Rabaptin-5 regulates receptor expression and functional activation in mast cells

Abstract

Rab5 is a small GTPase that regulates early endocytic events and is activated by RabGEF1/Rabex-5. Rabaptin-5, a Rab5 interacting protein, was identified as a protein critical for potentiating RabGEF1/Rabex-5's activation of Rab5. Using Rabaptin-5 shRNA knockdown, we show that Rabaptin-5 is dispensable for Rab5-dependent processes in intact mast cells, including high affinity IgE receptor (FcepsilonRI) internalization and endosome fusion. However, Rabaptin-5 deficiency markedly diminished expression of FcepsilonRI and beta1 integrin on the mast cell surface by diminishing receptor surface stability. This in turn reduced the ability of mast cells to bind IgE and significantly diminished both mast cell sensitivity to antigen (Ag)-induced mediator release and Ag-induced mast cell adhesion and migration. These findings show that, although dispensable for canonical Rab5 processes in mast cells, Rabaptin-5 importantly contributes to mast cell IgE-dependent immunologic function by enhancing mast cell receptor surface stability.

Figure 1

Rabaptin-5 knock-down does not impair canonical Rab5 processes in BMCMCs. (A) Total cell lysates from BMCMCs treated with control (shC) or Rabaptin-5 (shR) targeted shRNA constructs were resolved by SDS-PAGE and probed with the indicated primary antibodies. (B) BMCMCs generated as in panel A were processed for confocal microscopy as described in Document S1, (“Immunofluorescence”) and stained with α-EEA1 (red) and α-Rab5 (green) antibodies and phalloidin (blue) to identify the actin cytoskeleton. The regions outlined by dashed boxes are shown magnified, and as individual channels, above the overlay. (C) BMCMCs generated as in panel A were transiently transfected with the indicated GFP constructs by electroporation for 12 to 24 hours and processed for immunofluorescence as in panel B with GFP fluorescence in green and α-EEA1 in red. (D) Endosome sizes from individual shC- or shR-treated BMCMCs prepared as in panel C were measured as described in Document S1, “Immunofluorescence,” averaged, pooled from 3 separate experiments, and compared (***P < .001 vs corresponding “control” untransfected cells). (E) BMCMCs generated as in panel A were sensitized with IgE, stimulated with biotinylated α-IgE Abs for the indicated times, and surface α-IgE was assessed by flow cytometry. The bar graph shows the mean plus or minus SEM of percentage FcϵRI internalization determinations from 4 separate batches of BMCMCs (***P < .001 vs MFI at 30 minutes). (F) BMCMCs generated as in panel A were pulsed with 0.5 mg/mL of Cy 5-labeled BSA for 20 minutes, washed, and chased with unlabeled BSA. Associated fluorescence was measured at the indicated times after washout by flow cytometry. To prevent endosome acidification, some cells were pretreated with 50 μM of bafilomycin for 10 minutes before the BSA pulse. Graphs represent data transformed to show fold increases above unlabeled cells pooled from 3 pairs of shC- or shR-treated BMCMCs. Linear regression analysis was performed to generate lines of best fit with the slopes and 95% confidence intervals noted. Scale bars in panels B and C represent 7.5 μm.

Figure 2

Rabaptin-5 knock-down decreases surface FcϵRIα expression in BMCMCs. (A) Surface FcϵRIα expression was analyzed by flow cytometry on control (shC) or Rabaptin-5 (shR) shRNA-treated BMCMCs. Representative histograms comparing control (black filled histogram) and Rabaptin-5-deficient (unfilled histogram) BMCMCs. Gray filled histogram indicates streptavidin only. Bar graph depicts surface expression of the indicated mast cell receptors assayed by flow cytometry, pooled from 5 different pairs of shC- or shR-treated BMCMCs. (B) Total or surface FcϵRIα levels from control or Rabaptin-5–deficient BMCMCs were analyzed by flow cytometry. Bar graph indicates total FcϵRIα expression relative to shC-treated BMCMCs from 5 separate experiments. (C) The subcellular distribution of FcϵRIα in shC- or shR-treated BMCMCs was assessed by confocal microscopy as described in Document S1, “Immunofluorescence.” Bar represents 7.5 μm. (D) Localization of Rab5 (green), Rabaptin-5 (red), and FcϵRIα (blue) was examined in control BMCMCs by confocal microscopy. Magnified images of the regions outlined by dashed boxes are shown in the upper right corner of each panel. Bar represents 7.5 μm. In panels A and B: +P < .05, ++P < .01, +++P < .001, compared with hypothetical value of 100.

Figure 3

Rabaptin-5 deficiency does not alter intracellular FcϵRI localization to the trans-Golgi. (A) BMCMCs were processed for confocal microscopy as in Figure 1 and stained with antibodies to calnexin (red; endoplasmic reticulum) and GCC185 (green; trans-Golgi network) to localize intracellular FcϵRI (blue). Magnified regions are outlined by dashed boxes and shown in the upper right corner of each panel. (B) The relative subcellular localization of GCC-185 (green) and FcϵRI (red) was examined in control (shControl) and Rabaptin-5-deficient (shRabaptin-5) BMCMCs. Large panels show overlay of GCC-185 and FcϵRI immunostaining. Magnified images of the regions outlined by dashed boxes are shown in the upper right corner of each panel. Bars represent 7.5 μm.

Figure 4

Rabaptin-5 deficiency decreases FcϵRIα half-life. (A) FcϵRIα half-life was measured in control (shC) or Rabaptin-5 (shR) shRNA-treated BMCMCs by exposing cells to 100 μg/mL of brefeldin A (BFA), or 1.5 μg/mL of cycloheximide (CHX) for the indicated times. Surface FcϵRIα was then assessed by flow cytometry. Expression relative to time 0 was calculated for the indicated time points. Data from 3 separate pairs of shC- or shR-treated BMCMCs were pooled and fit to exponential decay curves. (B) Bar graph indicates shR-treated BMCMC surface FcϵRIα levels relative to shC-treated BMCMCs at the indicated times from data pooled from 3 separate pairs of shC- or shR-treated BMCMCs (*P < .05, **P < .01 by Student t test compared with percentage difference at time 0).

Figure 5

Rabaptin-5 deficiency does not influence FcϵRI recycling but does alter surface stability of β1 integrin. (A) BMCMCs were induced to adhere to fibronectin (FN)-coated coverslips as described in Document S1, “Immunofluorescence,” labeled with Alexa 647-labeled α-FcϵRIα Fabs on ice, transferred to 37°C, and then fixed and processed for confocal microscopy at the indicated times. Bar represents 7.5 μm. (B) FcϵRI recycling was assessed in shC- or shR-treated BMCMCs using biotinylated α-FcϵRIα Fabs as described in Document S1, “FcϵRI recycling.” Bar graph represents data pooled from 3 separate pairs of shC- or shR-treated BMCMCs. (C) Transferrin recycling in shC- or shR-treated BMCMCs was performed as described in Document S1, “Transferrin recycling.” Bar graph represents data pooled from 4 separate pairs of shC- or shR-treated BMCMCs. (D) shC- or shR-treated BMCMCs were exposed to 50 μM of primaquine (left bar graph) or vehicle (right bar graph) for the indicated times, and then surface β1 integrin levels were assessed by flow cytometry. Data were pooled from 3 separate pairs of shC- or shR-treated BMCMCs. +P < .05; ++P < .01 for shC- versus shR-treated cells; *P < .05, **P < .01, ***P < .001, versus t = 5 minutes time point (B,C) or time 0 (D).

Figure 6

Rabaptin-5 deficiency diminishes mast cell IgE-dependent responses to specific antigen. (A) Control (shC) or Rabaptin-5 (shR) shRNA-treated BMCMCs were cultured with various concentrations of Alexa 647-labeled IgE (IgE-647) for the indicated times and then assessed for associated fluorescence. Top panel bar graph represents total associated fluorescence; bottom panel bar graph, fold increase in associated fluorescence compared with time 0 (incubated for 1 hour at 4°C). (B) shC- or shR-treated BMCMCs were cultured with low amounts of IgE-647 (25 ng/mL) in the presence (gray bars) or absence (black bars) of 100 μg/mL of brefeldin A, and associated fluorescence was assessed at the indicated times. +P < .05 for vehicle versus BFA; *P < .05 versus time 0 in the same treatment group. (C) C57BL/6-Kit^{W−sh/W−sh} mice were injected intraperitoneally with GFP⁺ shC- or shR-treated BMCMCs; 6 weeks later, peritoneal cells were harvested, stained with α-IgE, analyzed by flow cytometry, and MFIs of GFP⁺ cells were pooled to generate the bar graphs. (D) Ag-induced adhesion to fibronectin (FN) was assessed in shC- or shR-treated BMCMCs. Cells were sensitized with 1 μg/mL of DNP-specific IgE overnight, washed, and placed in FN-coated wells in the presence of the indicated concentrations of Ag and allowed to adhere for 1 hour. Data were pooled from triplicate determinations and are representative of the similar results that were obtained in each of the 5 experiments that were performed. (E) Migration to Ag through FN-coated transwells was assessed for shC- or shR-treated BMCMCs. Cells were prepared as in panel D, then placed in a transwell with the indicated concentrations of Ag and allowed to migrate for 6 hours. Migrated cells were counted by flow cytometry. Data were pooled from duplicate determinations and are representative of similar results that were obtained in the 3 experiments that we performed. (F) IL-6 produced from shC- or shR-treated BMCMCs, prepared as in panel D that were stimulated with the indicated concentrations of Ag for 6 hours; IL-6 was quantified by ELISA. (A,C-F) +P < .05, ++P < .01, +++P < .001 for shC- versus shR-treated cells.