors (EphB2, EphB3 and di€erentiated and non-metastatic mammary tumors of EphB4) and their ligands (ephrin-B1 and ephrin-B2) c-Myc expressing mice (Andres et al., 1994). Progres- have been recently implicated in the formation of the sion of the malignant process in the H-Ras tumors circulatory system of the mouse embryo (Adams et al., correlated with the transition of EphB4 expression 1999; Gerety et al., 1999; Wang et al., 1998). Ephrin-B2 from myoepithelial cells to the anaplastic tumor cells of is expressed in arterial endothelial cells, whereas EphB4 epithelial origin (Nikolova et al., 1998). Expression of has complementary expression and is con®ned to EphB4 mRNA was also detected in all human breast venous endothelial cells. EphB4 and ephrin-B2 may, carcinoma cell lines examined, including MCF7, MDA- therefore, de®ne boundaries between arterial and MB-231, SKBR, T47D, BT20 and BT474 (Bennett et venous endothelial cells. However, cell-cell interactions al., 1994; Berclaz et al., 1996). between Eph receptors and ephrins are not restricted to It therefore seems that a positive correlation is the borders between arteries and veins: ephrin-B1 is emerging between the level of expression of Eph widely expressed in both arterial and venous endothe- receptors in tumor tissue and the observed degree of lial cells and EphB3 is expressed in veins and some malignancy. As for the ephrin ligands, too little data arteries. Finally, expression of ephrin-B2 and EphB2 in exists to establish if such a correlation exists. Indeed, mesenchyme adjacent to vessels suggest that Eph the expression patterns of ephrins in tumors have only family proteins also mediate interactions between begun to be characterized, and it is clear that mesenchymal cells and endothelial cells. Indeed, mice additional studies are necessary. Upregulation of lacking ephrin-B2, and a proportion of double mutants ephrin-A1 and ephrin-B2 has been reported in

Oncogene Eph receptors and ephrin ligands VC Dodelet and EB Pasquale 5617 melanomas (Easty et al., 1999; Vogt et al., 1998), but ephrin-B ligands and the ligand-independent activation in a mouse mammary tumor model, ephrin-B2 potential of the Eph receptors, conditions in tumor expression, which is normally restricted to the luminal tissue where ligand expression is low and receptor epithelial cells, was lost at the onset of tumorigenesis expression is high may be the most favorable for tumor (Nikolova et al., 1998). This last observation may be of growth and metastasis. great interest since transmembrane ephrin B ligands Eph receptors may also in¯uence cell-cell attachment have been shown to inhibit the transforming activity of by interacting with certain cell adhesion molecules. oncogenic tyrosine kinases (Bruckner et al., 1997). Loss of E-cadherin, the main cell adhesion molecule of early embryonic and adult epithelial cells, has been shown to a€ect the expression and subcellular localiza- tion of several Eph receptors and ephrins (Orsulic and Mechanisms of tumorigenesis Kemler, 2000; Zantek et al., 1999). Reciprocally, cadherin function may be regulated by Eph receptors Molecular control of integrin activity ± enhanced cell and ephrins, since ectopic expression of ephrin-B1 or motility, invasion and metastasis activated EphA4 in early Xenopus embryos disrupted In spite of this wealth of information correlating cell cadherin dependent cell adhesion (Jones et al., 1998; transformation with increased expression of the Eph Winning et al., 1996). The molecular mechanisms receptors, the molecular mechanisms underlying the involved in this cross-regulation are still undetermined. speci®c roles of the Eph family in tumor formation, However, because tissue disorganization and abnormal progression, and metastasis have only started to be cell adhesion are hallmarks of the more advanced understood. Recent work has highlighted the ability of stages of cancer, the inappropriate expression and Eph receptors to a€ect cell-matrix attachment by regulation of an Eph receptor would be expected to modulating integrin activity. Activation of endogenous increase tumor metastatic potential. EphA2 in PC-3 prostate carcinoma cells with ephrin- Modulation of cell adhesion is only one aspect, A1 ligand was shown to induce transient inhibition of albeit a crucial one, of cell motility. The general integrin-mediated cell adhesion (Miao et al., 2000). process of cellular guidance in which the Eph receptors Rapid recruitment of the protein tyrosine phosphatase have been implicated also involves reorganization of SHP2 to activated EphA2 lead to dephosphorylation cytoskeletal elements to allow for changes in cell and thus inactivation of focal adhesion kinase (FAK). morphology during movement. The small GTPases of Zou et al. demonstrated that the small GTPase R-Ras, the Rac/Rho family are prime candidates for mediating which regulates integrin binding (Zhang et al., 1996), these e€ects, and a link between the Eph receptors and can be tyrosine phosphorylated by EphB2 (Zou et al., these GTPases has recently been established. Stimula- 1999). This phosphorylation most probably leads to an tion of retinal ganglion cells by ephrin-A5 was shown inability of R-Ras to interact with downstream to induce growth cone collapse in a Rho dependent e€ectors, and ultimately to decreased integrin activa- manner (Wahl et al., 2000). Inhibition of Rho or of the tion and cell adhesion. downstream Rho e€ector, Rho kinase (ROCK), However, the density of ephrin ligand may ulti- dramatically reduced ephrin-A5 induced collapse and mately determine the e€ects of Eph signaling on even allowed for the continuing advance of growth integrin-mediated adhesion. In an attempt to under- cones in the presence of ligand. Since Rho proteins are stand the mechanism whereby Eph receptors guide cell known regulators of the actin cytoskeleton (reviewed in migration in response to ligand gradients, Huynh-Do Mackay and Hall, 1998; Zohn et al., 1998) and have et al. (1999) plated cells expressing EphB1 on surfaces been shown, in vitro, to be essential for cellular coated with de®ned extracellular matrix components invasion (Banyard et al., 2000), their activation by and varying densities of ephrin-B1 ligand. Lower Eph receptors may very well contribute to increased densities of ligand stimulated integrin-mediated attach- tumor invasion and metastasis. ment in an Eph receptor dependent fashion, whereas higher densities did not, despite similar levels of Promotion of angiogenesis ± enhanced tumor growth activation of the receptor, as evidenced by the level of tyrosine phosphorylation (Huynh-Do et al., 1999). Although it is now clear that the Eph receptors and Interestingly, the oligomeric state of activating ligand ephrins play a signi®cant role in the formation of the seems to determine the nature of the signaling complex embryonic vasculature, their precise contribution to that is recruited to the receptor (Stein et al., 1998). pathogenic neovascularization is not well understood. Thus it is probable that, in vivo, cell contact induced However, the existing data does allow us to put forth a Eph receptor activation may in¯uence directional cell few interesting hypotheses. Since ephrins have been motility by modulating integrin function either posi- shown, in in vitro experiments, to promote the tively or negatively, dependent on the ratio of receptor organization and assembly of endothelial cells into to ligand densities. Overexpression of Eph receptors capillary-like structures and to stimulate sprouting may therefore impart tumors with increased sensitivity from existing vessels (Daniel et al., 1996; Pandey et to ligand-induced stimulation, favoring decreased cell al., 1995a), it would seem likely that these molecules adhesion, increased cell motility, and a concomitantly may be involved in similar processes during tumor higher degree of tissue invasiveness. Alternatively, the growth. Proliferation of the tumor mass is dependent presence of ligand may be unnecessary since over- on an adequate blood supply, and many tumors secrete expression of Eph receptors can in and of itself angiopoietic factors for new vessel recruitment (Folk- promote receptor activation (Zisch et al., 1997), and man, 1995; Hanahan et al., 1996; Yancopoulos et al., may thus be sucient to promote these e€ects. In fact, 1998). A complex interplay could occur between Eph given the anti-oncogenic e€ects observed for the receptors/ephrins expressed by tumor cells and en-

Oncogene Eph receptors and ephrin ligands VC Dodelet and EB Pasquale 5618 dothelial cells. For example, secreted ephrins may prognostic tumor markers and targets for therapeutic function as di€usible chemoattractants for endothelial intervention in cancer. cells (as has been reported for ephrin-A1 (Pandey et al., 1995a)), and tumor expressed Eph receptors may act as contact-dependent organizing molecules to appropriately guide the incoming vessels. Acknowledgments Many other possibilities exist but, even if function The authors' work is supported by grants from the NIH (to remains elusive, the fact that blood vessels and tumor EB Pasquale), the University of California Breast Cancer cells express Eph receptors and ephrins makes these Program (EB Pasquale), and the National Sciences and molecules stand out as attractive candidates as Engineering Research Council of Canada (to VC Dodelet).

References

Adams RH, Wilkinson GA, Weiss C, Diella F, Gale NW, Flenniken AM, Gale NW, Yancopoulos GD and Wilkinson Deutsch U, Risau W and Klein R. (1999). Genes Dev., 13, DG. (1996). Dev. Biol., 179, 382 ± 401. 295 ± 306. Folkman J. (1995). Nat. Med., 1, 27 ± 31. Andres AC, Reid HH, Zurcher G, Blaschke RJ, Albrecht D Frisen J, Yates PA, McLaughlin T, Friedman GC, O'Leary and Ziemiecki A. (1994). Oncogene, 9, 1461 ± 1467. DD and Barbacid M. (1998). Neuron, 20, 235 ± 243. Banyard J, Anand-Apte B, Symons M and Zetter BR. (2000). Gale NW, Holland SJ, Valenzuela DM, Flenniken A, Pan L, Oncogene, 19, 580 ± 591. Ryan TE, Henkemeyer M, Strebhardt K, Hirai H, Bennett BD, Wang Z, Kuang WJ, Wang A, Groopman JE, Wilkinson DG, Pawson T, Davis S and Yancopoulos Goeddel DV and Scadden DT. (1994). J. Biol. Chem., 269, GD. (1996). Neuron, 17, 9 ± 19. 14211 ± 14218. Gerety SS, Wang HU, Chen ZF and Anderson DJ. (1999). Berclaz G, Andres AC, Albrecht D, Dreher E, Ziemiecki A, Mol. Cell., 4, 403 ± 414. Gusterson BA and Crompton MR. (1996). Biochem. Hanahan D, Christofori G, Naik P and Arbeit J. (1996). Eur. Biophys. Res. Commun., 226, 869 ± 875. J. Cancer, 32A, 2386 ± 2393. Birgbauer E, Cowan CA, Sretavan DW and Henkemeyer M. Henkemeyer M, Orioli D, Henderson JT, Saxton TM, Roder (2000). Development, 127, 1231 ± 1241. J, Pawson T and Klein R. (1996). Cell, 86, 35 ± 46. BohmeB,HoltrichU,WolfG,LuziusH,GrzeschikKH, Hirai H, Maru Y, Hagiwara K, Nishida J and Takaku F. Strebhardt K and Rubsamen-Waigmann H. (1993). (1987). Science, 238, 1717 ± 1720. Oncogene, 8, 2857 ± 2862. Hock B, Bohme B, Karn T, Yamamoto T, Kaibuchi K, Bruckner K and Klein R. (1998). Curr. Opin. Neurobiol., 8, Holtrich U, Holland S, Pawson T, Rubsamen-Waigmann 375 ± 382. H and Strebhardt K. (1998). Proc. Natl. Acad. Sci. USA, Bruckner K, Pasquale EB and Klein R. (1997). Science, 275, 95, 9779 ± 9784. 1640 ± 1643. Holland SJ, Gale NW, Mbamalu G, Yancopoulos GD, Buchert M, Schneider S, Meskenaite V, Adams MT, Canaani Henkemeyer M and Pawson T. (1996). Nature, 383, 722 ± E, Baechi T, Moelling K and Hovens CM. (1999). J. Cell. 725. Biol., 144, 361 ± 371. Holzman LB, Marks RM and Dixit VM. (1990). Mol. Cell. Cheng HJ and Flanagan JG. (1994). Cell, 79, 157 ± 168. Biol., 10, 5830 ± 5838. Cowan CA, Yokoyama N, Bianchi LM, Henkemeyer M and Huynh-Do U, Stein E, Lane AA, Liu H, Cerretti DP and Fritzsch B. (2000). Neuron, 26, 417 ± 430. Daniel TO. (1999). EMBO J., 18, 2165 ± 2173. Craven SE and Bredt DS. (1998). Cell, 93, 495 ± 498. Imondi R, Wideman C and Kaprielian Z. (2000). Develop- Daniel TO, Stein E, Cerretti DP, St John PL, Robert B and ment, 127, 1397 ± 1410. Abrahamson DR. (1996). Kidney Int. Suppl., 57, S73 ± S81. Jones TL, Chong LD, Kim J, Xu RH, Kung HF and Daar Davy A, Gale NW, Murray EW, Klingho€er RA, Soriano P, IO. (1998). Proc. Natl. Acad. Sci. USA, 95, 576 ± 581. Feuerstein C and Robbins SM. (1999). Genes Dev., 13, Kalo MS and Pasquale EB. (1999). Cell Tissue Res., 298, 1± 3125 ± 3135. 9. Dodelet VC, Pazzagli C, Zisch AH, Hauser CA and Pasquale Kiyokawa E, Takai S, Tanaka M, Iwase T, Suzuki M, Xiang EB. (1999). J. Biol. Chem., 274, 31941 ± 31946. YY, Naito Y, Yamada K, Sugimura H and Kino I. (1994). Donoghue MJ and Rakic P. (1999). J. Neurosci., 19, 5967 ± Cancer Res., 54, 3645 ± 3650. 5979. Labrador JP, Brambilla R and Klein R. (1997). EMBO J., Dottori M, Hartley L, Galea M, Paxinos G, Polizzotto M, 16, 3889 ± 3897. Kilpatrick T, Bartlett PF, Murphy M, Kontgen F and Lin D, Gish GD, Songyang Z and Pawson T. (1999). J. Biol. Boyd AW. (1998). Proc. Natl. Acad. Sci. USA, 95, 13248 ± Chem., 274, 3726 ± 3733. 13253. Linnemann T, Geyer M, Jaitner BK, Block C, Kalbitzer HR, Drescher U. (1997). Curr. Biol., 7, R799 ± R807. Wittinghofer A and Herrmann C. (1999). J. Biol. Chem., Drescher U, Kremoser C, Handwerker C, Loschinger J, 274, 13556 ± 13562. Noda M and Bonhoe€er F. (1995). Cell, 82, 359 ± 370. Mackay DJ and Hall A. (1998). J. Biol. Chem., 273, 20685 ± Easty DJ, Guthrie BA, Maung K, Farr CJ, Lindberg RA, 20688. Toso RJ, Herlyn M and Bennett DC. (1995). Cancer Res., Maru Y, Hirai H and Takaku F. (1990). Oncogene, 5, 445 ± 55, 2528 ± 2532. 447. Easty DJ, Hill SP, Hsu MY, Fallow®eld ME, Florenes VA, Maru Y, Hirai H, Yoshida MC and Takaku F. (1988). Mol. Herlyn M and Bennett DC. (1999). Int. J. Cancer, 84, Cell. Biol., 8, 3770 ± 3776. 494 ± 501. McBride JL and Ruiz JC. (1998). Mech. Dev., 77, 201 ± 204. Eph-Nomenclature-Committee. (1997). Cell, 90, 403 ± 404. Mellitzer G, Xu QL and Wilkinson DG. (1999). Nature, 400, Feldheim DA, Vanderhaeghen P, Hansen MJ, Frisen J, Lu 77 ± 81. Q, Barbacid M and Flanagan JG. (1998). Neuron, 21, Miao H, Burnett E, Kinch M, Simon E and Wang BC. 1303 ± 1313. (2000). Nature Cell. Biol., 2, 62 ± 69. Flanagan JG and Vanderhaeghen P. (1998). Annu. Rev. Neurosci., 21, 309 ± 345.

Oncogene Eph receptors and ephrin ligands VC Dodelet and EB Pasquale 5619 Monschau B, Kremoser C, Ohta K, Tanaka H, Kaneko T, Vogt T, Stolz W, Welsh J, Jung B, Kerbel RS, Kobayashi H, Yamada T, Handwerker C, Hornberger MR, Loschinger Landthaler M and McClelland M. (1998). Clin. Cancer J, Pasquale EB, Siever DA, Verderame MF, Muller BK, Res., 4, 791 ± 797. Bonhoe€er F and Drescher U. (1997). EMBO J., 16, Wahl S, Barth H, Ciossek T, Aktories K and Mueller BK. 1258 ± 1267. (2000). J. Cell. Biol., 149, 263 ± 270. Nakamoto M, Cheng HJ, Friedman GC, McLaughlin T, Wang HU, Chen ZF and Anderson DJ. (1998). Cell, 93, Hansen MJ, Yoon CH, O'Leary DD and Flanagan JG. 741 ± 753. (1996). Cell, 86, 755 ± 766. Wicks IP, Wilkinson D, Salvaris E and Boyd AW. (1992). Nikolova Z, Djonov V, Zuercher G, Andres AC and Proc. Natl. Acad. Sci. USA, 89, 1611 ± 1615. Ziemiecki A. (1998). J. Cell. Sci., 111, 2741 ± 2751. Winning RS, Scales JB and Sargent TD. (1996). Dev. Biol., Orioli D, Henkemeyer M, Lemke G, Klein R and Pawson T. 179, 309 ± 319. (1996). EMBO J., 15, 6035 ± 6049. Xu Q, Alldus G, Holder N and Wilkinson DG. (1995). Orsulic S and Kemler R. (2000). J. Cell. Sci., 113, 1793 ± Development, 121, 4005 ± 4016. 1802. Xu Q, Mellitzer G, Robinson V and Wilkinson DG. (1999). Pandey A, Lindberg RA and Dixit VM. (1995a). Curr. Biol., Nature, 399, 267 ± 271. 5, 986 ± 989. Yancopoulos GD, Klagsbrun M and Folkman J. (1998). Pandey A, Shao H, Marks RM, Polverini PJ and Dixit VM. Cell, 93, 661 ± 664. (1995b). Science, 268, 567 ± 569. Zantek ND, Azimi M, Fedor-Chaiken M, Wang B, Bracken- Pasquale EB. (1997). Curr. Opin. Cell Biol., 9, 608 ± 615. bury R and Kinch MS. (1999). Cell Growth Di€er., 10, Smith A, Robinson V, Patel K and Wilkinson DG. (1997). 629 ± 638. Curr. Biol., 7, 561 ± 570. Zhang JH, Cerretti DP, Yu T, Flanagan JG and Zhou R. Stein E, Lane AA, Cerretti DP, Schoecklmann HO, Schro€ (1996). J. Neurosci., 16, 7182 ± 7192. AD, Van Etten RL and Daniel TO. (1998). Genes Dev., 12, Zisch AH, Stallcup WB, Chong LD, Dahlin-Huppe K, 667 ± 678. Voshol J, Schachner M and Pasquale EB. (1997). J. Takahashi H and Ikeda T. (1995). Oncogene, 11, 879 ± 883. Neurosci. Res., 47, 655 ± 665. Torres R, Firestein BL, Dong H, Staudinger J, Olson EN, Zohn IM, Campbell SL, Khosravi-Far R, Rossman KL and Huganir RL, Bredt DS, Gale NW and Yancopoulos GD. Der CJ. (1998). Oncogene, 17, 1415 ± 1438. (1998). Neuron, 21, 1453 ± 1463. Zou JX, Wang B, Kalo MS, Zisch AH, Pasquale EB and Vanderhaeghen P, Lu Q, Prakash N, Frisen J, Walsh CA, Ruoslahti E. (1999). Proc. Natl. Acad. Sci. USA, 96, Frostig RD and Flanagan JG. (2000). Nat. Neurosci., 3, 13813 ± 13818. 358 ± 365.

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