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First published online 8 August 2006
doi: 10.1242/jcs.03077

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Activation of Tie2 by angiopoietin-1 and angiopoietin-2 results in their release and receptor internalization

¹ Division of Molecular and Cellular Biology Research, Sunnybrook and Women's Research Institute, 2075 Bayview Avenue, Research Building, S-218, Toronto, Ontario M4N 3M5, Canada
² Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada
³ Heart and Stroke/Richard Lewar Centre of Excellence, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 3E2, Canada

View larger version (20K): [in a new window]   Fig. 1. Ang1 and Ang2 activate Tie2 in a concentration-dependent manner. (A,B) HUVECs were stimulated with the indicated concentrations of Ang1 pre-clustered with 10 µg/ml anti-polyhistidine antibody (A) or Ang2 (B) for 15 minutes at 37°C. Top panels are representative immunoblots. Intensities of Tie2 tyrosine phosphorylation were quantified by densitometry and plotted versus Ang1 or Ang2 concentration (lower panels). The value at 800 ng/ml was designated 100%. (C) Comparison of Tie2 tyrosine phosphorylation when HUVECs are stimulated with Ang1 (800 ng/ml), Ang1 (800 ng/ml) pre-clustered with the anti-polyhistidine (anti-his) antibody (10 µg/ml), Ang2 (800 ng/ml), or the anti-polyhistidine antibody (10 µg/ml) alone. (D) EA.hy926 cells were stimulated with Ang1 (800 ng/ml, pre-clustered) or Ang2 (800 ng/ml) for 15 minutes at 37°C. Tie2 was immunoprecipitated and immunoblotted with an anti-phospho Tyr1102 and Tyr1108 antibody (top panel) or an anti-Tie2 antibody (lower panel).

View larger version (21K): [in a new window]   Fig. 2. Turnover of Tie2 in HUVECs. [35S]methionine and [35S]cysteine labelled HUVECs were washed and incubated at 37°C for the indicated times in either HUVEC medium alone, or containing pre-clustered Ang1 (800 ng/ml), or Ang2 (800 ng/ml). The cells were lysed, Tie2 immunoprecipitated with an anti-Tie2 antibody, and immunoprecipitates resolved by SDS-PAGE. (A) Representative autoradiograph. (B) Semi-logarithmic plot of the relative Tie2 intensities versus time in unstimulated cells (), in cells stimulated with Ang1 () or Ang2 (). Each point on the graph is an average value from three to nine independent experiments.

View larger version (27K): [in a new window]   Fig. 3. Ang1 and Ang2 do not influence Tie2 synthesis. [35S]methionine and [35S]cysteine labelled HUVECs were incubated for various times together with pre-clustered Ang1 (800 ng/ml), or Ang2 (800 ng/ml). Tie2 immunoprecipitates were resolved by SDS-PAGE and the dried gel exposed to a phosphorimager screen. (A) Representative autoradiograph. (B) The intensities of the bands were quantified and plotted against time. Time=12 hours was designated 100%. All other values were expressed as a percentage of the 12-hour time point. Unstimulated cells (), Ang1 (), Ang2 (). Results are mean ± s.e.m. of three independent experiments.

View larger version (20K): [in a new window]   Fig. 4. Tie2 protein levels decrease in Ang1 and Ang2 stimulated HUVECs. (A) HUVECs were stimulated with pre-clustered Ang1 (800 ng/ml) or Ang2 (800 ng/ml) for the indicated times. Total cell lysates were analyzed by western immunoblotting using a monoclonal Tie2 antibody to detect total receptor levels and an anti-ß-actin antibody to verify that equal amounts of protein were analyzed. Time 0 represents cells stimulated with medium alone. (B) The intensities of the bands in A were quantified and expressed as a percentage of the t=0 value and plotted against time, Ang1 () Ang2 (). Results are mean ± s.e.m. of three independent experiments.

View larger version (23K): [in a new window]   Fig. 5. Ang1 and Ang2 induce differential internalization of Tie2 in HUVECs. Surface biotinylation of HUVECs was used to assess the amount of Tie2 remaining at the cell surface upon Ang1 and Ang2 stimulation. (A) Surface biotinylated HUVECs were lysed and incubated with streptavidin conjugated to agarose beads. Non-biotinylated cells (Non), biotinylated (Biotin), biotinylated followed by stripping (Biotin + Stripping) and total cell lysate (Cell Lysate) proteins were resolved by SDS-PAGE and immunoblotted using a monoclonal anti-Tie2 antibody. (B) HUVECs were incubated with pre-clustered Ang1 (800 ng/ml), or Ang2 (800 ng/ml) for the indicated times at 37°C. Cell surface proteins were biotinylated and isolated with streptavidin agarose followed by immunoblotting as described in (A), t=0 represents cells stimulated with media alone. (C) The intensities of the bands in B were quantified and plotted against time, Ang1 () Ang2 (). Results are mean ± s.e.m. of four independent experiments.

View larger version (42K): [in a new window]   Fig. 6. Binding and release of 125I-Ang1 and 125I-Ang2 to HUVECs. Electrophoretic mobility of 125I-Ang1 and Ang1 (A) and 125I-Ang2 and Ang2 (B). Aliquots of 125I-Ang1 and 125I-Ang2 and 50 ng Ang1 and Ang2 were resolved by SDS-PAGE under reduced (R) and non-reduced (NR) conditions. To visualize 125I-Ang1 and 125I-Ang2, the gel was dried and exposed to X-ray film. To visualize Ang1 and Ang2, proteins were transferred to PVDF membranes and immunoblotted using an anti-polyhistidine antibody. Numbers indicate relative molecular masses. (C) 125I-Ang1 and 125I-Ang2 were allowed to bind to HUVECs at 0°C. The cells were washed to remove unbound ligands and then transferred to 37°C. At the indicated times, the cells were solubilized and cell-associated radioactivity counted and plotted against time. Radioactivity at t=0 was designated 100%, 125I-Ang1 () 125I-Ang2 (). (D) Time-dependent appearance of radioactivity in the medium collected from cells bound with 125I-Ang1 () and 125I-Ang2 (). Radioactivity was expressed as a percentage of the total radioactivity contained within the sample. (E) Medium was collected at the indicated times from HUVECs bound by 125I-Ang1 and immunoprecipitated with an anti-polyhistidine antibody. Immunoprecipitates were resolved by SDS-PAGE and exposed to X-ray film. Cold refers to cells incubated at 0°C for 120 minutes. Numbers on the left represent relative molecular masses. (F) Media was collected at the indicated times from cells bound with 125I-Ang2 and processed as in E. White line indicates that intervening lanes have been spliced out. (G) Ang1 and Ang2 were allowed to bind to HUVECs. Cells were washed and incubated at 37°C in fresh binding medium. At the indicated times, the medium was immunoprecipitated and immunoblotted using an anti-polyhistidine antibody. As controls for the position of Ang1 and Ang2, 50 ng of each ligand were resolved alongside the media samples. Cold refers to media collected from cells incubated at 0°C for 3 hours.

View larger version (24K): [in a new window]   Fig. 7. Potential mechanisms of angiopoietin release. (A) HUVECs were treated with either Ang1 (800 ng/ml cross-linked, white bars), Ang2 (800 ng/ml) (black bars), Mock (cross-linking antibody alone, hatched bars) or left untreated (grey bars). At the indicated times, the media was collected and the amount of soluble Tie2 (sTie2) quantified using a Human Tie2 Immunoassay Kit. (B) HUVECs were incubated with Ang1 (800 ng/ml) for 90 minutes at 0°C in the presence or absence of 100 µM pervanadate. The cells were washed to remove unbound Ang1 and incubated for 30 minutes at 37°C in fresh medium alone or containing 100 µM pervanadate. Ang1 was immunoprecipitated from the media and immunoblotted using an anti-polyhistidine antibody. (C) The cells from B were washed, surface proteins were biotinylated and isolated with streptavidin agarose, followed by immunoblotting for Tie2. As a control for Tie2 internalization, cells incubated in media alone in the absence of Ang1 or pervanadate were included (Non). White line indicates that intervening lanes have been spliced out.