DNA Constructs

HA-CXCR4, FLAG-ubiquitin, HA-CXCR4-YFP, HA-arrestin-3, and HA-arrestin-2 constructs were described previously (Bhandari et al., 2007). Primers used for generating all constructs are listed in Supplemental Table S1. For STAM-1 truncation mutants (1-195, 1-269, 1-390, 391-540, 337-540, 270-540, 212-540, 144-540), full-length STAM-1 in 3×FLAG-pCMV-10 was amplified by polymerase chain reaction (PCR) using primers flanking various regions of STAM-1 as indicated above and harboring 5′ and 3′ HindIII and XbaI restriction enzyme sites, respectively. PCR fragments were digested and ligated into the HindIII and XbaI sites of 3×FLAG pCMV-10 (Sigma-Aldrich). For STAM-1-ΔGAT, the region encompassing amino acid residues 343-377 was deleted by two-step PCR with mutually annealing overlapping primers and flanking primers based on 3×FLAG-pCMV-10. Amplified product was digested and ligated into HindIII and XbaI sites of 3×FLAG-pCMV-10 and pGEX-4T2 (GE Healthcare). For STAM-1-GAT, amino acid residues 296-380 were amplified by PCR from full-length FLAG-STAM-1 and cloned into the HindIII and XbaI sites of 3×FLAG-pCMV-10 and EcoRI and XhoI sites of pGEX-4T2. For arrestin-2-(25-161) constructs, amino acid residues 25-161 were amplified by PCR from HA-arrestin-2-(1-161) and cloned into HindIII and XbaI sites of 3×FLAG pCMV-10 and SmaI and XhoI sites of pGEX4T2 respectively. For YFP-STAM-1, full-length STAM-1 was amplified from FLAG-STAM-1 and cloned into the HindIII and KpnI sites of pEYFP-C1 vector (Clontech, Mountain View, CA). The sequence of all constructs was verified by sequencing.

GST-Fusion Protein Binding Assays

Escherichia coli BL21 cells transformed with GST-fusion protein constructs or empty vector (pGEX-4T2) were grown overnight in Luria Broth containing 100 μg/ml ampicillin. The next day, cultures were diluted (3.7%) and grown to an OD₆₀₀ ~ 0.35–0.40 at 37°C followed by induction with 0.1 mM isopropyl-1-thio-β-d-galactopyranoside for 1 h at 18°C. Cells were then pelleted by centrifugation and resuspended in 1 ml of binding buffer (20 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 1 mM dithiothreitol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 μg/ml pepstatin-A), followed by sonication and centrifugation. Clarified lysates were incubated with glutathione-Sepharose 4B resin for 1 h, washed, and resuspended in binding buffer. Samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and stained with Gel-Code blue to estimate the protein amounts by comparing the samples to known amounts of purified bovine serum albumin (Fraction V; Roche Diagnostics, Indianapolis, IN). For binding assays, equimolar amounts of purified GST-fusion proteins were incubated with 100 μl of clarified cell lysate of HEK293 cells expressing the desired construct for 2–4 h at 4°C. For binding experiments using purified arrestin-2, GST fusion proteins were incubated with 500 ng of arrestin-2 in 100 μl of binding buffer for 1 h at 4°C. After incubation, samples were washed three times with binding buffer, eluted in 2× sample buffer by boiling for 10 min and bound proteins were detected by SDS-PAGE followed by immunoblotting.

Degradation Assay

HEK293 cells stably expressing HA-CXCR4 or HeLa cells expressing endogenous levels of CXCR4 grown on 10-cm dishes were transfected with 100 nM STAM-1, AMSH, or GAPD siRNA using Lipofectamine 2000 transfection reagent (Invitrogen). To assess the role of STAM-1 and arrestin-2 minigene constructs on CXCR4 degradation, HEK293 cells grown on 10-cm dishes were cotransfected with 1 μg of HA-CXCR4 and 9 μg of FLAG-STAM-1-GAT, FLAG-arrestin-2-(25-161) or empty vector (pCMV-10) using TransIT-LT1 transfection reagent (Mirus, Madison, WI). Twenty-four hours later, cells were passaged onto poly-l-lysine (0.1 mg/ml; Sigma-Aldrich) coated 24-well plates (HEK293 cells) or six-well plates (HeLa cells) and grown for an additional 18–24 h. Cells were washed once and incubated with DMEM containing 10% FBS and 50 μg/ml cyclohexamide to stop protein synthesis for 15 min at 37°C. Cells were then incubated with the same medium containing vehicle (0.5% bovine serum albumin [BSA]) or 30 nM CXCL12 for 1, 2, and 3 h. Cells were washed and collected in 300 μl of 2× sample buffer and then sonicated. Receptor amounts were determined by SDS-PAGE followed by immunoblotting using an anti-HA mAb or anti-CXCR4 antibody, as described previously (Marchese, 2009). To assess EGFR degradation, HeLa cells grown on six-well plates were transfected with 3 μg of FLAG-STAM-1-GAT, FLAG-arrestin-2-(25-161), or empty vector (pCMV-10) using TransIT-LT1 transfection reagent. Forty-eight hours after transfection cells were incubated with DMEM containing 10% FBS and 50 μg/ml cyclohexamide to stop protein synthesis for 15 min at 37°C. Cells were then incubated with the same medium containing vehicle (0.5% BSA) or 100 ng/ml EGF for 1 h. Cells were processed as described above for CXCR4 degradation.

Coimmunoprecipitation Studies

HeLa cells were transiently transfected with HA-Arrestin-2, HA-arrestin-3, or empty vector alone (pcDNA3) using TransIT-LT1 transfection reagent. Forty-eight hours later, cells were collected in 1.5-ml immunoprecipitation buffer [20 mM Na₂PO₄, pH 6.5, 150 mM NaCl, 1% (vol/vol) Triton-X 100, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 μg/ml pepstatin A] and incubated at 4°C for 30 min. Cells were sonicated, centrifuged, and the clarified lysates were incubated with an anti-HA mAb or isotype control antibody to immunoprecipitate HA-tagged arrestin-2/3 followed by immunoblotting to detect bound endogenous STAM-1 and HRS. Endogenous arrestins were immunoprecipitated from HeLa cells using an anti-arrestin2/3 mouse monoclonal or isotype control antibody followed by immunoblotting to detect bound endogenous STAM-1 and HRS. To assess the effect of the STAM-1-GAT minigene on the interaction between STAM-1 and arrestin-2, lysates from HeLa cells transfected with HA-arrestin-2 and FLAG-STAM-1-GAT or pCMV were incubated with an anti-HA or isotype control antibody and immunoprecipitates were analyzed for the presence of endogenous STAM-1. To assess the effect of the arrestin-2-(25-161) minigene on the interaction between STAM-1 and arrestin-2, HeLa cells transfected with T7-STAM-1, HA-arrestin-2, and FLAG-arrestin-2-(25-161) or pCMV were incubated with an anti-T7 polyclonal antibody and immunoprecipitates were analyzed for the presence of HA-arrestin-2 and endogenous HRS.

Confocal Immunofluorescence Microscopy

HEK293 cells transiently transfected with HA-CXCR4-YFP were passaged onto poly-l-lysine–coated coverslips and allowed to grow for 24 h. HeLa cells were used to examine the distribution of endogenous CXCR4. Cells were washed once with warm DMEM containing 20 mM HEPES, pH 7.5, and incubated in the same medium for 3–4 h at 37°C. Cells were treated with 30 nM CXCL12 or vehicle for 30 min, fixed with 3.7% paraformaldehyde, and then permeabilized with 0.05% (wt/vol) saponin for 10 min, similar to a protocol we have described previously (Bhandari et al., 2007). Cells were coincubated with STAM-1, EEA1, or β-arrestin2/3 antibodies. Endogenous CXCR4 in HeLa cells was stained with rat anti-CXCR4 mAb. In brief, after permeabilization and fixation, cells were incubated with 1% BSA in 0.05% saponin-phosphate-buffered saline (PBS) for 30 min at 37°C, followed by incubating with primary antibody for 1 h at 37°C. Primary antibodies for STAM-1 and EEA1 were used at 1:100 dilution and against CXCR4 and β-arrestin2/3 was used at a 1:50 dilution. Cells were washed five times with 0.05% saponin-PBS, followed by incubating with appropriate Alexa-Fluor–conjugated secondary antibodies for 30 min at 37°C. Finally, cells were washed with PBS and fixed again in 3.7% formaldehyde-PBS and mounted onto glass slides using mounting media containing 4,6-diamidino-2-phenylindole. Samples were analyzed using an LSM 510 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY) equipped with a Plan-Apo 63×/1.4 oil lens objective. Images were acquired using a 1.4-megapixel cooled extended spectra range RGB digital camera set at 512 × 512 resolution. Acquired images were analyzed using ImageJ, version 1.41o (National Institutes of Health, Bethesda, MD), and the amount of colocalization between proteins was determined using MetaMorph 7.6 (Molecular Devices, Downingtown, PA).

Ubiquitination Assays

For CXCR4 ubiquitination, HEK293 cells stably expressing HA-CXCR4 grown on 10-cm dishes were transfected with 3 μg of FLAG-ubiquitin. Eight hours later, cells were transfected either with 10 μg of FLAG-STAM-1-GAT, FLAG-Arr2-(25-161), or empty vector (pCMV). The next day, cells were passaged onto 6-cm dishes and allowed to grow for an additional 24 h. The next day, cells were serum starved in DMEM containing 20 mM HEPES for 3 h and then treated with 30 nM SDF for 30 min, washed once on ice with cold PBS, and collected in 1 ml of lysis buffer [50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% (wt/vol) sodium deoxycholate, 1% (vol/vol) NP-40, 0.1% (wt/vol) SDS, 20 mM N-ethylmaleimide (NEM), and 10 μg/ml each of leupeptin, aprotinin, and pepstatin A]. Samples were transferred into microcentrifuge tubes and placed at 4°C for 30 min, followed by sonification and centrifugation to pellet cellular debris. Clarified cell lysate was incubated with an anti-HA polyclonal antibody and the immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting using an anti-FLAG antibody conjugated to HRP.

To detect HRS ubiquitination, HEK293 cells stably expressing HA-CXCR4 were transfected with 3 μg of FLAG-ubiquitin. Eight hours later, cells were cotransfected with 8 μg of FLAG-STAM-1-GAT or empty vector (pCMV-10) and 2 μg of T7-tagged HRS. Twenty-four hours later, cells were passaged onto poly-l-lysine–coated 6-cm dishes, and the next day cells were serum starved for 4–5 h in DMEM containing 20 mM HEPES and were treated with 30 nM SDF or vehicle alone for 30 and 60 min. Cells were washed with cold PBS and collected in 1 ml of ubiquitination buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Triton-X 100, 5 mM EDTA, 20 mM NEM, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 μg/ml pepstatin-A), incubated for 30 min at 4°C, sonicated, and clarified by centrifugation. HRS was immunoprecipitated using an anti-HRS polyclonal antibody and immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting to detect ubiquitinated HRS using an anti-FLAG antibody conjugated to HRP.

For STAM-1 ubiquitination experiments, HeLa cells grown in six-well dishes were cotransfected with 3 μg of T7-STAM-1 and 40 ng of HA-ubiquitin. Eight hours later, cells were transfected with 3 μg of FLAG-STAM-1-GAT or empty vector (pCMV-10). Twenty-four hours later, cells were passed onto poly-l-lysine–coated 6-cm dishes, and the next day cells were serum starved and treated and processed as described above for HRS ubiquitination using a modified ubiquitination buffer (20 mM NaPO₄, pH 6.5, 150 mM NaCl, 1% Triton-X 100, 20 mM NEM, and protease inhibitor cocktail). Tagged STAM-1 was immunoprecipitated using an anti-T7 goat polyclonal antibody and immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting to detect ubiquitinated STAM-1 using an anti-HA mAb.

Internalization and Recycling Assays

For measuring internalization and recycling of CXCR4, HEK293 cells grown on 10-cm dishes were cotransfected with FLAG-CXCR4 (1 μg) and 100 nM STAM-1 or GAPD siRNA using Lipofectamine 2000 transfection reagent. The next day, cells were passaged onto poly-l-lysine–coated 24-well plates and grown for an additional 24 h. Cells were serum starved for 3–4 h; placed on ice; washed once with DMEM containing 0.1% BSA, 20 mM HEPES, and 1 mM Ca²⁺; and then incubated in the same medium containing the calcium-dependent M1 anti-FLAG antibody for 1 h on ice, which labels cell surface receptors only. Cells were washed and incubated in the same medium containing vehicle or 30 nM CXCL12 for 45 min at 37°C. To remove surface bound antibody, cells were washed three times with Ca²⁺- and Mg²⁺-free PBS containing 0.04% EDTA. Cells were incubated in DMEM containing 1 mM Ca²⁺ and the CXCR4 antagonist AMD3100 (10 μM) to block any further internalization for 30 and 60 min at 37°C. The amount of receptor/antibody that recycled back to the cell surface was quantified by incubating cells with an alkaline-phosphatase conjugated goat anti-mouse immunoglobulin (Ig)G antibody. In brief, cells were washed once with PBS containing 1 mM Ca²⁺ and then fixed with 3.7% paraformaldehyde for 5 min on ice. After fixation, cells were washed three times and incubated with alkaline phosphatase-conjugated goat anti-mouse antibody diluted in PBS containing 1% BSA for 1 h at room temperature. Cells were then washed with PBS and incubated in p-nitrophenyl phosphate diluted in diethanolamine buffer (Bio-Rad Laboratories) for 5–15 min. Reactions were stopped by adding 0.4 N NaOH and an aliquot was used to measure the absorbance at 405 nm. Percentage of receptor recycling was calculated by dividing the amount of receptor internalized by the amount of receptors recovered after incubation at different time intervals. To calculate the percentage of receptor internalization, the amount of receptor remaining on the cell surface was divided by the total number of receptors present on the cell surface before treatment with agonist.

Role of STAM-1 in CXCR4 Trafficking

Because these data suggest that STAM-1 has a role in endosomal sorting of CXCR4, we examined agonist promoted degradation of CXCR4 in cells that were depleted of STAM-1 by RNA interference. HEK293 cells stably expressing HA-CXCR4 were transfected with control or STAM-1 siRNA, followed by treatment with CXCL12 (30 nM) for 3 h, and receptor degradation was assessed by immunoblot analysis, as described previously (Marchese et al., 2003). As shown in Figure 4A, siRNA-mediated depletion of STAM-1 lead to a moderate, but statistically significant, increase in CXCR4 degradation, compared with control siRNA-treated cells, suggesting that STAM-1 negatively regulates agonist promoted degradation of CXCR4. As the amount of receptor that is degraded is in part a function of the rate of receptor internalization and recycling, we also examined the effect of depleting STAM-1 on CXCR4 internalization and recycling. Cell surface FLAG-tagged CXCR4 was labeled with the M1 anti-FLAG antibody on ice in the presence of 1 mM Ca²⁺, because the M1 antibody binds to the FLAG epitope in a calcium-dependent manner. Cells were washed to remove unbound antibody, and the media were replaced with DMEM containing CXCL12 (30 nM) in the continued presence of 1 mM Ca²⁺ and placed at 37°C for 45 min to allow for internalization of the M1/CXCR4 complexes to take place. Antibody remaining on the surface, mostly representing uninternalized receptor, was removed by incubating cells with PBS containing EDTA (0.04%), a calcium-chelating agent. The amount of antibody (i.e., receptor) that recycled back to the cell surface was quantified by cell surface enzyme-linked immunosorbent assay (ELISA) in parallel wells that were incubated at 37°C for 30 and 60 min. In control siRNA treated cells, ~20% of internalized CXCR4 recycled back to the cell surface after 30 and 60 min, similar to what we observed in STAM-1–depleted cells, suggesting that STAM-1 depletion had no effect on recycling of CXCR4 (Figure 4B). In addition, agonist-promoted internalization of CXCR4 was similar in STAM-1–depleted cells, compared with control siRNA-treated cells, suggesting that STAM-1 is not involved in CXCR4 internalization (Figure 4C). We also examined the role of AMSH on agonist promoted degradation of CXCR4. AMSH is a deubiquitinating enzyme that interacts with STAM-1 and negatively regulates endosomal sorting of the EGFR (McCullough et al., 2004). As shown in Figure 4D, siRNA-mediated depletion of AMSH did not effect agonist promoted degradation of CXCR4 in HeLa cells, suggesting that AMSH does not regulate endosomal sorting of activated CXCR4. However, CXCR4 levels were elevated in vehicle treated cells transfected with AMSH siRNA (Figure 4D), suggesting that AMSH may regulate degradation of constitutively internalized CXCR4, similar to what has been reported recently (Sierra et al., 2010). Together, our data suggest that STAM-1 negatively regulates CXCR4 degradation likely through a mechanism that directly attenuates endosomal sorting.

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Figure 4.

STAM-1 negatively regulates CXCR4 degradation. HEK293 cells stably expressing HA-CXCR4 were transfected with control (GAPD) and STAM-1 siRNA as described in Materials and Methods. Cells were treated with vehicle (PBS containing 0.01% BSA) or 30 nM CXCL12 for 3 h and receptor levels were determined by immunoblotting followed by densitometric analysis. Bars represent the average amount of CXCR4 degraded ± SEM from three independent experiments. *p < 0.05, unpaired t test. (B) CXCR4 recycling was measured in HEK293 cells transfected with FLAG-CXCR4 and siRNA as described in A. Surface receptors were labeled with the M1 anti-FLAG antibody followed by treatment with 30 nM CXCL12 for 45 min in DMEM containing 0.1% BSA, 20 mM HEPES, pH 7.4, and 1 mM Ca²⁺. Antibody remaining on the cell surface was stripped by two rapid washes with Ca²⁺/Mg²⁺ free PBS containing 0.04% EDTA. Cells were then incubated in DMEM containing 1 mM Ca²⁺ and 10 μM AMD3100 (CXCR4 antagonist) and incubated at 37°C for 30 and 60 min. The amount of antibody reappearing on the cell surface was quantified by ELISA, as described in Materials and Methods, and used as an indicator of receptor recycling. Bars represent the percentage of internalized receptor that recycled ± SEM from three independent experiments. (C) Bars represent the percentage of cell surface receptors internalized in cells treated with CXCL12 compared with vehicle treated cells. The error bars represent SEM from three independent experiments. (D) HeLa cells were transfected with GAPD and AMSH siRNA and treated and analyzed as in A. Data represent the mean ± SEM from three independent experiments.

Mapping the Arrestin-2 Binding Site on STAM-1

We recently reported that arrestin-2 positively regulates CXCR4 sorting into the degradative pathway. To gain insight into the function of the arrestin-2/STAM-1 interaction on CXCR4 trafficking, we initially set out to determine the mechanism of the interaction. To accomplish this we mapped the arrestin-2 binding region on STAM-1 by truncation mutagenesis. STAMs contain multiple domains, characterized by the presence of an amino-terminal Vps27, Hrs, STAM homology (VHS) domain, UIM, SH3 (Src homology) domain, immunoreceptor based tyrosine activation motif (ITAM), and a GGA and TOM1 homologous (GAT) domain that partially overlaps with the ITAM (Prag et al., 2007; Ren et al., 2009). We created several STAM-1 N-terminal and C-terminal truncation mutants, according to its domain organization, tagged with the FLAG epitope on the amino-terminal end (Figure 5A). GST-arrestin-2 and GST immobilized on glutathione-Sepharose resin were incubated with lysates expressing the various STAM-1 truncation mutants, and bound proteins were detected by immunoblotting. The results from these experiments are summarized in Figure 5A and the data are shown in Supplemental Figure S1. The arrestin-2 binding region was determined to reside between amino acid residues 296-380 on STAM-1. This region encompasses the GAT domain, which has been shown to form two tandem coiled-coil domains (amino-acid residues 301–377) (Prag et al., 2007; Ren et al., 2009). To further confirm that the GAT domain mediates binding to arrestin-2, deletion of the GAT domain abrogated STAM-1 binding to arrestin-2 (Figure 5B), and the GAT domain alone fused to GST was able to bind to arrestin-2 (Figure 5C).

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Figure 5.

The STAM-1 GAT domain is both necessary and sufficient for arrestin-2 binding. (A) STAM-1 truncation mutants are represented schematically. Binding or no binding to GST-arrestin-2 is represented by + and − symbols, respectively, on the right as assessed by data shown in Supplemental Figure S1. (B) Equimolar amounts (~600 nM) of GST-arrestin-2 and GST were incubated with lysates from HEK293 cells transiently transfected with FLAG-tagged full-length-STAM-1 or STAM-1-ΔGAT. (C) Equimolar amounts (~117 nM) of (GST-STAM-1,) GST-STAM-1-GAT and GST were incubated with lysates from HEK293 cells transiently transfected with FLAG-tagged arrestin-2. In B and C, bound proteins were detected by immunoblotting, followed by staining blots with Ponceau-S to assess the amount of GST fusion protein used in the binding assay. Shown are representative blots from one of three independent experiments.

To determine whether the interaction between STAM-1 and arrestin-2 is important for CXCR4 trafficking, we initially expressed the GAT domain as a minigene in cells and assessed whether it disrupted the arrestin-2/STAM-1 interaction. HeLa cells transfected with FLAG-S1-GAT and HA-arrestin-2 were subjected to immunoprecipitation using an anti-HA antibody followed by immunoblotting to detect the presence of endogenous STAM-1 in the immunoprecipitates. As shown in Figure 6A, expression of the GAT domain disrupted the arrestin-2/STAM-1 interaction. To determine the function of the arrestin-2/STAM-1 interaction on lysosomal targeting of CXCR4, we examined the effect of expressing the GAT domain on CXCR4 degradation. Remarkably, expression of the GAT domain significantly accelerated CXCR4 degradation after agonist treatment as compared with empty vector (Figure 6, B and C). Together, these data suggest that the STAM-1/arrestin-2 interaction negatively regulates CXCR4 sorting to lysosomes. Because the STAM-1 GAT domain has been shown recently to bind to HRS and is predicted to be required for the assembly of ESCRT-0 (Ren et al., 2009), it is conceivable that arrestin-2 binding to STAM-1 displaces its interaction with HRS and promotes disassembly of ESCRT-0, which somehow negatively regulates the amount of CXCR4 that is targeted for lysosomal degradation.

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Figure 6.

Expression of the GAT domain disrupts the arrestin-2/STAM-1 interaction and accelerates CXCR4 degradation. (A) Lysates from HeLa cells cotransfected with HA-arrestin-2 and FLAG-STAM-1-GAT (S1-GAT) or empty vector (pCMV) were incubated with anti-HA and IgG control antibodies. Immunoprecipitates were analyzed by immunoblotting to detect bound endogenous STAM-1, and lysates were analyzed to assess expression of the various constructs. Shown are representative blots from one of three independent experiments. (B) HA-CXCR4 degradation was assessed in HEK293 cells expressing FLAG-STAM-1-GAT or empty vector (pCMV) as described in Materials and Methods. (C) Graphical representation of percentage of receptor degraded. Error bars represent SEM from three independent experiments. Data were analyzed by two-way ANOVA and followed by a Bonferroni's post hoc test. (*p < 0.0001). Shown are representative blots from one of three independent experiments.

Mapping the STAM-1 Binding Site on Arrestin-2

To gain greater insight into this process, we next set out to identify the STAM-1 binding region on arrestin-2 by truncation mutagenesis. Schematic representations of the arrestin-2 truncation mutants used are shown in Figure 7A; most have been described previously (Bhandari et al., 2007). GST-STAM-1 and GST were incubated with lysates prepared from HEK293 cells expressing various HA-tagged arrestin-2 truncation mutants. The results from these binding experiments are summarized in Figure 7A, and the data are shown in Supplemental Figure S2. Both the N- and C-terminal regions of arrestin-2 bound to GST-STAM1, but not GST, although binding to the N-terminal region seemed to be stronger, suggesting that it represented the main binding region. Further deletion of this region revealed that the STAM-1 binding site on arrestin-2 is between amino acid residues 1–161 (Supplemental Figure S2B). We next determined whether expression of this region as a minigene in cells also disrupted the arrestin-2/STAM-1 interaction. However, when expressed in cells the arrestin-2-(1-161) minigene completely blocked CXCR4 degradation (data not shown). The N-terminal lysine residues within arrestin-2 are predicted to serve as phosphosensors and recognize phosphates attached to receptors (Kern et al., 2009), analogous to what has been observed for arrestin-1 (Vishnivetskiy et al., 2000); therefore, the arrestin-2-(1-161) construct may bind to CXCR4 and have a dominant-negative effect on CXCR4 internalization. To rule out any effects at the level of internalization, the first 24 amino acids from the N terminus of arrestin-2 were deleted to create arrestin-2-(25-161), and we initially tested the ability of this mutant to bind to STAM-1. As shown in Figure 7B, GST fused to arrestin-2-(25-161), but not GST alone, efficiently bound to FLAG-STAM-1 expressed in cells. A FLAG-tagged construct of arrestin-2-(25-161) when expressed in HEK293 cells also bound to GST-STAM-1-GAT, suggesting that the STAM-1/GAT domain binding site on arrestin-2 is located between amino acid residues 25-161 (Figure 7C). We next examined whether expression of arrestin-2-(25-161) disrupted the STAM-1/arrestin-2 interaction and modulated CXCR4 degradation. Expression of FLAG-arrestin-2-(25-161) markedly disrupted the interaction between arrestin-2 and STAM-1 (Figure 8A) and significantly accelerated agonist promoted degradation of CXCR4 (Figure 8, B and C), similar to what was observed with the STAM-1 GAT domain (Figure 6). Together these data further indicate that the interaction between STAM-1 and arrestin-2 attenuates CXCR4 trafficking into the degradative pathway.

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Figure 7.

Mapping of the STAM-1 binding domain on arrestin-2. (A) Arrestin-2 truncation mutants used in the binding studies are represented schematically. Binding between GST-STAM-1 and HA-tagged arrestin-2 truncation mutants is shown as weak (+), intermediate (++), and strong (+++) on the right. (B) Equimolar amounts (~234 nM) of GST-arrestin-2, GST-Arr2-(25-161), and GST were incubated with lysates from HEK293 cells transiently transfected with FLAG-tagged STAM-1 and empty vector (pCMV-10). (C) Equimolar amounts (~276 nM) of GST-STAM-1, GST-STAM-1-GAT, and GST alone were incubated with lysates from HEK293 cells transiently transfected with FLAG-Arr-2-(25-161). In B and C, bound proteins were detected by immunoblotting using an anti-FLAG antibody and blots were stained with Ponceau-S to assess the amount of GST-tagged protein used in the binding assay. Shown are representative blots from one of three independent experiments.

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Figure 8.

Expression of Arr2-(25-161) disrupts the STAM-1/arrestin-2 interaction and accelerates CXCR4 degradation. (A) Lysates were prepared from HeLa cells cotransfected with T7-STAM-1, HA-arrestin-2 and increasing amounts (0, 1, and 2.5 μg) of FLAG-Arr2 (25-161). Lysates were divided into equal aliquots and incubated with either an anti-T7 polyclonal antibody or protein G agarose alone (control). Immunoprecipitates were analyzed by immunoblotting to detect bound HA-arrestin-2 and endogenous HRS and lysates were analyzed to assess the expression of the various constructs. Shown are representative blots from one of three independent experiments. (B) HA-CXCR4 degradation was assessed in HEK293 cells expressing FLAG-Arr2-(25-161) or empty vector (pCMV) as described in Materials and Methods. (C) Graphical representation of percent receptor degraded. Error bars represent SEM from three independent experiments. Data were analyzed by two-way ANOVA and followed by a Bonferroni's post hoc test. (*p < 0.0001). Shown are representative blots from one of three independent experiments.

This work was supported by National Institutes of Health grant GM-75159 (to A. M.) and by American Heart Association predoctoral fellowship 0910098G (to R. M.).