Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses

Abstract

Eph family receptor tyrosine kinases (including EphA3, EphB4) direct pathfinding of neurons within migratory fields of cells expressing gradients of their membrane-bound ligands. Others (EphB1 and EphA2) direct vascular network assembly, affecting endothelial migration, capillary morphogenesis, and angiogenesis. To explore how ephrins could provide positional labels for cell targeting, we tested whether endogenous endothelial and P19 cell EphB1 (ELK) and EphB2 (Nuk) receptors discriminate between different oligomeric forms of an ephrin-B1/Fc fusion ligand. Receptor tyrosine phosphorylation was stimulated by both dimeric and clustered multimeric ephrin-B1, yet only ephrin-B1 multimers (tetramers) promoted endothelial capillary-like assembly, cell attachment, and the recruitment of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) to receptor complexes. Cell-cell contact among cells expressing both EphB1 and ephrin-B1 was required for EphB1 activation and recruitment of LMW-PTP to EphB1 complexes. The EphB1-binding site for LMW-PTP was mapped and shown to be required for tetrameric ephrin-B1 to recruit LMW-PTP and to promote attachment. Thus, distinct EphB1-signaling complexes are assembled and different cellular attachment responses are determined by a receptor switch mechanism responsive to distinct ephrin-B1 oligomers.

Figure 1

Ephrin-B1/Fc dimers and multimers elicit different responses. (A) In vitro angiogenesis assay (Martin et al. 1997) HRMEC were plated on Matrigel-coated dishes in defined medium in the absence (NA), or presence of PMA (20 ng/ml), ephrin-B1/Fc (at the indicated concentrations, ng/ml) or a control Fc fusion protein, ORF/Fc (Beckmann et al. 1994) (500 ng/ml). Fc fusion proteins were presented as either dimers, or as preclustered multimers (+ anti-Fc). Cells were photographed 8 hr after plating. (Top) Complexes recovered with EphB1 (anti-EphB1) or nonimmune (anti-NI) antibodies from HRMEC stimulated for 15 min with indicated concentrations of ephrin-B1/Fc dimers (ephrin-B1/Fc), preclustered ephrin-B1/Fc multimers (ephrin-B1/Fc plus anti-Fc) or control fusion dimers (ORF/Fc, 500 ng/ml) or multimers (ORF/Fc plus anti-Fc ng/ml), were evaluated by immunoblot for receptor recovery (anti-EphB1) and activation (anti-pTyr). (B) P19 cells were plated on fibronectin coated dishes in the presence of the indicated concentrations of dimeric or multimeric forms of ephrin-B1/Fc or ORF/Fc, as described in Materials and Methods. The percentage of total cells attached after 90 min is displayed. (Mean ±s.e.m. of three independent determinations). In the insert, EphB1 receptors were immunoprecipitated from P19 cells plated on fibronectin-coated dishes and stimulated with 500 ng/ml of the indicated agents for 20 min. EphB1 recovery (anti-EphB1) and activation (anti-pTyr) were evaluated by immunoblot.

Figure 1

Ephrin-B1/Fc dimers and multimers elicit different responses. (A) In vitro angiogenesis assay (Martin et al. 1997) HRMEC were plated on Matrigel-coated dishes in defined medium in the absence (NA), or presence of PMA (20 ng/ml), ephrin-B1/Fc (at the indicated concentrations, ng/ml) or a control Fc fusion protein, ORF/Fc (Beckmann et al. 1994) (500 ng/ml). Fc fusion proteins were presented as either dimers, or as preclustered multimers (+ anti-Fc). Cells were photographed 8 hr after plating. (Top) Complexes recovered with EphB1 (anti-EphB1) or nonimmune (anti-NI) antibodies from HRMEC stimulated for 15 min with indicated concentrations of ephrin-B1/Fc dimers (ephrin-B1/Fc), preclustered ephrin-B1/Fc multimers (ephrin-B1/Fc plus anti-Fc) or control fusion dimers (ORF/Fc, 500 ng/ml) or multimers (ORF/Fc plus anti-Fc ng/ml), were evaluated by immunoblot for receptor recovery (anti-EphB1) and activation (anti-pTyr). (B) P19 cells were plated on fibronectin coated dishes in the presence of the indicated concentrations of dimeric or multimeric forms of ephrin-B1/Fc or ORF/Fc, as described in Materials and Methods. The percentage of total cells attached after 90 min is displayed. (Mean ±s.e.m. of three independent determinations). In the insert, EphB1 receptors were immunoprecipitated from P19 cells plated on fibronectin-coated dishes and stimulated with 500 ng/ml of the indicated agents for 20 min. EphB1 recovery (anti-EphB1) and activation (anti-pTyr) were evaluated by immunoblot.

Figure 2

Ephrin-B1/Fc multimers recruit LMW–PTP to EphB1 and EphB2 complexes. EphB1 immunoprecipitates from HRMEC (left) or P19 cells (center) or EphB2 immunoprecipitates (from HRMEC, right) were recovered from cells exposed to no addition (NA), ORF/Fc or ephrin-B1/Fc (500 ng/ml) as either dimers (1−anti-Fc) or as preclustered multimers (+anti-Fc). Receptor complex immunoblots were analyzed to show receptor recovery (anti-EphB1), left and center, or anti-EphB2, right, receptor activation (130 kD, anti-pTyr), an 18-kD tyrosine phosphoprotein (anti-pTyr) that is recognized by antibodies to LMW–PTP (anti-LMW–PTP). The 25-kD band in all samples is immunoglobulin light chain from the immunoprecipitation. LMW–PTP is recruited to both EphB1 and EphB2 receptor complexes only by ephrin-B1/Fc multimers (+anti-Fc).

Figure 3

Reconstitution of EphB1 binding of LMW–PTP requires Y929 and an active EphB1 tyrosine kinase. (A) A yeast two-hybrid system (Stein et al. 1996) was used to analyze interactions between LMW–PTP and the wild-type EphB1 cytoplasmic domain (EphB1cy), truncations of indicated subdomains (EphB1cy/ΔJM, EphB1cy/ΔCterm) or point mutants (EphB1cyY929F, or EphB1cyK652R). Two-hybrid dependent growth (+) on histidine deficient selection media was eliminated (−) by deletion of the carboxy-terminal domain, inactivation of tyrosine-kinase function (K652R), or point mutation of Y929 (Y929F). (B) Recombinant GST fusions comprising the wild-type EphB1 cysoplasmic domain (GST–EphB1cy), a kinase-inactive mutant (GST–EphB1cyK652R) or GST–EphB1cy–Y929F/HA, were expressed in Sf9 cells, immobilized on glutathione-sepharose, incubated in kinase buffer in the presence (+) or absence (−) of ATP (Stein et al. 1996) as described in Materials and Methods. Glutathione–Sepharose-immobilized, phoshphorylated (+ATP), or unphosphorylated (−ATP) GST fusion proteins were incubated with HRMEC extracts or recombinant LMW–PTP protein, washed extensively, then analyzed by immunoblot, by use of the indicated antibodies. (C) P19 cells were transfected with the indicated ratios of pSRα expression constructs encoding wild-type EphB1 (pSRα-hEphB1/HA) or mutant (pSRα-hEphB1–Y929F/HA). Cells were stimulated with 500 ng/ml of ephrin-B1/Fc (dimers) or precomplexed ephrin-B1/Fc (multimers, +anti-Fc), or no addition, as indicated. (Top) Cells were stimulated with ephrin-B1/Fc multimers (+anti-Fc) and EphB1 immunoprecipitates (anti-HA) were analyzed for EphB1 activation (anti-pTyr) and recruitment of LMW–PTP (anti-LMW–PTP) by immunoblot. (Left) Transfected cells were exposed to indicated agonists in solution and attachment to fibronectin coated dishes was assayed (Materials and Methods). (Right) Alternatively, nitrocellulose-coated dishes were precoated with either fibronectin alone (FN), or in combination with ephrin-B1/Fc dimers or multimers (+anti-Fc), and cell attachment was assayed (Materials and Methods).

Figure 4

Ephrin-B1/Fc tetramers recruit LMW–PTP to EphB1 and promote attachment. (A) Ephrin-B1/Fc dimers (ephrin-B1/Fc, 50 μg) or preclustered multimers (ephrin-B1/Fc plus 50 μg of anti-Fc 5 μg) were separated by exclusion chromatogrpahy in PBS on a Superose 6 column (Pharmacia) precalibrated with standards of 445 kD (ferritin), 272 kD (urease), and 150 kD (IgG) as indicated. Fractions containing 500 ng/ml of protein from the indicated A₂₇₈ peaks (A,B,C) were analyzed for activity to promote tyrosine phosphorylation of EphB1 and recruitment of LMW–PTP to EphB1 complexes (B) and to promote attachment of HRMEC and P19 cells (C). Fractions containing complexes of size consistent with tetramers (one anti-Fc molecule complexing two ephrin-B1/Fc dimers) promoted LMW–PTP recruitment and attachment.

Figure 5

Endogenous ligand activation of EphB1 requires cell–cell contact and promotes LMW–PTP recruitment. Equal numbers of HRMEC (5 × 10⁵ cells) were plated on Matrigel-coated 35 (p35)-, 60-, (p60)-,100-, (p100)-, or 150 (p150)-mm-diam. dishes, representing 1×, 3.1×, 8.6×, or 19.5× unit surface areas, in medium supplemented with 1% bovine albumin. Cells were incubated for 2 hr at 37°C, then stimulated for 15 min with either no addition (NA), control Fc fusion (ORF/Fc, 250 ng/ml), preclustered ORF/Fc (250 ng/ml) plus anti-Fc (25 ng/ml), ephrin-B1/Fc (250 ng/ml), preclustered ephrin-B1/Fc (250 ng/ml) plus anti-Fc (25 ng/ml), or phorbol myristate acetate (PMA, 2 ng/ml). Immunoprecipitated EphB1 receptor complexes were analyzed for receptor activation (anti-pTyr, 130-kD band) and recruitment of the 18-kD LMW–PTP (anti-pTyr and anti-LMW–PTP). The 25-kD band is immunoglobulin light chain from the immunoprecipitation. Notably, PMA-stimulated cell density-dependent activation of EphB1 and recruitment of LMW–PTP to receptor complexes.

Figure 6

EphB1/Fc blocks juxtacrine activation of EphB1 and EphB2 by PMA and blocks capillary-like endothelial assembly. (A) HRMEC were plated on Matrigel-coated 35-mm dishes and stimulated as above with agonists added in the absence (−) or presence (+) or a competitive EphB1 ectodomain antagonist, EphB1/Fc (500 ng/ml). As before, EphB1 immunoprecipitates were analyzed for receptor recovery (anti-EphB1), activation (anti-pTyr, 130kD), and recruitment of LMW–PTP (anti-pTyr and anti-LMW–PTP, 18 kD). EphB1/Fc blocked the PMA-stimulated EphB1 activation and LMW–PTP recruitment. (B) EphB1 (left) or EphB2 (right) immunoprecipitates were recovered from HRMEC plated on Matrigel at high density and stimulated for 15 min with no addition (NA), PMA (20 ng/ml), or preclustered ephrin-B1/Fc (250 ng/ml) plus anti-Fc (25 ng/ml), as above, in the absence (−) or presence (+) or EphB1/Fc (500 ng/ml). (C) Cells were plated as in Fig. 1 in medium containing no addition (no addition), PMA (20 ng/ml) or preclustered ephrin-B1/Fc (250 ng/ml) plus anti-Fc (25 ng/ml), in the absence (NA) or presence (+EphB1/Fc, 500 ng/ml) of receptor ectodomain competitor. EphB1/Fc blocked capillary-like endothelial assembly responses to PMA and clustered ephrin-B1/Fc.