Silencing MS4A4 alters surface expression of KIT

We next examined the expression of surface KIT using flow cytometry, given the key role that the KIT ligand, stem cell factor (SCF), plays in maintaining MC viability and function. A marked reduction in KIT on the cell surface was observed with all shMS4A4 constructs (Figure 1H). Examination of total KIT protein expression in fixed and permeabilized LAD-2 cells revealed that silencing MS4A4 also resulted in a comparable reduction in both surface and total KIT expression compared with control cells (compare Figure 1, I to ​toJ).J). This suggests that the reduced receptor expression could be due to increased internalization and degradation. Expression of another MC surface marker, the intercellular adhesion molecule 1 (ICAM-1 [CD54]), was unaffected (Figure 1K). The reduction in surface KIT expression correlated with relative MS4A4 mRNA expression after transduction with all shMS4A4 constructs (compare Figure 1, E and ​andH).H). Therefore the reduction in KIT surface expression appeared to be directly related to the reduced expression of MS4A4. Because there was no reduction in KIT mRNA expression (Figure 1L) as determined by quantitative PCR and no correlation between surface KIT and relative KIT mRNA expression (Figure 1M), it would appear that MS4A4 contributes to surface KIT expression by regulating trafficking and/or degradation pathways rather than down-regulating c-KIT transcription.

Measurement of cell viability and apoptosis indicated that three of the shMS4A4 constructs reduced cell viability and increased surface annexin V expression. However, shMS4A4v4 had no cytotoxic effects (Figure 1N), silenced MS4A4 with equal efficiency to the other constructs (Figure 1E), and also reduced KIT expression to the same degree (Figure 1H). Therefore shMS4A4v4 was selected for further studies.

MS4A4 regulates KIT endocytosis and recycling to the plasma membrane

To examine whether MS4A4 promotes trafficking of KIT to the plasma membrane, we removed SCF from the cultures to stop ligand-induced KIT internalization. Measurement of surface KIT on LAD-2 cells in the continuous presence of SCF in culture revealed a marked reduction in surface KIT in MS4A4-silenced cells compared with control cells (Figure 2A). However, removal of SCF for 16 h led to partial restoration of surface KIT expression in the MS4A4-silenced cells compared with control cells (Figure 2B). Subsequent induction of KIT internalization by exposing the SCF-deprived cells to 100 ng/ml SCF for 60 min demonstrated that control and MS4A4-silenced LAD-2 cells internalized KIT with similar efficacy (Figure 2C). Thus the greatest difference in surface expression of KIT between scrambled control and MS4A4-silenced cells was apparent when cells were continuously exposed to SCF, as shown in Figure 2, D and E (“no starving”). These data indicate that, in the presence of SCF, surface KIT expression is held in dynamic equilibrium between internalization and recycling of KIT to the cell surface, with either or both processes being retarded with MS4A4 silencing.

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FIGURE 2:

Silencing MS4A4 alters KIT internalization, recycling to the plasma membrane and signaling in LAD-2 cells. (A) Flow cytometry histogram showing surface KIT expression at day 7 postinfection with shMS4A4. (B) Flow cytometry histogram showing surface KIT expression after 16 h of SCF withdrawal. (C) As in B, but followed by stimulation with 100 ng/ml SCF for 1 h. Isotype, black; scramble, blue; shMS4A4, red). (D) Average flow cytometry data from SCF exposure experiments, Values are geometric mean fluorescence intensity (MFI; error bars, SEM; n = 3). (E) Average flow cytometry data calculated as percentage expression normalized to scramble control (error bars, SEM; n = 3). (F) Average flow cytometry data from KIT internalization assays after exposure to 100 ng/ml SCF. SCF was withdrawn for 16 h before experiment (n = 5, p = 0.0008, two-way ANOVA scramble vs. shMS4A4). *p < 0.05, Sidak's posttest. (G) KIT kinetics assay using SCF pulse chase with 100 ng/ml SCF. The x-axis is the time spent at 37°C during chase (n = 3). (H) KIT degradation assay using pulse chase with 100 ng/ml SCF in the presence of 10 μM cycloheximide and staining for surface and intracellular KIT. (I) As in F, after exposure to 10 ng/ml SCF. BFA was included to eliminate repopulation of surface KIT from newly synthesized pools (n = 4, p = 0.047, two-way ANOVA scramble vs. shMS4A4). *p < 0.05, Sidak's posttest. (J) KIT kinetics assay using pulse chase with 10 ng/ml SCF. The x-axis is the time spent at 37°C during chase (n = 3). *p < 0.05, Sidak's posttest. (K) KIT degradation assay by using pulse chase with 10 ng/ml SCF in the presence of 10 μM cycloheximide and staining for surface and intracellular KIT. (L) Immunoblots for pKIT Y^568/570 (Invitrogen antibody), pAKT S⁴⁷³, and pERK T²⁰²Y²⁰⁴ in scramble shRNA control and shMS4A4- treated LAD-2 cells for the indicated time after stimulation with 10 ng/ml SCF. (M) Analysis of fluorescence intensity (LI-COR system) of phosphorylated KIT (M), AKT (N), and ERK (O) after correction against loading control normalized to percentage of scramble control at 2 min (mean + SEM, n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA with Sidak's posttest.

To assess the effect of MS4A4 silencing on KIT turnover, we undertook kinetic studies using flow cytometry. In the presence of 100 ng/ml SCF, KIT internalization followed a typical decay curve. Calculation of the half-life (t_1/2) of surface receptor expression by nonlinear regression indicated a t_1/2 of KIT internalization of 6.17 ± 0.85 min in control LAD-2 cells and 4.01 ± 0.42 min in MS4A4-silenced cells (Figure 2F; n = 5, p = 0.025, paired t test for t_1/2). Because loss of surface expression of receptors could involve changes in the rate of recycling as well as endocytosis of receptors, we also used a pulse-chase method that eliminates repeated cycling of receptors. By use of a previously published pulse-chase procedure (Roepstorff et al., 2009), we found that although there was increased endocytosis of KIT with MS4A4 silencing (Figure 2F), there was no apparent recycling of KIT with 100 ng/ml SCF in either scramble control or shMS4A4-treated cells (Figure 2G). We also determined whether KIT was degraded more readily with MS4A4 silencing by using the pulse-chase method in the presence of cycloheximide (10 μM) and found no alteration in KIT degradation after stimulation with 100 ng/ml SCF when MS4A4 was silenced (Figure 2H).

Because trafficking of RTKs can depend on the concentration of ligand (Goh and Sorkin, 2013), we next studied the kinetics of KIT internalization with a lower concentration (10 ng/ml) of SCF. The rate and extent of KIT internalization were less with 10 ng/ml SCF (Figure 2I) than with 100 ng/ml SCF (Figure 2F), but the enhancement of KIT internalization with MS4A4 silencing was more pronounced at the lower concentration of SCF (Figure 2I). If high concentrations of ligand decrease efficiency of receptor recycling and increase degradation, this could account for the greater effect of MS4A4 silencing at the lower SCF concentration. To address this possibility, we examined KIT kinetics using the pulse-chase method with 10 ng/ml SCF. As expected and consistent with the internalization assays in the continuous presence of SCF (Figure 2, F and I), 10 ng/ml SCF resulted in less overall internalization than 100 ng/ml SCF (compare Figure 2, G and J). Silencing MS4A4 resulted in more rapid endocytosis, with maximal internalization evident at 5 min compared with 10 min in scramble control cells (Figure 2J). In addition, there was a consistent increase in surface expression of KIT at 15 min when compared with 10 min in control cells, which was not evident with MS4A4 silencing (Figure 2J). This increase in surface KIT expression most likely represents KIT recycling. Despite more rapid endocytosis and reduced recycling of KIT with MS4A4 silencing, there was no significant difference in KIT degradation with 10 ng/ml SCF (Figure 2K; p = 0.3997, two-way analysis of variance [ANOVA] scramble vs. shMS4A4, n = 3). These data are consistent with the conclusion that MS4A4 contributes to both stabilization of KIT at the cell surface and recycling and repopulation of KIT in the plasma membrane and thus regulates the steady-state dynamics of KIT at the cell surface.

KIT signaling is altered with MS4A4 silencing

We next examined whether the effects of MS4A4 silencing on KIT internalization altered MC signaling through KIT. An early signaling event in the KIT activation cascade in MCs is trans-phosphorylation of Tyr-568/570 residues of KIT (Blume-Jensen et al., 1991; Pawson, 2004; Ronnstrand, 2004) to initiate Src family kinase–mediated signaling pathways (Linnekin et al., 1997; Abram and Courtneidge, 2000). These events most likely occur at the plasma membrane within lipid raft microdomains (Kovarova et al., 2001; Arcaro et al., 2007; Jahn et al., 2007). SCF (100 ng/ml) induced rapid phosphorylation of KIT Tyr-568/570 in control LAD-2 cells, and such phosphorylation was reduced in MS4A4-silenced cells (Supplemental Figure S2). Paradoxically, later signaling events, including the activating phosphorylations of AKT (Ser-473) and ERK (Thr-202/Tyr-204), were not significantly different despite diminished expression and phosphorylation of KIT in MS4A4-treated cells (Supplemental Figure S2).

We also examined KIT-mediated signaling with 10 ng/ml SCF because the differences in kinetics of KIT internalization were more apparent at this concentration of SCF when analyzed by the pulse-chase procedure (Figure 2J). SCF at 10 ng/ml also induced rapid phosphorylation of KIT (Tyr-568/570) within 2 min, which was significantly reduced in shMS4A4-treated cells (Figure 2, L and M). A significant reduction in phosphorylation of KIT was also evident at 10 min with shMS4A4 treatment (Figure 2, L and M). Of interest, phosphorylation of KIT appeared to increase at 10 min when compared with 5 min in scramble control cells but not in MS4A4-silenced cells (Figure 2, L and M) and might be a consequence of KIT recycling. In contrast to reduced KIT phosphorylation, phosphorylation of AKT (Ser-473) was enhanced with MS4A4 silencing, which was particularly evident at 5 min (Figure 2, L and N). We did not detect any significant difference in ERK phosphorylation with shMS4A4 treatment (Figure 2, L and O). Taken together, these data indicate that silencing MS4A4 enhances AKT signaling despite reduced KIT phosphorylation.

MS4A4 colocalizes with KIT after SCF exposure

Because MS4A4 may facilitate KIT receptor surface expression by influencing both receptor internalization and receptor recycling pathways, we explored whether MS4A4 and KIT colocalized to the plasma membrane and intracellular organelles. We performed confocal microscopy on LAD-2 cells transfected with enhanced green fluorescent protein (EGFP)–tagged MS4A4 in both fixed and live cells after SCF stimulation. Transfected MS4A4 localized to a juxtanuclear region, as well as in distinct vesicles throughout the body and periphery of the cell, with evidence of some plasma membrane localization (Figure 3A), although this localization was somewhat variable among cells. MS4A4 exhibited low colocalization with endogenous KIT in the absence of SCF, which was observed in both vesicles and the plasma membrane (Figure 3A). However, after exposure to SCF, MS4A4 and KIT colocalized to intracellular vesicles, especially in the centrosomal region (Figure 3, A and B). Particularly striking was the widespread intracellular distribution of KIT in the absence of SCF stimulation, which may be indicative of continuous turnover of KIT in nonstimulated LAD-2 cells.

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FIGURE 3:

MS4A4 colocalizes with KIT and EEA1 in LAD-2 cells. (A) Confocal micrographs of LAD-2 cells transfected with MS4A4:EGFP chimeric protein (green) immunostained for KIT (APC) (red) in the absence (top) or after stimulation with SCF (bottom). (B) Manders coefficient of colocalization for MS4A4 and KIT. (C) Confocal micrographs of LAD-2 cells transfected with MS4A4:EGFP (left) or cells immunostained with anti-MS4A4 followed by AF488-labeled anti-mouse secondary (right). (D) Immunofluorescence confocal micrographs of LAD-2 cells immunostained for mouse anti-MS4A4 (secondary, AF488) and rabbit anti-EEA1 (secondary, AF594) in the absence of SCF (top) or with SCF stimulation for 10 min (bottom). Scale bars, 5 μm. (E) Manders coefficient of colocalization for MS4A4 and EEA1. For B and E, bars are the mean + SEM from the volume of 16 stacks of images from two separate experiments). *p < 0.05, ANOVA with Sidak's posttest.

We next determined whether the intracellular structures associated with MS4A4 were early endosomes (EEs). Transfection of MS4A4 into LAD-2 cells displayed some localization, which appeared to be the endoplasmic reticulum (Figure 3C). Indeed, cotransfection of MS4A4 and Sec61B (a marker of the endoplasmic reticulum) in HEK293T cells demonstrated a degree of colocalization in some cells (Supplemental Figure S3A). We therefore performed immunofluorescence (IF) for endogenous MS4A4 using a mouse polyclonal antibody (Ab) and found that the distribution of native MS4A4 was comparable to that of overexpressed MS4A4, except that native MS4A4 displayed a more punctate morphology (Figure 3C). We confirmed that anti-MS4A4 was immunoreactive to MS4A4 by performing IF on MS4A4-transfected HEK293T cells (Supplemental Figure S3, B–D). Having validated Ab specificity, we performed IF on LAD-2 cells costained with the EE marker EEA1 and determined that MS4A4 and EEA1 colocalized to early endosomes surrounding an MS4A4⁺ juxtanuclear compartment (Figure 3D). The colocalization of MS4A4 and EEA1 was significantly decreased after 5 min of stimulation with SCF, although this decrease was no longer significant at later time points (Figure 3E). The relevance of this decreased colocalization at early time points after SCF stimulation is not clear but could be due to rapid MS4A4 cycling to the plasma membrane in response to SCF. However, more study is required to determine whether this observation is relevant to the function of MS4A4.

MS4A4 colocalizes with Rab5 in endosomes

The concerted action of Rab family proteins and their effector proteins regulates almost all membrane and protein trafficking in eukaryotic cells (Mizuno-Yamasaki et al., 2012). An important component of endocytic trafficking and recycling pathways is Rab5, which is a key regulator of early endocytic events and the sorting endosome (Woodman, 2000; Stenmark, 2009) and participates in receptor trafficking in mast cells (Kalesnikoff et al., 2007; Rios et al., 2008; Kageyama-Yahara et al., 2011). Rab5 facilitates vesicle fusion and is critical for the formation of EEs by fusion of endocytic vesicles (for review, see Woodman, 2000). Thus constitutively active Rab5 (Q79L) promotes enlarged EE formation, whereas dominant-negative Rab5 (S34N) perturbs endocytic vesicle fusion and endosomal formation (Stenmark et al., 1994).

Because MS4A4 appeared to traffic through the endocytic system, the passage of MS4A4 through Rab protein–regulated endocytic trafficking pathways was examined by cotransfection of fluorescent protein–tagged MS4A4 and various Rab proteins and their variants in live LAD-2 cells. With respect to Rab5, there was substantial colocalization of MS4A4 in enlarged endosomes (up to 2 μm in diameter) with both wild-type Rab5 (Figure 4A, left) and constitutively active Rab5 (Q79L; Figure 4A, middle). However, cotransfection of MS4A4 with constitutively active Rab5 (Q79L) led to formation of very large endosomes (>4 μm in diameter), most of which were not positive for MS4A4 (Figure 4A, middle). Consequently colocalization of MS4A4 with constitutively active Rab5 decreased compared with wild-type Rab5 (Figure 4B). This observation may be related to the decrease in colocalization between MS4A4 and EEA1 in response to SCF (Figure 3E). MS4A4 localization to enlarged early endosomes was dependent on active Rab5 since cotransfection of dominant-negative Rab5 (S34N) and MS4A4 showed no evidence of distinct endosomes. Instead, fluorescence for Rab5 (S34N) was more diffuse, and both MS4A4 and Rab5 (S34N) displayed some localization to a juxtanuclear region of the cell (Figure 4A, right). This distribution of MS4A4 with cotransfection of dominant-negative Rab5 (S34N) resembled the distribution of single transfection of MS4A4 into LAD-2 cells (compare Figure 4A to Figure 3B). Cotransfection of MS4A4 and Rab5 variants in HEK293T cells, which do not express KIT, exhibited limited colocalization of MS4A4 and WT Rab5 (Supplemental Figure S4A). However, cotransfection of MS4A4 and the constitutively active Rab5 (Q79L) showed marked colocalization (Supplemental Figure S4B). Therefore the pattern of MS4A4 colocalization with Rab5 variants in MC may differ from that of HEK293T cells, which do not natively express MS4A4 or KIT and are not dependent on growth factors for survival. Collectively these data indicate that MS4A4 trafficking in the endosomal compartments, but not colocalization with Rab5, appears to be dependent on active Rab5. In addition, when dominant-negative Rab5 (S34N) was transfected into LAD-2 cells, KIT no longer translocated to EEA1-positive EEs after stimulation with SCF, and staining of both KIT and EEA1 was diffuse (Supplemental Figure S5).

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FIGURE 4:

MS4A4 traffics through endocytic recycling pathways. (A) Confocal micrographs of live LAD-2 cells cotransfected with MS4A4:EGFP and mRFP Rab5 (left), constitutively active mCherry Rab5 (Q79L; middle), or dominant-negative mCherry Rab5 (S34N; right). (B) Pearson's coefficient in colocalized volumes of MS4A4 with wild-type Rab5, constitutively active Rab5, and dominant-negative Rab5. *p < 0.05. (C) Confocal micrographs of live LAD-2 cells cotransfected with MS4A4:EGFP and DsRed Rab7 (left), DsRed Rab9 (middle), or DsRed Rab11 (right). Scale bars, 5μm. (D) Pearson's coefficient in colocalized volumes of MS4A4 with Rab5, Rab7, Rab9, or Rab11.

MS4A4 colocalizes with components of EEs and recycling compartments but not late endosomes in MCs

Because MS4A4 traffics with active Rab5 and colocalizes with KIT in EEs, we next examined the association of MS4A4 with other compartments of the endocytic pathways. Cargo that is not directed through recycling pathways remains in EEs and, during maturation into late endosomes (LEs), regulation switches from Rab5- to Rab7-mediated events such as lysosomal fusion and subsequently protein degradation (Poteryaev et al., 2010; Huotari and Helenius, 2011). Thus Rab7 localizes to LE and lysosomes. Using cotransfection of MS4A4 and Rab7 in LAD-2 cells, we determined that MS4A4 did not colocalize with Rab7 under any condition (Figure 4, C and D). Similarly, Rab9, which is also a LE marker and may regulate endosomes to trans-Golgi network trafficking of certain cargo (Stenmark, 2009), also localized to different cellular compartments than MS4A4 (Figure 4, C and D).

Because MS4A4 does not apparently migrate to LE compartments, we examined whether MS4A4 migrated instead to the endocytic recycling compartment (ERC), where Rab11 mediates receptor recycling back to the plasma membrane (Maxfield and McGraw, 2004; Grant and Donaldson, 2009). MS4A4 and Rab11 colocalized in LAD-2 cells within a juxtanuclear region. possibly indicative of an MS4A4⁺/Rab11⁺ compartment (Figure 4, C and D). In total, our cotransfection data demonstrate that MS4A4 localizes to components of EE pathways and the recycling pathway rather than the LE and degradation pathways.

Gene silencing of MS4A4 results in altered KIT trafficking and enlarged EEs

On the basis of the foregoing experiments, we hypothesized that MS4A4 promotes trafficking of KIT toward the ERC from EEs and that this results in reduced KIT degradation and greater steady-state KIT expression at the plasma membrane. This was tested by silencing of MS4A4 with shMS4A4, or scrambled shRNA as controls, in LAD-2 cells. We found that KIT was recruited to EEA1-positive EEs after stimulation with SCF in both control and MS4A4-silenced cells (Figure 5, A and B). However, KIT colocalizations with Rab5 and EEA1 were both reduced with gene silencing of MS4A4 (Figure 5, C and D), whereas colocalization of KIT with Rab9⁺ LEs was increased (Figure 5E). In addition, EEA1⁺ EEs were significantly larger in the MS4A4-silenced cells, compared with the control cells, after SCF stimulation (p < 0.0001, ANOVA; Figure 5, B and F). A plausible explanation for enlarged EEs is that the EE burden is increased in the absence of MS4A4 because of more rapid endocytosis and reduced recycling. The reduction in KIT recycling may also promote more rapid transit of KIT into the late endosomal/endolysosomal compartments, where it has been reported that oncogenic KIT can signal through AKT pathways (Obata et al., 2014).

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FIGURE 5:

Silencing MS4A4 alters endocytic KIT trafficking. (A) Immunofluorescence confocal microscopy of scramble shRNA–treated (top) and shMS4A4-treated (bottom) LAD-2 cells stained with rabbit anti-EEA1 (secondary AF488; green) and mouse anti-KIT (APC; red) in the absence of SCF. White arrowhead shows occasional enlarged endosomes in shMS4A4-treated cells. Right, superimposed z-projection of the stack of images. Scale bars, 10 μm. (B) Immunofluorescence confocal microscopy after stimulation with SCF for 10 min. Yellow arrowheads show enlarged endosomes positive for KIT. Orange arrowheads show very large endosomes in shMS4A4-treated cells that are negative for KIT immunofluorescence. Scale bars, 10 μm. (C–E). Manders coefficient of colocalization of KIT and Rab5 (C), KIT and EEA1 (D), and KIT and Rab9 (E) before and after stimulation with SCF. Scramble shRNA, blue bars; shMS4A4, red bars. (Bars are mean + SEM from the volume of 16 stacks of images from two separate experiments.) (F). Endosome size from the volume of image stacks calculated using Imaris software. Two observers obtained comparable measure­ments. Scramble shRNA, blue circles; shMS4A4, red circles. For C–F, *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA.

MS4A4 expression affects KIT internalization pathways and PLCγ1 phosphorylation

RTKs are internalized by both clathrin-mediated and clathrin-independent endocytosis (reviewed in Goh and Sorkin, 2013). We found that SCF stimulated increased colocalization of KIT with clathrin, but this increase was unaffected by silencing MS4A4 over the time course tested (10 min; Figure 6, A and B). However, the SCF stimulated increase in colocalization of KIT with the lipid raft protein caveolin-1 (Lajoie and Nabi, 2010) was reduced (Figure 6, C and D). We therefore examined whether MS4A4 itself colocalized with caveolin-1, especially since the mouse homologue of MS4A4, Ms4a4b, is recruited to lipid rafts after T-cell activation (Xu et al., 2006). Stimulation with SCF did not significantly affect MS4A4 and clathrin colocalization (Figure 7, A and B). However, MS4A4 and caveolin-1 colocalization rapidly increased with stimulation using SCF (Figure 7, C and D), suggesting that MS4A4 was recruited along with KIT to lipid raft microdomains in human LAD-2 cells.

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FIGURE 6:

Silencing MS4A4 alters KIT colocalization with caveolin-1 but not clathrin. (A) Immunofluorescence confocal microscopy of scramble shRNA–treated (left) and shMS4A4- treated (right) LAD-2 cells stained with rabbit anti-clathrin HC (secondary, AF488; green) and mouse anti-KIT (APC, red) left unstimulated or stimulated with SCF for 2 min. Scale bars, 5 μm. (B) Manders coefficient of colocalization of KIT and clathrin HC at times indicated (minutes) after SCF stimulation. (C) Immunofluorescence confocal microscopy of scramble shRNA–treated (left) and shMS4A4-treated (right) LAD-2 cells stained with rabbit anti–caveolin-1 (secondary, AF488; green) and mouse anti-KIT (APC, red) left unstimulated or stimulated with SCF for 2 min. Scale bars, 5 μm. (D) Manders coefficient of colocalization of KIT and caveolin-1 with SCF stimulation time course. For B and D, bars are mean + SEM from the volume of 16 stacks of images from two separate experiments. **p < 0.01, ***p < 0.001, two-way ANOVA.

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FIGURE 7:

MS4A4 colocalizes preferentially with caveolin-1 over clathrin after stimulation with SCF promoting PLCγ1 phosphorylation. (A) LAD-2 human mast cells immunostained with mouse anti-MS4A4 and rabbit anti-clathrin HC, followed by anti-mouse AF488 and anti-rabbit AF594 before (top) and after SCF stimulation (bottom). No increase in colocalization was observed with stimulation. (B) Manders coefficient of colocalization of MS4A4 and clathrin HC with SCF stimulation time course. (C) LAD-2 human mast cells immunostained with mouse anti-MS4A4 and rabbit anti–caveolin-1 demonstrated an increase in colocalization with SCF stimulation (bottom) compared with untreated cells (top). Scale bars, 5 μm (A, C). (D) Manders coefficient of colocalization of MS4A4 and caveolin-1 with SCF stimulation time course. For B and D, bars are the mean + SEM from the volume of 15 stacks of images from two separate experiments. **p < 0.01, ***p < 0.001, two-way ANOVA. (E) Coimmunoprecipitation using anti-MS4A4 or IgG control as pull down. Immunoblots for clathrin HC and caveolin-1. (F) Immunoblot of PLCγ1 Y⁷⁸³ phosphorylation in response to SCF stimulation over time. β-Actin was used as a control. (G) Densitometry of phosphorylated PLCγ1 (Y783) after correction. Average data from three experiments. Error bars, SEM. **p < 0.01.

We next investigated MS4A4 regulation of lipid raft–associated signaling events, in particular PLCγ1, for several reasons. As noted earlier, shMS4A4 reduces phosphorylation of KIT Y^568/570 (Figure 2I), and this phosphorylation is critical for recruitment of SH2 domain– containing proteins such as Lyn and PLCγ1 by KIT (van der Geer et al., 1994; Linnekin et al., 1997; Abram and Courtneidge, 2000; Pawson, 2004; Ronnstrand, 2004). KIT signaling through Lyn kinase is promoted by lipid rafts (Arcaro et al., 2007; Jahn et al., 2007). In addition, we found that MS4A4 not only colocalized (Figure 7C) but also coimmunoprecipitated with caveolin-1 (Figure 7E). On examining PLCγ1 activation, we found that silencing MS4A4 blunted the phosphorylation of PLCγ1 (Figure 7, F and ​andG).G). These data are compatible with our thesis that MS4A4 promotes recruitment of KIT to lipid raft microdomains, altering signaling and internalization pathways.

Human mast cell culture

LAD-2 cells were cultured as described (Kirshenbaum et al., 2003; Radinger et al., 2010) in StemPro-34 medium containing 13 ml of StemPro-34 Nutrient Supplement and l-glutamine (2 mM) and penicillin (100 U/ml)/streptomycin (100 μg/ml; all from GIBCO, Grand Island, NY) with 100 ng/ml recombinant human SCF added (Peprotech, Rocky Hill, NJ). Half of the medium supplemented with SCF was changed every 7 d. HLMCs were obtained by lung resection for bronchial carcinoma and purified using immunoaffinity magnetic selection using anti-CD117 (BD Biosciences, San Jose, CA) as described (Sanmugalingam et al., 2000) and were cultured as described (Cruse et al., 2008). All human subjects gave written informed consent, and the study was approved by the Leicestershire Research Ethics Committee, United Kingdom. Human peripheral blood–derived mast cells were cultured from CD34⁺ progenitors as described (Radinger et al., 2010). The donors provided an informed consent, and cells were obtained under a protocol (NCT00001756) approved by the National Institute of Allergy and Infectious Diseases, National Institutes of Health Internal Review Board.

MS4A4 cloning

Total RNA was isolated from 2 × 10⁶ human lung mast cells and LAD-2 cells using the RNEasy kit, which was used according to the manufacturer's instructions (Qiagen, Valencia, CA). Primers were designed to amplify the full open reading frame of MS4A4 isoforms 1 and 2. cDNA was amplified as described (Cruse et al., 2010). The primers used for cloning were as follows. For isoform 1, forward, GGGG­AATTCATGCATCAGACCTACAGCAGAC, and reverse, GGGGG­ATCCTCAAACCTCATTAAGTGGTGTGG. For isoform 2, forward, GGGGAATTCATGACAACCATGCAAGGAATGGAAC, and reverse, GGGGGATCCTCAAACCTCATTAAGTGGTGTGG.

Only isoform 2 primers amplified a product. The resulting product was gel purified and cloned into the pGEM T Easy Vector System (Promega) and then positively selected and sequenced as described (Cruse et al., 2010). MS4A4 clones were then PCR amplified with high-fidelity DNA polymerase to remove the stop codon and restriction sites SacI and SacII added to the 5′ and 3′ ends using the following primers: forward, GGGG­AG­CTCATGACAACCATGCAAGGAATGGAAC, and reverse, GGGCC­GCGGAACCTCATTAAGTGGTGTGGGAG.

The resulting clones without stop codons were subcloned into pEGFP-N1 to attach EGFP to the C terminus of MS4A4 isoform 2. Final clones were sequenced to confirm correct subcloning.

Lentivirus production and transduction of mast cells

For gene silencing, MISSION shRNA constructs and lentiviruses were used (Sigma-Aldrich) as described (Cruse et al., 2013). The following constructs were purchased:

Scramble shRNA control: SHC002

shMS4A4v1: TRCN0000063624

CCGGCCGTGTATATCGGGTACACAACTCGAGTTGTGTACCCGATATACACGGTTTTTG

shMS4A4v2: TRCN0000063625

CCGGGAGAAGTTCTTGAAGGGAGAACTCGAGTTCTCCC­TTCAAGAACTTCTC­TTTTTG

shMS4A4v3: TRCN0000063626

CCGGCGAGGTAGTCTAGGAATGAATCTCGAGATTCATTCC­TAGACTACCT­CGTTTTTG

shMS4A4v4: TRCN0000063627

CCGGCCCACACCACTTAATGAGGTTCTCGAGAACCTCATTAAGTGGTGTGGGTTTTTG

Plasmid DNA was purified, and lentivirus containing the shRNA plasmids was produced as described (Cruse et al., 2013).

Plasmids

Wild-type monomeric red fluorescent protein (mRFP) Rab5 (pmRFP-C3) construct (Vonderheit and Helenius, 2005) was a gift from Ari Helenius (ETH Zurich; Addgene plasmid 14437). The constitutively active mCherry-Rab5CA(Q79L) (PmCherry-C1; Addgene plasmid 35138) and dominant-negative mCherry-Rab5DN(S34N) (PmCherry-C1; Addgene plasmid 35139) were gifts from Sergio Grinstein (University of Toronto; Bohdanowicz et al., 2012). The DsRed-Rab7 WT (pDsRed-C1; Addgene plasmid 12661), DsRed-Rab9 WT (pDsRed-C1; Addgene plasmid 12677), and DsRed-Rab11 WT (pDsRed-C1; Addgene plasmid 12679) were gifts from Richard Pagano (Mayo Clinic; Choudhury et al., 2002).

Transfection of LAD-2 cells

Transfections with cDNA were carried out using the Nucleofector II machine (Amaxa, Anaheim, CA) as described (Cruse et al., 2013). Confocal microscopy with cotransfected live cells was carried out at 16 h posttransfection. For colocalization of MS4A4 and Rab5 variants in Figure 4A, imaging was performed at 6 h posttransfection due to apparent cytotoxicity of dominant-negative Rab5 expression with the longer incubation.

Quantitative real-time PCR

Primers were designed for MS4A4. Quantitative real-time PCR (qRT-PCR) performed in the United Kingdom (Figure 1C) was carried out using the Full Velocity SYBR Green QRT-PCR system (Stratagene, La Jolla, CA) as described (Cruse et al., 2008). Products were also run on a 1.5% agarose gel to confirm that the products were the expected length. Bands were then excised from the gel and sequenced (Protein and Nucleic Acids Chemistry Laboratory, University of Leicester, Leicester, United Kingdom).

qRT-PCR in the United States (Figure 1, D, E, and L) was carried out as described (Cruse et al., 2013). Total RNA was isolated using the RNeasy Plus kit (Qiagen), which eliminates genomic DNA. Total RNA was reverse transcribed into cDNA using the QuantiTect reverse transcription system (Qiagen), which includes a DNase step to further ensure genomic DNA elimination. The qPCR was carried out using the Quantifast SYBR green kit (Qiagen) on the ABI 7500 machine. Primers were the same for all qPCR: forward, GCATGGGAATAACAATGATGTG; and reverse, TCCTAGACTACCTCGGACCAG.

Immunoblotting

LAD-2 cells were incubated overnight in LAD-2 medium without SCF. Cells were then washed twice in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer containing 0.04% bovine serum albumin (BSA) and resuspended at 2.5 × 10⁶ cells/ml HEPES buffer containing 0.04% BSA. The cells were challenged with SCF for the indicated time points and then immediately lysed as described (Cruse et al., 2013). Immunoblots were then carried out on the total cell lysates as described (Cruse et al., 2013). The acquired images were analyzed, and densitometry was performed using ImageJ software, version 1.32. For signaling experiments in Figure 2, IRDye (680RD or 800CW)–labeled secondary antibodies (1:20,000) were used to label both phosphorylated and total proteins simultaneously. Membranes were visualized and quantified using an Odyssey CLx imager (LI-COR Biosciences, Lincoln, NE).

Immunoprecipitation

LAD-2 cells were deprived of SCF for 16 h before experiments. Cells were then either left unstimulated or stimulated with SCF for 10 min, followed by plunging into ice. Cells were washed in ice-cold phosphate-buffered saline (PBS) and lysed for 1 h in 1% Triton X immunoprecipitation buffer as described (Cruse et al., 2013). The lysates were then centrifuged at 20,000 × g for 10 min to remove insoluble material. A 50-μl amount of protein A/G UltraLink resin (Thermo Scientific, Waltham, MA) was used to preclear the lysates for 30 min at 4°C with rotation. After preclearing, the lysates were centrifuged at 15,000 × g for 2 min to remove the resin. A 5-μg amount of mouse polyclonal anti-MS4A4 or mouse IgG control was added to the lysates and incubated overnight at 4°C with rotation. Then 50 μl of protein A/G UltraLink resin was added to the lysates and incubated for 3 h at 4°C. The lysates were washed five times in immunoprecipitation buffer. The coimmunoprecipitations were then eluted from the resin with boiling in SDS running buffer as described (Cruse et al., 2013).

Flow cytometry

To assay surface expression of receptors, we used flow cytometric analysis on the FACSCalibur machine (BD Biosciences).

KIT internalization assays

KIT internalization assays were performed on LAD-2 HuMC transduced with either shMS4A4 or scramble shRNA control as described (Cruse et al., 2013). Before the assay, LAD-2 HuMCs at day 7 postinfection with shRNA constructs were washed to remove SCF and incubated overnight (16 h) in cytokine-free LAD-2 HuMC medium. Brefeldin A (BFA) was added and incubated for 2 h at 37°C before experiments in which it was used. Cells were then washed twice in prewarmed (37°C) HEPES buffer (10 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.4 mM Na₂HPO₄7H₂O, 5.6 mM glucose, 1.8 mM CaCl₂2H₂O, and 1.3 mM MgSO₄7H₂O with 0.04% BSA) and resuspended at 5 × 10⁵ LAD-2 HuMCs/ml in HEPES buffer with or without BFA. Time zero was plunged into ice, and the remaining time points were stimulated with recombinant human SCF and mixed. At the indicated time points, cells were plunged into ice. Samples were maintained on ice and washed twice with ice-cold PBS/1% BSA and stained for surface KIT expression. Surface KIT expression was assessed as described in Flow cytometry.

KIT recycling and degradation assays

LAD-2 cells were incubated overnight (16 h) at 37°C in StemPro medium without SCF. KIT recycling and degradation were assessed using a method modified from Roepstorff et al. (2009) in which cells were kept ice cold throughout the initial steps. Cells were washed twice in cold HEPES buffer containing 0.04% BSA by centrifugation at 4°C, resuspended in the HEPES/BSA buffer, and maintained on ice. The cell suspensions were incubated with either 10 ng/ml or 100 ng/ml SCF for 1 h on ice. After the incubation, cells were washed three times in the cold HEPES/BSA buffer by centrifugation at 4°C, resuspended in the HEPES/BSA buffer, and divided into precooled fluorescence-activated cell sorting (FACS) tubes (5 × 10⁵ LAD-2 cells in 1 ml/tube) for each time point. Tubes were transferred to a 37°C water bath and transferred back to the ice-water bath at the indicated time points. The 0-min samples were never removed from the ice-water bath. Samples were centrifuged at 4°C and washed once in the HEPES/BSA buffer. Cells were then maintained on ice, and surface staining of KIT was performed as described in Flow cytometry.

For each set of stimulations, a subset of cells was incubated for 30 min in 10 μM cycloheximide in 0.1% DMSO before dividing into FACS tubes and warming to 37°C for the indicated time points. Cycloheximide was present for the duration of the assays to prevent repopulation with newly synthesized KIT. At each time point, cells were immersed in an ice-water bath for 10 min before centrifugation and washed once in cold PBS. Cells were then fixed in ice-cold 4% paraformaldehyde (PFA) for 30 min on ice and washed twice in cold PBS. Cells were permeabilized in PBS containing 0.1% saponin for 30 min on ice before staining for total KIT in PBS containing 0.1% saponin and analysis by flow cytometry.

Confocal microscopy and image analysis

Confocal microscopy was carried out within the Biological Imaging Section of the Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, on a Leica TCS SP8 confocal microscope. For immunofluorescence analyses, cells were fixed in ice-cold 4% PFA for 30 min on ice, washed twice in cold PBS, and permeabilized in PBS containing 0.1% Triton X for 30 min on ice before staining. Images were collected using a 63× oil immersion objective (numerical aperture [NA] 1.4). Fluorochromes were excited using an argon laser at 488 nm for EGFP and Alexa 488, a helium–neon laser at 568 for RFP, mCherry, DsRed, and Alexa 568, a helium–neon laser at 594 nm for Alexa 594, and a helium–neon laser at 633 for APC and Alexa 647. 4′,6-Diamidino-2-phenylindole was excited using an ultraviolet 405 laser. Sequential images at 200-nm intervals were collected sequentially and superimposed. Imaris software was used for colocalization analyses using automatic thresholding. For live-cell imaging, LAD-2 HuMCs were cotransfected with pEGFP-N1:MS4A4 and red fluorescent–tagged Rab proteins (see Plasmids). Cells were plated into eight-well chambered coverglass in HEPES buffer (described earlier) and imaged on a heated stage (37°C) flushed with 5% CO₂. A 63× glycerol immersion objection (NA 1.3) was used, which was also heated to 37°C. Images were collected simultaneously with detector slits configured to minimize any cross-talk between the channels. Images were acquired using Leica LAS AF software and processed using Bitplane Imaris and Huygens Essential software. For colocalization analyses, the Bitplane Imaris colocalization analysis tool was used on unprocessed image stacks.

Apoptosis assays

For the analysis of apoptosis. Fluorescein isothiocyanate–conjugated annexin V (BD PharMingen) and propidium iodide were used as described (Cruse et al., 2010).

Mast cell proliferation and migration assays

Mast cell proliferation was determined by incubation of transduced LAD-2 mast cells in complete StemPro medium with 100 ng/ml SCF in 12-well plates (5 × 10⁵ cells/well). Duplicate wells were counted by flow cytometry at the indicated time points, and nonviable cells were identified by staining with propidium iodide and eliminated from the analysis. Mast cell migration was assessed as described (Cruse et al., 2006, 2011).

We thank the Biological Imaging Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, for expert advice in the acquisition and analysis of the confocal imaging. We also thank E. C. Chan (Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health) for providing cDNA from maturing MC cultures. Financial support was provided by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases and National Heart, Lung, and Blood Institute within the National Institutes of Health. Work in Leicester was supported in part by the National Institute for Health Research Leicester Respiratory Biomedical Research Unit. The views expressed are those of the authors and not necessarily those of the National Health Service, National Institute for Health Research, or the Department of Health.