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J. Biol. Chem., Vol. 277, Issue 49, 46974-46979, December 6, 2002

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An Ephrin Mimetic Peptide That Selectively Targets the EphA2 Receptor^*

Mitchell Koolpe, Monique Dail, and Elena B. Pasquale

From the Burnham Institute, La Jolla, California 92037

Received for publication, August 20, 2002, and in revised form, September 24, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Eph receptor tyrosine kinases represent promising disease targets because they are differentially expressed in pathologic versus normal tissues. The EphA2 receptor is up-regulated in transformed cells and tumor vasculature where it likely contributes to cancer pathogenesis. To exploit EphA2 as a therapeutic target, we used phage display to identify two related peptides that bind selectively to EphA2 with high affinity (submicromolar K_D values). The peptides target the ligand-binding domain of EphA2 and compete with ephrin ligands for binding. Remarkably, one of the peptides has ephrin-like activity in that it stimulates EphA2 tyrosine phosphorylation and signaling. Furthermore, this peptide can deliver phage particles to endothelial and tumor cells expressing EphA2. In contrast, peptides corresponding to receptor-interacting portions of ephrin ligands bind weakly and promiscuously to many Eph receptors. Bioactive ephrin mimetic peptides could be used to selectively deliver agents to Eph receptor-expressing tissues and modify Eph signaling in therapies for cancer, pathological angiogenesis, and nerve regeneration.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Originally identified as regulators of neural development, the Eph family of receptor tyrosine kinases and their ephrin ligands are also critical for vascular development and pathological forms of angiogenesis (1-3). For example, the EphA2 receptor and ephrin-A1, a ligand for EphA2, are coordinately expressed in the vasculature of human tumors and mouse xenograft tumors grown from human cancer cells (4). The EphA2 receptor plays a critical role in tumor necrosis factor  (TNF)¹-induced neovascularization because TNF up-regulates ephrin-A1, which causes receptor activation in blood vessels (5). Similarly, the homeobox transcription factor Hox B3 promotes angiogenesis by up-regulating ephrin-A1 (6). Furthermore, EphA2 signaling is required for the formation of endothelial capillary tubes in vitro (4, 7) and promotes the formation of blood vessel-like structures by melanoma cells (8). The expression of EphA2 appears to be restricted to "activated" adult blood vessels as this receptor has not been detected in either embryonic or adult quiescent blood vessels (4, 9, 10). Ephrin-A1 has also not been detected in adult blood vessels, although it is present in the embryonic vasculature (11).

In addition to being present in tumor endothelial cells, EphA2 and ephrin-A1 are up-regulated in the transformed cells of a wide variety of tumors including breast, prostate, colon, skin, and esophageal cancers (4, 12-15). Many factors increase EphA2 expression in cancer cells including the H-Ras oncogene, E-cadherin, members of the p53 family of transcriptional regulators, DNA damage, and loss of estrogen receptors and c-Myc (16-18).

Because the tumor vasculature is discontinuous and leaky in nature, it is possible to utilize the up-regulation of EphA2 and ephrin-A1 to deliver cancer-eradicating agents to both blood vessels and tumor cells (19). Indeed, systemically administered biological agents can easily penetrate into tumors from the blood circulation (20). Selectively targeting EphA2 and ephrin-A1 is a challenging task because these proteins belong to large families of closely related proteins (21). One approach that has been successfully used to identify peptides that exhibit selectivity for their targets is to screen random peptide libraries displayed on the surface of filamentous bacteriophage (22-25). Therefore, we used a phage display approach to search for peptides that bind selectively to the extracellular domains of EphA2 and ephrin-A1. Remarkably the two peptides identified bind selectively to EphA2, but not other Eph receptors, and antagonize ephrin binding. Intriguingly, at least one of the peptides has bioactive properties in that it stimulates EphA2 tyrosine phosphorylation and signaling. In addition, this peptide delivers phage particles to EphA2-expressing cells and thus can have therapeutic value in delivering agents to tissues that express EphA2. Thus, Eph receptor-binding peptides with different agonistic and drug targeting activities can have important therapeutic applications.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Phage Display-- An M13 phage library (New England Biolabs, Beverly, MA) was used for panning on EphA2. A histidine-tagged mouse EphA2 Fc fusion protein (R&D Systems, Minneapolis, MN) was incubated overnight at 4 °C in nickel-nitrilotriacetic acid-coated ELISA plates at concentrations of 1-10 µg/ml in Tris-buffered saline (TBS) (150 mM NaCl, 50 mM Tris-HCl, pH 7.5). Wells were blocked with 0.5% bovine serum albumin (BSA) in TBS and rinsed with binding buffer (TBS, 1 mM CaCl₂, 0.1% Tween 20).

In round 1 of EphA2 panning, 1.7 × 10¹¹ plaque-forming units (PFUs) of the phage library in 100 µl of binding buffer were incubated for 1 h at room temperature in an EphA2-coated well. Phage remaining bound after washing were eluted with 100 µl of 0.2 M glycine-HCl, pH 2.2 or 100 µg of ephrin-A1 Fc. The entire eluate was used to infect early log phase ER2738 host bacteria and amplified. The phage were concentrated and stored according to the manufacturer's recommendations. In rounds 2 and 3, 2 × 10¹¹ PFU-amplified phage pool from the previous round were added to an EphA2 Fc-coated well and a BSA-coated control well. The phage were panned as described for round 1, except that the Tween concentration in the wash buffer was 0.5%, and eluted phage were titered to assess enrichment.

Phage Binding to Cells-- Phage binding to cells was quantitated either by incubating 1 × 10⁹ PFUs for 60-90 min at 37 °C with 1 × 10⁶ MDA-MB-435 cells in a 0.5-ml suspension or by adding 1 × 10¹⁰ PFUs directly to confluent monolayers of human umbilical vein endothelial (HUVE) cells in 24-well tissue culture plates. The phage were diluted into either Dulbecco's modified Eagle's medium with 1% BSA (MDA-MB-435 cells) or endothelial basal medium-2 (EBM-2) (Clonetics Products, BioWhittaker, Inc., Walkersville, MD) with 1% BSA and 10 mM HEPES (HUVE cells).

ELISA Assays-- Phage binding to Eph receptor-coated plates was quantified using an anti-phage antibody conjugated to horseradish peroxidase (M13 phage detection kit, Amersham Biosciences). For peptide competition assays with phage clones, nickel-nitrilotriacetic acid microtiter wells coated with EphA2 Fc were incubated for 1 h at room temperature with phage clones diluted between 1:600 and 1:9000 in binding buffer (100 µl/well). Unbound phage were washed away, and competing peptides were added for 1 h. Alternatively, peptides and phage were co-incubated together.

Ephrin binding to Eph receptor-coated plates was quantified using alkaline phosphatase (AP) fusion proteins of ephrin-A5 and ephrin-A6. Diluted cell culture supernatants containing ephrin-A5-AP and ephrin-A6-AP were co-incubated with peptides in microtiter wells coated with EphA2 Fc or EphA4 Fc. Ephrins remaining bound after washing were detected by measuring AP activity.

Biotinylated peptides were captured on streptavidin-coated microtiter plates (Pierce) and incubated with Eph receptor Fc fusion proteins. Bound Eph receptors were detected with anti-human Fc antibody conjugated to AP (Promega, Madison, WI). Alternatively, the immobilized biotinylated peptides were incubated with diluted cell culture supernatants containing EphA2-AP. Substrates were 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) for horseradish peroxidase and p-nitrophenyl phosphate for AP. Absorbance at 405 or 450 nm was measured using an ELISA plate reader.

Synthetic Peptides-- Biotinylated peptides, containing a carboxyl-terminal GSGSK linker, were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry, purified by high pressure liquid chromatography, and verified by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Plasmids-- The ephrin-A5-AP and ephrin-A6-AP plasmids have been described (26). To construct the EphA2 AP (EphA2-AP) plasmid, the globular amino-terminal region of human EphA2 (amino acids 1-219, GenBank^TM M36395) was amplified by PCR and cloned into the APtag-2 vector (27). The expression plasmid was transiently transfected into 293T cells using Superfect transfection reagent (Qiagen). Cell culture supernatants containing the AP fusion proteins were centrifuged to eliminate cell debris, supplemented with 20 mM HEPES, and stored frozen at -20 °C.

Immunoprecipitation and Immunoblotting-- HUVE cells were grown in microvascular endothelial cell medium-2 (EGM-2 MV) (Clonetics) with 10% fetal calf serum and serum-starved in EBM-2 for 2 h prior to stimulation with 2 µg/ml ephrin-A1 Fc or Fc protein in the presence or absence of YSA peptide. For some experiments, ephrin-A1 Fc was cross-linked by preincubation with 0.2 µg/ml anti-human Fc antibodies for 30 min on ice. After stimulation, the cells were lysed in modified RIPA buffer (4). Cell lysates were immunoprecipitated with 5 µg of anti-EphA2 antibody (Upstate, Lake Placid, NY), separated by SDS-polyacrylamide gel electrophoresis and probed by immunoblotting with peroxidase-conjugated anti-phosphotyrosine antibody (Transduction Laboratories, San Diego, CA) or anti-EphA2 antibody followed by a secondary anti-mouse IgG peroxidase-conjugated antibody (Amersham Biosciences). Alternatively, non-serum starved HUVE cells were stimulated with 2 µg/ml ephrin-A1 Fc or Fc protein in the presence or absence of YSA peptide. Cell lysates were used to immunoprecipitate EphA2 as described above or probed by immunoblotting with anti-phopho-p44/p42 MAPK antibody (Cell Signaling Technology, Beverly, MA) followed by an anti-mouse IgG peroxidase-conjugated antibody (Amersham Biosciences) and reprobed with an anti-ERK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by a secondary anti-rabbit IgG peroxidase-conjugated antibody (Amersham Biosciences).

BIAcore Analysis-- EphA2 Fc was covalently coupled to activated biosensor chips, and the equilibrium binding of peptides at various concentrations was determined by measuring changes in surface plasmon resonance using the Biacore 3000. The chips were regenerated by washing with 1 M Na₂CO₃, pH 10.5.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification of Peptides That Target EphA2-- An M13 phage library displaying random 12-mer peptides was panned on the extracellular domain of EphA2 fused to human Fc (Fig. 1A). Bound phage was eluted with a low pH solution to maximize phage recovery or with ephrin-A1 to improve recovery of peptides that interact with the ligand-binding site of EphA2. After several rounds of selection on EphA2, the screen yielded ~17-fold (low pH elution) and 115-fold (ephrin-A1 elution) enrichment of phage binding to EphA2 versus phage binding to BSA. In contrast, panning on ephrin-A1 did not result in phage enrichment and was not pursued further.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Identification of two peptides that bind selectively and with high affinity to EphA2. A an M13 phage library was panned on wells coated with EphA2 Fc or BSA as a control. EphA2-bound phage were recovered by low pH elution or ephrin-A1 ligand elution, amplified, and used in successive rounds of panning. Phage recovered from the first round of panning (R1*) was not quantitated to avoid losing possible rare clones. PFUs per microliter of phage are shown for R2 and R3. Peptide sequences of the EphA2-binding clones isolated are shown. B, immobilized SWL and YSA peptides were used to capture Fc fusion proteins of EphA receptors. C, changes in surface plasmon resonance units over the surface of Biacore biosensor chips coated with EphA2 Fc were measured for various concentrations of peptides. Dissociation constants were determined by non-linear regression.

Nineteen of 20 individual phage clones from the pool eluted with low pH bind specifically to EphA2 Fc when compared with ephrin-A1 Fc, which was used as a negative control (data not shown). All 19 clones display the same peptide: SWLAYPGAVSYR (SWL peptide). Furthermore, nine of 10 phage clones from the pool eluted with ephrin-A1 bind specifically to EphA2 (data not shown). Seven of these clones display the SWL peptide and two display the peptide YSAYPDSVPMMS (YSA peptide). The corresponding synthetic SWL and YSA peptides also bind to EphA2 but, surprisingly, not to other EphA receptors (Fig. 1B). Equilibrium binding data obtained by surface plasmon resonance indicate that the YSA peptide binds to EphA2 with higher affinity (K_D = 186 nM ± 7) than the SWL peptide (K_D = 678 nM ± 23) (Fig. 1, C and D).

The YSA Peptide Targets EphA2 on the Cell Surface and Stimulates Activation of the Receptor-- The YSA phage exhibit 50-fold higher binding than wild type phage to MDA-MB-435 human breast cancer cells overexpressing the EphA2 extracellular domain on their surface (Fig. 2A). The SWL phage instead exhibit 7-fold higher binding. In the case of untransfected MDA-MB-435 cells, which express only low levels of endogenous EphA2, the YSA phage show a 2.5-fold higher binding than wild type phage, whereas the SWL phage does not show specific binding. The lower binding affinity of the SWL peptide may account for its inability to mediate phage binding unless EphA2 expression is high. The YSA phage also exhibit 12-fold higher binding than wild type phage to HUVE cells, which express moderate levels of EphA2. In contrast, the SWL phage does not specifically bind to these cells. Hence, the YSA peptide is a promising targeting agent for tumor and endothelial cells. Surprisingly, in addition to binding to EphA2 in HUVE cells the YSA peptide also stimulates tyrosine phosphorylation of the receptor in the absence of ephrin and does not decrease EphA2 phosphorylation in the presence of ephrin (Fig. 2B). Furthermore, the YSA peptide activates a previously described EphA2 signaling pathway that suppresses MAP kinase activation (28). Thus, the YSA peptide is an agonist for EphA2.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   The YSA peptide delivers phage particles to cells expressing EphA2 and stimulates receptor signaling. A, equal amounts of wild type control phage (WT) and phage displaying the SWL and YSA peptides were incubated with MDA-MB-435 human breast cancer cells overexpressing the extracellular and transmembrane domains of EphA2 fused to enhanced green fluorescent protein (MDA EphA2-EGFP) (4), untransfected MDA-MB-435 cells (MDA WT), or HUVE cells (HUVEC). Binding was performed using MDA cells in suspension and adherent HUVE cells. PFUs corresponding to total eluted phage are shown. Error bars show standard deviation from duplicate platings. B, serum-starved HUVE cells were treated for 20 min with Fc, ephrin-A1 Fc cross-linked with anti-Fc antibodies, or ephrin-A1 Fc. Where indicated, the cells were preincubated with 10 µM or 50 µM YSA peptide for 20 min prior to the Fc or ephrin-A1 Fc treatment. EphA2 was immunoprecipitated (IP) and probed by immunoblotting (IB) with anti-phosphotyrosine (PTyr) or anti-EphA2 antibodies. C, non-serum-starved HUVE cells were treated for 20 min with Fc or ephrin-A1 Fc. Where indicated, the cells were preincubated with 10 µM or 50 µM YSA peptide for 20 min prior to the Fc treatment. Cell lysates were probed by immunoblotting (IB) with anti-phospho-p42/p44 MAP kinase antibodies (P-MAPK), which detect the phosphorylated activated forms of Erk1 and Erk2 MAP kinases, or with anti-Erk2 (MAPK) antibodies as a control. Erk2 (p42 MAP kinase) is the major phosphorylated form present. EphA2 was immunoprecipitated (IP) and probed by immunoblotting with anti-phosphotyrosine (PTyr) or anti-EphA2 antibodies.

The YSA and SWL Peptides Inhibit Ephrin-A Binding to EphA2-- The YSA and SWL peptides inhibit, in a concentration-dependent manner, the binding of A-ephrins to immobilized EphA2 but not EphA4 (Fig. 3, A and B). This suggests that the peptides bind to the surfaces of EphA2 that interact with the ephrins. ELISA assays confirmed that the YSA and SWL peptides bind to the globular ligand-binding domain of EphA2, which is at the amino terminus of the receptor and contains two distinct ephrin-binding regions (29, 30) (Fig. 3C).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   The SWL and YSA peptides antagonize binding of ephrin-A ligands and bind to the EphA2 ligand-binding domain. AP fusion proteins of ephrin-A5 (A) and ephrin-A6 (B) were incubated in wells coated with EphA2 Fc or EphA4 Fc before adding peptides at the indicated concentrations. C, an AP fusion protein of the ligand-binding domain of human EphA2 (EphA2-AP) was incubated in wells coated with ephrin-A1 Fc, BSA as a control, or peptides. The control peptide was a 12-mer peptide of unrelated sequence. Error bars in all panels show standard deviation from duplicate measurements.

Peptides Corresponding to Ephrin Sequences Bind to Eph Receptors Promiscuously and Weakly-- The YSA and SWL peptides, which have related sequences, have some similarity with a high affinity Eph receptor-binding interface (the G-H loop of the A-ephrins) (Fig. 4A) (31). When their sequences are considered in reverse order, the peptides are also similar to a lower affinity receptor binding interface (the A-A' -strand) (Fig. 4B). Twelve-mer synthetic peptides corresponding to the G-H loop of ephrin-A3 (A3 peptide, Fig. 4A) and the A-A' -strand of ephrin-A5 (A5 peptide, Fig. 4B) indeed bind EphA2, albeit more weakly than the YSA peptide (Fig. 4C). Even a longer A5 peptide containing additional receptor-binding residues (VADRYAVYWNSSNPR) exhibits a similar weak binding (data not shown). Interestingly, the A3 and A5 peptides bind promiscuously to all EphA receptors, similar to A-ephrins and even to EphB4 (Fig. 4, D and E) (21).

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Peptides corresponding to A-ephrin sequences bind promiscuously to Eph receptors. A, alignment of the sequences of the SWL and YSA peptides, the G-H loop of the A-ephrins, and an "A3 peptide" that was synthesized based on the alignment. B, alignment of the reverse sequences of the SWL and YSA peptides, the A-A' -strand of ephrins, and an "A5 peptide" that was synthesized based on the alignment. Identical amino acids are in dark gray; amino acids with similar characteristics are in light gray. C, biotinylated A3, A5, and YSA peptides immobilized on streptavidin-coated wells were used to capture EphA2 Fc. Absorbance readings at 405 nm at 5 and 20 min are shown. D and E, biotinylated A3 and A5 peptides immobilized on streptavidin-coated wells were used to capture Fc fusion proteins of EphA receptors. Error bars in all panels show standard deviation from duplicate measurements.

To further characterize the binding site of the YSA and SWL peptides, we performed competition experiments with phage-displayed peptides. Synthetic SWL peptide competes with YSA phage bound to immobilized EphA2 (Fig. 5A), and conversely YSA peptide competes with SWL phage (data not shown). This indicates that the YSA and SWL peptides bind to the same or overlapping sites on EphA2. The A3 peptide does not inhibit phage binding (Fig. 5, A and B), suggesting that the A3 peptide, which presumably binds to the high affinity ephrin-binding site, may bind too weakly to compete. Indeed, the A3 peptide also does not compete with ephrin-A5 for binding to EphA2 (Fig. 5C). In contrast, the A5 peptide enhances both phage and ephrin-A5 binding to EphA2 (Fig. 5, B and C). Taken together, these results suggest that binding of the A5 peptide to the low affinity ephrin-binding site of EphA2 enhances binding of ephrins and YSA and SWL peptides to the high affinity site.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   The ephrin-derived A5 peptide enhances binding of ephrin-A5 and YSA and SWL peptides to EphA2. A, phage clones displaying the YSA peptide were incubated with immobilized EphA2 Fc, unbound phage was washed away, and synthetic YSA, SWL, A3, A5 peptides, or a control peptide of unrelated sequence were incubated at the indicated concentrations. B, phage clones displaying the YSA or SWL peptide were co-incubated with synthetic YSA, SWL, A3, and A5 peptides at the indicated concentrations in wells coated with EphA2 Fc. C, an ephrin-A5 AP fusion protein was co-incubated with the YSA, SWL, A3, and A5 peptides in wells coated with EphA2 Fc. Error bars show standard deviation from duplicate (A and B) or triplicate (C) measurements.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have used phage display to isolate novel peptides that selectively bind to the EphA2 receptor, a cell-surface protein present in pathologic angiogenic vasculature, including tumor vasculature, and in many types of cancer cells (2). Thus, targeting this receptor should allow therapeutic intervention in cancer and other diseases. The YSA peptide could be used to deliver cytotoxic agents to blood vessels of diseased tissues. Indeed, vascular-targeted peptides coupled to chemotherapeutic drugs, toxins, or proapoptotic peptides can decrease tumor growth, suppress clinical arthritis, or destroy prostate tissue (32-36). The YSA peptide has the added benefit in that it stimulates EphA2 activation, which likely mediates internalization of the receptor and the peptide (37-39). Therefore, toxic or apoptotic substances could be delivered intracellularly to selectively kill cells (34). Furthermore, activation of EphA2 signaling induced by the YSA peptide should reduce proliferation, invasiveness, and metastatic behavior of EphA2-expressing cancer cells (28, 37, 38). This is consistent with the finding that EphA2 activation correlates with decreased malignancy of breast and prostate cancer cells and reverses the transforming effects of EphA2 overexpression (18, 37, 38). Intriguingly, EphA2 activation could sensitize cells to apoptotic stimuli when the YSA peptide is used to deliver cytotoxic agents (17).

Phage-displayed peptides isolated by panning on receptors often bind within ligand-binding sites (40-43) and can mimic natural receptor-binding motifs of ligands (42, 44, 45). Our evidence strongly suggests that the YSA and SWL peptides bind to the interface of EphA2 that mediates the initial high affinity heterodimerization with ephrins (31). First, the peptides interact with the ligand-binding globular domain of EphA2 and antagonize ephrin binding. Second, at least one of the peptides causes EphA2 phosphorylation and signaling, similar to ephrins. Third, the YSA and SWL peptide, as ephrin-A5, bind to a site whose affinity is regulated by the ephrin-derived A5 peptide (Fig. 5, B and C). This effect of the A5 peptide, which presumably binds to the low affinity tetramerization interface of EphA2 (31), also suggests a previously unrecognized allosteric regulation between the two ephrin-binding sites of an Eph receptor.

The YSA and SWL peptides bind selectively only to EphA2. Therefore these peptides have features that confer specificity and are not shared by the ephrins and the ephrin-derived peptides. However, the two related YSA and SWL peptides do show similarity to receptor-binding sequences of A-ephrins, including a conserved xx motif (where  is an aromatic amino acid and x is a non-conserved amino acid) present in both the dimerization and tetramerization interfaces. Understanding the structural determinants of peptide-binding promiscuity versus specificity and high affinity may allow the rational design of new peptides that bind with high affinity to specific Eph receptors.

Conceivably, phage display could also be used to isolate peptides that specifically bind to each of the fifteen known Eph receptors (cbweb.med.harvard.edu/eph-nomenclature). Different Eph receptors have been implicated in various types of cancer and should be investigated as possible therapeutic targets (2). Indeed, we have already isolated peptides that bind EphB4 (data not shown), another receptor expressed in blood vessels and up-regulated in tumors (2). Eph receptors could also be targets for promoting nerve regeneration, where ephrin mimetic peptides could desensitize regenerating nerves to the repulsive effects of ephrins up-regulated at injury sites (46-48).

Bioactive peptides that selectively target an Eph receptor also represent unique reagents for developmental studies to interfere with the activity of a specific receptor, a feat not possible by using the naturally occurring ligands. In addition, such peptides will be useful to discern the signal transduction mechanisms of different Eph receptors in cells expressing multiple receptors. Small-molecule ephrin mimetics with exclusive specificity have great potential value as tools to characterize Eph receptor function and as therapeutics to change such function in disease.

	ACKNOWLEDGEMENTS

We thank Fernando Ferrer, Christian Lombardo, Arnold Satterthwait, Fatima Valencia, Steven Kridel, and Jason Hoffman for technical help and advice; John Flanagan for the AP vector; and Keith Murai for comments on the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant CA82713 (to E. B. P.), Department of Defense postdoctoral fellowship DAMD17-01-1-0168 (to M. K.) awarded and administered by the United States Army Medical Research Acquisition Activity, and an American Heart Association predoctoral fellowship (to M. D.). We assert that the content of this manuscript does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3131; Fax: 858-646-3199; E-mail: elenap@burnham.org.

Published, JBC Papers in Press, September 25, 2002, DOI 10.1074/jbc.M208495200

	ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; PFU, plaque-forming units; AP, alkaline phosphatase; HUVE, human umbilical vein endothelial; MAP, mitogen-activated protein.

	REFERENCES

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