Figure 6.
Figure 6. Intracellular clustering determinants sensitize or desensitize cells toward extracellular ephrinB2. (A) Model showing possible synergistic response between extracellular (ephrinB2-Fc) and intracellular (dimerizer) clustering determinants. EphB2-1FKBP forms dimers in the presence of AP20187. Both ephrin ligand and AP20187 together can induce functional oligomers. (B) Western blots of anti-Flag–immunoprecipitated EphB2-1FKBP overexpressed in HeLa cells using anti-phospho-EphB2 antibodies; blot was stripped and reblotted for total EphB2. Cells were stimulated with control (ethanol) or AP20187 (250 nM), in the absence or presence of 5 ng/ml (+) or 10 ng/ml (++) unclustered ephrinB2-Fc for 10 min. Numbers below the blots indicate fold change in phosphorylation compared with Fc/ethanol control (1.0), normalized to total receptor protein levels (representative blot of three experiments). (C) Quantification of collapse responses of cells expressing low levels of the 1FKBP isoform induced by the indicated combinations of stimuli (as in B; for description of the assay see Fig. 4 A; mean cell area ± SEM from n = 8–11 cells per condition; Statistical significance was determined as in Fig. 4 A. Green stars, dimerizer alone versus dimerizer plus ephrinB2-Fc; **, P < 0.01. (D) Model showing intracellular steric hindrance (using myr-FRB-mCherry) of extracellular (ephrinB2-Fc–induced) clustering. In the presence of heterodimerizer AP21967 the coexpressed inhibitory construct myr-FRB-mCherry couples to the wtEphB2-3FKBP receptor, keeping it as monomer. EphrinB2-Fc can still bind to the ectodomain of the receptor. (E) Western blot of immunoprecipitated wtEphB2-3FKBP (carrying a Flag epitope tag) using the phospho-specific anti-EphB2 antibody; blot was stripped and reblotted for total EphB2. HeLa cells transfected with both wtEphB2-3FKBP and myr-FRB-mCherry were stimulated with ephrinB2-Fc in the presence or absence of AP21967 (representative blot of two separate experiments). (F) Quantification of cell collapse assays. The presence of AP21967 prevents EphB2-3FKBP–transfected cells from collapse in response to ephrinB2-Fc. Mean cell area ± SEM from n = 9 cells per condition tested; P < 0.001, Mann-Whitney nonparametric test.

Intracellular clustering determinants sensitize or desensitize cells toward extracellular ephrinB2. (A) Model showing possible synergistic response between extracellular (ephrinB2-Fc) and intracellular (dimerizer) clustering determinants. EphB2-1FKBP forms dimers in the presence of AP20187. Both ephrin ligand and AP20187 together can induce functional oligomers. (B) Western blots of anti-Flag–immunoprecipitated EphB2-1FKBP overexpressed in HeLa cells using anti-phospho-EphB2 antibodies; blot was stripped and reblotted for total EphB2. Cells were stimulated with control (ethanol) or AP20187 (250 nM), in the absence or presence of 5 ng/ml (+) or 10 ng/ml (++) unclustered ephrinB2-Fc for 10 min. Numbers below the blots indicate fold change in phosphorylation compared with Fc/ethanol control (1.0), normalized to total receptor protein levels (representative blot of three experiments). (C) Quantification of collapse responses of cells expressing low levels of the 1FKBP isoform induced by the indicated combinations of stimuli (as in B; for description of the assay see Fig. 4 A; mean cell area ± SEM from n = 8–11 cells per condition; Statistical significance was determined as in Fig. 4 A. Green stars, dimerizer alone versus dimerizer plus ephrinB2-Fc; **, P < 0.01. (D) Model showing intracellular steric hindrance (using myr-FRB-mCherry) of extracellular (ephrinB2-Fc–induced) clustering. In the presence of heterodimerizer AP21967 the coexpressed inhibitory construct myr-FRB-mCherry couples to the wtEphB2-3FKBP receptor, keeping it as monomer. EphrinB2-Fc can still bind to the ectodomain of the receptor. (E) Western blot of immunoprecipitated wtEphB2-3FKBP (carrying a Flag epitope tag) using the phospho-specific anti-EphB2 antibody; blot was stripped and reblotted for total EphB2. HeLa cells transfected with both wtEphB2-3FKBP and myr-FRB-mCherry were stimulated with ephrinB2-Fc in the presence or absence of AP21967 (representative blot of two separate experiments). (F) Quantification of cell collapse assays. The presence of AP21967 prevents EphB2-3FKBP–transfected cells from collapse in response to ephrinB2-Fc. Mean cell area ± SEM from n = 9 cells per condition tested; P < 0.001, Mann-Whitney nonparametric test.

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