The E3 ubiquitin ligase atrophin interacting protein 4 binds directly to the chemokine receptor CXCR4 via a novel WW domain-mediated interaction

Abstract

The E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) mediates ubiquitination and down-regulation of the chemokine receptor CXCR4. AIP4 belongs to the Nedd4-like homologous to E6-AP carboxy terminus domain family of E3 ubiquitin ligases, which typically bind proline-rich motifs within target proteins via the WW domains. The intracellular domains of CXCR4 lack canonical WW domain binding motifs; thus, whether AIP4 is targeted to CXCR4 directly or indirectly via an adaptor protein remains unknown. Here, we show that AIP4 can interact directly with CXCR4 via a novel noncanonical WW domain-mediated interaction involving serine residues 324 and 325 within the carboxy-terminal tail of CXCR4. These serine residues are critical for mediating agonist-promoted binding of AIP4 and subsequent ubiquitination and degradation of CXCR4. These residues are phosphorylated upon agonist activation and phosphomimetic mutants show enhanced binding to AIP4, suggesting a mechanism whereby phosphorylation mediates the interaction between CXCR4 and AIP4. Our data reveal a novel noncanonical WW domain-mediated interaction involving phosphorylated serine residues in the absence of any proline residues and suggest a novel mechanism whereby an E3 ubiquitin ligase is targeted directly to an activated G protein-coupled receptor.

Figure 1.

The carboxy-terminal tail of CXCR4 interacts with AIP4. (A) Schematic representation of the carboxy-terminal tail of CXCR4 fused to GST. CXCR4 is a seven transmembrane domain spanning integral plasma membrane protein. The predicted cytoplasmic carboxy-terminal tail (amino acid residues 308-352) was fused to GST to create GST-C-tail. (B) The C-tail of CXCR4 interacts with AIP4. Whole cell lysates from HEK293 cells expressing FLAG-tagged AIP4 were incubated with GST-C-tail (57 nM) or GST (71 nM) alone. Input represents 0.4% of the lysate used in the binding reactions. Immunoblot was probed with the anti-FLAG M2 antibody to detect FLAG-AIP4. Blot was stained with Ponceau-S to determine the levels of the GST proteins used in the binding assay. Data from one of three independent experiments are shown. (C) The C-tail of CXCR4 binds directly to AIP4. Histidine-tagged AIP4 (HIS-AIP4; ∼5.2 pmol) was incubated with GST-C-tail (57 nM) or GST (71 nM) alone. Input represents ∼0.2% of the purified HIS-tagged AIP4 used in the binding assay. Immunoblot was probed with an anti-HIS antibody to detect HIS-AIP4. A GelCode blue stained gel indicating the levels of the GST proteins used in the binding assay is shown. Data from one of three independent experiments are shown.

Figure 2.

Agonist mediated interaction between CXCR4 and AIP4. (A) Agonist enhances the interaction between CXCR4 and AIP4. Serum-starved HEK293 cells cotransfected with FLAG-AIP4 and HA-CXCR4 or pcDNA3.0 were treated with either vehicle (DMEM containing 0.5% FBS) or CXCL12 (30 nM) for 15 min. The clarified whole cell lysates (WCL) were immunoprecipitated (IP) by using an anti-HA antibody and analyzed by SDS-PAGE and immunoblotting to detect bound FLAG-AIP4. Immunoprecipitates were also immunoblotted (IB) for HA-CXCR4 and WCL were immunoblotted to detect the expression of FLAG-AIP4 and HA-CXCR4. The HA antibody in the CXCR4 IP recognizes multiple bands, which are likely differentially glycosylated forms of the receptor. Immunoblots were stripped and reprobed for actin to assess loading. The bar graph represents average FLAG-AIP4 binding normalized to the level of receptor in the IPs. Error bars represent the SEM from three independent experiments. Binding of FLAG-AIP4 to CXCR4 in the presence of CXCL12 was significantly increased over its binding to the unstimulated receptor. *p < 0.05, unpaired t test. (B) FRET analysis of fluorescent protein-tagged CXCR4 and AIP4 in live cells. Shown are images of a representative cell treated with CXCL12. Prebleach and postbleach donor (HA-CXCR4-YFP; yellow) and acceptor (AIP4-CFP; cyan) images are shown. Arrows indicate cell periphery where FRET was observed. (C) In total, 20 cells without CXCL12 (−) and 17 cells with CXCL12 (+) treatment were analyzed from three independent experiments. The average F/F₀ ratio for AIP4-CFP, where F₀ and F are the donor emissions before and after progressive photobleaching, respectively, is plotted against time. Fluorescence intensity was measured using the whole cell for the analysis. The error bars represent the SE. The data were subject to an unpaired t test (p < 0.0001).

Figure 3.

The WW domains of AIP4 mediate the interaction with CXCR4. (A) Schematic representation of AIP4. AIP4 has a C2 domain, a PRR, four tandemly linked WW domains, and a catalytic HECT-domain. GST was fused to the amino terminus of full-length AIP4 (GST-AIP4), to AIP4 lacking the WW domains (GST-AIP4ΔWW) and to the WW domains alone (GST-WW-I-IV). (B and C) CXCR4 interacts with the WW domains of AIP4. (B) Whole cell lysates (WCL) from HEK293 cells transiently expressing HA-CXCR4 and empty vector (pcDNA3) were incubated with 1 μg of GST-AIP4, GST-WW-I-IV, or GST alone. (C) WCL from HEK293 cells stably expressing HA-CXCR4 were incubated with equimolar amounts (∼22 pM) of GST-AIP4, GST-AIP4ΔWW, GST-WW-I-IV, or GST alone. Samples were resolved by SDS-PAGE, followed by immunoblotting with an anti-HA antibody (top) to detect bound HA-CXCR4. Coomassie- (B) and GelCode blue (C)-stained gels (bottom) indicate the level of the GST fusion proteins used in the binding experiments. Input represents ∼0.6% (B) and 0.2% (C) of the lysate that was used for the binding experiment. Data from one of three experiments are shown. Receptor levels were determined by densitometry and average receptor binding was expressed as the percentage of control wild-type AIP4 binding (WT) ± SEM from three independent experiments (C, right). Please note that the SEM for ΔWW binding is ± 0.39. Data were analyzed by a one-way ANOVA, followed by Bonferroni's post hoc test. Binding to the ΔWW mutant was significantly different from WT and WW-I-IV binding. *p < 0.05. (D) The C-tail interacts directly with AIP4 WW domains. Increasing amounts of HIS-tagged WW-I-IV were incubated with equal amounts of GST-C-tail or GST alone. Bound HIS-WW-I-IV (60% of sample) was detected by immunoblotting with a HIS antibody conjugated to HRP. A known amount of HIS-WW-I-IV (3 ng) was also immunoblotted. The asterisk represents nonspecific binding of the HIS-HRP antibody to GST-C-tail. HIS-WW-I-IV binding was determined by densitometric analysis and normalization to GST-C-tail levels. Data are the mean HIS-WW-I-IV binding ± SE from four experiments. The data were analyzed by a one-way ANOVA followed by Bonferoni's post hoc test; p < 0.05 between 100 and 500 ng.

Figure 4.

Identification of residues within WW domains I and II that are critical for binding to CXCR4. (A) CXCR4 interacts with AIP4 WW domains I and II. Whole cell lysates (WCL) from HEK293 cells expressing HA-CXCR4 were incubated with GST alone (∼110 nM), GST-AIP4 (∼25 nM), GST-WW-I-IV (∼55 nM) or GST fusions of each of the four individual WW domains (∼100 nM). Immunoblot was probed with an anti-HA antibody to detect bound HA-CXCR4. A Coomassie-stained gel is shown to indicate the level of GST proteins used in the binding assay. Input represents 1.5% of the lysate used in the binding assay. Shown are data from one of three independent experiments. (B) Alignment of the WW domains from AIP4 and several members of the Nedd4-like family of E3 ligases. The four WW domains of AIP4 are shown aligned. Residues that are either identical or conserved to both WW domains I and II are high-lighted black. Residues that are unique to each of the four WW domains are boxed and shaded gray. Sequence of the Pin1 WW domain is also shown and the critical arginine residue shown to be important for phosphorylation mediated binding to Pin1 is shaded. The WW domains from other members of the Nedd4-like HECT domain family are shown (WWP1, WWP2, Nedd-4, and Rsp5). The conserved tryptophan residues from which the WW domain derives its name are boxed. A space (-) has been inserted to maximize the alignment. The three conserved β sheets that are connected by two loops are indicated above the sequence. Residues are numbered relative to their appearance in the full-length protein. The single-letter amino acid code is used. Sequences were obtained from the GenBank flat files under the following accession numbers: NP_113671 (AIP4), AAC51324 (WWP1), AAC51325 (WWP2), NP_011051 (yeast RSP5), P46934 (hNedd4), and AAC50492 (Pin1). (C–E) Identification of critical residues within WW domains I and II important for binding to CXCR4. WCL from HEK293 cells expressing HA-CXCR4 were incubated with the indicated GST-WW domain fusion protein (∼25–50 nM) or GST (∼45 nM) alone. The immunoblot (IB) was probed with an anti-HA antibody to detect bound receptor (top). IB was stripped and reprobed with an anti-GST antibody (C and E) or gel was stained with GelCode blue (D) to reveal level of GST fusion proteins used in the assay. Input represents 0.3% of the lysate used in the binding assay. Bound receptor levels were determined by densitometry and average receptor binding was expressed as the percentage of control GST-WW-I-IV wild-type (WT) binding ± SEM from three (D and E) or 5 (C) independent experiments. Data were analyzed by either a t test (E) or a one-way ANOVA and Bonferroni's post hoc test (C and D). Binding of the double mutants (W313/W345A and Q297/N329A) and the quadruple mutant (W313/Q297/W345/N329A) to CXCR4 was significantly different from wild-type binding. C, *p < 0.01; D, *p < 0.05; and E, *p < 0.011. (F) Purified HIS-tagged WWI-IV WT, Q297/N329A, W313/W345A, and 4A (∼70 nM) were incubated with equimolar amounts of either GST-C-tail WT or GST alone. Immunoblot was probed with an anti-HIS antibody to detect HIS-WWI-IV. The input represents ∼2% of the purified His-WWI-IV (WT or mutants) used in the binding assay. The immunoblot was stripped and reprobed with an anti-GST antibody to detect the levels of GST proteins used in the binding assay. HIS-WWI-IV binding was determined by densitometric analysis, and data represent the mean ± SE from three independent experiments. Data were analyzed by a one-way ANOVA and Bonferroni's post hoc test. The binding of HIS-WWI-IV mutants to GST-C-tail was significantly different from that of wild-type (WT). *p < 0.01.

Figure 4.

Identification of residues within WW domains I and II that are critical for binding to CXCR4. (A) CXCR4 interacts with AIP4 WW domains I and II. Whole cell lysates (WCL) from HEK293 cells expressing HA-CXCR4 were incubated with GST alone (∼110 nM), GST-AIP4 (∼25 nM), GST-WW-I-IV (∼55 nM) or GST fusions of each of the four individual WW domains (∼100 nM). Immunoblot was probed with an anti-HA antibody to detect bound HA-CXCR4. A Coomassie-stained gel is shown to indicate the level of GST proteins used in the binding assay. Input represents 1.5% of the lysate used in the binding assay. Shown are data from one of three independent experiments. (B) Alignment of the WW domains from AIP4 and several members of the Nedd4-like family of E3 ligases. The four WW domains of AIP4 are shown aligned. Residues that are either identical or conserved to both WW domains I and II are high-lighted black. Residues that are unique to each of the four WW domains are boxed and shaded gray. Sequence of the Pin1 WW domain is also shown and the critical arginine residue shown to be important for phosphorylation mediated binding to Pin1 is shaded. The WW domains from other members of the Nedd4-like HECT domain family are shown (WWP1, WWP2, Nedd-4, and Rsp5). The conserved tryptophan residues from which the WW domain derives its name are boxed. A space (-) has been inserted to maximize the alignment. The three conserved β sheets that are connected by two loops are indicated above the sequence. Residues are numbered relative to their appearance in the full-length protein. The single-letter amino acid code is used. Sequences were obtained from the GenBank flat files under the following accession numbers: NP_113671 (AIP4), AAC51324 (WWP1), AAC51325 (WWP2), NP_011051 (yeast RSP5), P46934 (hNedd4), and AAC50492 (Pin1). (C–E) Identification of critical residues within WW domains I and II important for binding to CXCR4. WCL from HEK293 cells expressing HA-CXCR4 were incubated with the indicated GST-WW domain fusion protein (∼25–50 nM) or GST (∼45 nM) alone. The immunoblot (IB) was probed with an anti-HA antibody to detect bound receptor (top). IB was stripped and reprobed with an anti-GST antibody (C and E) or gel was stained with GelCode blue (D) to reveal level of GST fusion proteins used in the assay. Input represents 0.3% of the lysate used in the binding assay. Bound receptor levels were determined by densitometry and average receptor binding was expressed as the percentage of control GST-WW-I-IV wild-type (WT) binding ± SEM from three (D and E) or 5 (C) independent experiments. Data were analyzed by either a t test (E) or a one-way ANOVA and Bonferroni's post hoc test (C and D). Binding of the double mutants (W313/W345A and Q297/N329A) and the quadruple mutant (W313/Q297/W345/N329A) to CXCR4 was significantly different from wild-type binding. C, *p < 0.01; D, *p < 0.05; and E, *p < 0.011. (F) Purified HIS-tagged WWI-IV WT, Q297/N329A, W313/W345A, and 4A (∼70 nM) were incubated with equimolar amounts of either GST-C-tail WT or GST alone. Immunoblot was probed with an anti-HIS antibody to detect HIS-WWI-IV. The input represents ∼2% of the purified His-WWI-IV (WT or mutants) used in the binding assay. The immunoblot was stripped and reprobed with an anti-GST antibody to detect the levels of GST proteins used in the binding assay. HIS-WWI-IV binding was determined by densitometric analysis, and data represent the mean ± SE from three independent experiments. Data were analyzed by a one-way ANOVA and Bonferroni's post hoc test. The binding of HIS-WWI-IV mutants to GST-C-tail was significantly different from that of wild-type (WT). *p < 0.01.

Figure 5.

CXCR4 C-tail serine residues mediate the interaction with AIP4. (A) CXCR4 C-tail amino acid sequence. Shown is the amino acid sequence of the carboxy-terminal tail of CXCR4 and the various serine receptor mutants. The single-letter amino acid code is used. (B and C) CXCR4 serine receptor mutant binding to AIP4. (B) Whole cell lysates (WCL) from HEK293 cells expressing wild-type HA-CXCR4 and the indicated serine mutants were incubated with GST-WW-I-IV (∼17 nM) or GST (∼97 nM) alone. Bound receptor was detected by immunoblotting (top). Blots were stripped and reprobed with an anti-GST antibody (bottom). Input represents 0.3% of the lysate used in the binding assay. Shown are representative blots. (C) Bound receptor levels were determined by densitometry and average receptor binding was expressed as the percentage of control CXCR4 binding ± SEM from five independent experiments. Data were analyzed by a Kruskal–Wallis one-way ANOVA, followed by Dunn's post hoc test. Binding to S3245A was significantly different from WT and S330A binding. *p < 0.05. (D) FRET analysis between S324/5A and AIP4. Cells expressing HA-S324/5A-YFP and AIP4-CFP were treated and the FRET efficiency was calculated as described in Materials and Methods. Shown is the average FRET efficiency between HA-S324/5-YFP and AIP4-CFP from cells treated with (n = 23) or without (n = 26) CXCL12. Data from Figure 2 were used to calculate the FRET efficiency between HA-CXCR4-YFP and AIP4-CFP. Data were analyzed by a two-way ANOVA (p < 0.01), followed by a Bonferroni's post hoc test (*p < 0.0001, between CXCL12 treated wild-type and S324/5A cells).

Figure 6.

CXCR4 C-tail serine residues 324 and 325 are important for CXCR4 ubiquitination and degradation. (A) Ubiquitination of S324/5A is attenuated compared with wild-type CXCR4. HEK293 cells were transfected with the indicated CXCR4 constructs and FLAG-tagged ubiquitin. Cells were treated in the presence or absence of 100 nM SDF for 30 min at 37°C, followed by immunoprecipitation (IP) of the receptor and immunoblotting (IB) to detect the incorporation of epitope-tagged ubiquitin. Blots were then stripped and reprobed with an anti-HA mAb to assess receptor levels. Shown are representative blots from one of six independent experiments. Ubiquitinated CXCR4 levels were assessed by densitometric analysis and the –fold increase upon CXCL12 treatment compared with vehicle treatment was normalized to receptor levels present in the immunoprecipitates. Ubiquitination of CXCR4 was significantly increased compared with the S324/5A mutant. Unpaired t test: *p < 0.03. (B) Involvement of serine residues 324 and 325 in agonist-promoted degradation of CXCR4. HEK293 cells transfected with the indicated HA-tagged CXCR4 constructs were treated in the presence or absence of 10 nM SDF for 3 h at 37°C in DMEM containing 10% FBS and 50 μg/ml cycloheximide, essentially as described previously (Marchese and Benovic, 2001). Equal amounts of lysates were subject to SDS-PAGE and immunoblotting to detect receptor levels. Receptor levels were assessed by densitometric analysis and normalized to tubulin levels. The bars represent the average percent receptor degraded in cells treated with agonist compared with vehicle-treated cells from five independent experiments. Error bars represent the SE of the mean.

Figure 7.

CXCR4 phospho-mimetic mutants show enhanced binding to AIP4. (A) The amino acid sequence of the carboxy-terminal tail of CXCR4 and the various serine-to-aspartic acid receptor mutants. (B) CXCR4 phospho-mimetic mutant binding to GST-WW-I-IV. Whole cell lysates from HEK293 cells expressing wild-type HA-CXCR4 or the indicated serine mutants were incubated with GST-WW-I-IV (∼12 nM) or GST (∼38 nM) alone. Immunoblot was probed with an anti-HA antibody to detect bound receptor. Shown is an immunoblot to indicate the level of the GST proteins used in the binding assay. Input represents 0.3% of lysate used in the binding assay. Receptor levels were determined by densitometry and average receptor binding was expressed as the percentage of control CXCR4 binding ± SEM from four independent experiments. (C) Binding analysis using purified proteins. Purified HIS-tagged WW-I-IV (∼70 nM) was incubated with equimolar amounts of either the wild-type C-tail, S324/325A, and S324/325D fused to GST or GST alone. Immunoblot was probed with an anti-HIS antibody to detect bound HIS-WW-I-IV. The input represents ∼1% of the purified His-WWI-IV used in the binding assay. The immunoblot was stripped and reprobed with an anti-GST antibody to detect the levels of GST proteins used in the binding assay. HIS-WWI-IV binding was determined by densitometric analysis, and data represent the mean ± SE from three independent experiments. (D) Enhanced agonist-promoted degradation of CXCR4 phospho-mimetic mutant S324/5D. Degradation of HA-tagged CXCR4 was assessed in cells treated with 10 nM SDF for 30 min at 37°C, essentially as described previously (Marchese and Benovic, 2001). The bars represent the average percentage of receptor degraded in cells treated with agonist compared with vehicle-treated cells from four independent experiments. Error bars represent the SE of the mean. Degradation of S324/5D was significantly enhanced as compared with wild-type receptor degradation (average mean, 3.8 vs. 27.2%). Unpaired t test, *p < 0.03.

Figure 8.

CXCR4 C-tail serine residues 324 and 325 are phosphorylated upon agonist activation. HEK293 cells transiently expressing HA-tagged CXCR4-YFP (A, B, E, and F) or S324/5A-YFP (C and D) were incubated with either vehicle or CXCL12 for 15 min. Cells were fixed, permeabilized, and immunostained using an anti-phosphoserine 324/5 mAb (5E11). Images were captured under identical settings. To dephosphorylate receptors, permeabilized cells were treated with calf intestinal alkaline phosphatase (E) or λ phosphatase (F) before immunostaining with 5E11. HA-CXCR4-YFP- and 5E11-labeled CXCR4 images were pseudocolored as green and red, respectively, and merged using ImageJ software. Yellow in merged images represents S324/5 phosphorylated CXCR4. DIC images of the cells are shown (right). Representative images of cells analyzed from three independent experiments are shown. Bars, 10 μm.

Figure 9.

Time course of CXCR4 phosphorylation. HEK293 cells transiently expressing HA-CXCR4-YFP were incubated with vehicle or CXCL12 for 5 (A and B), 15 (C and D), or 30 (E and F) min. Cells were fixed, permeabilized, and immunostained with 5E11. HA-CXCR4-YFP- and 5E11-stained receptor images were pseudocolored as green and red, respectively, and merged using ImageJ software. Yellow in merged images represents S324/5 phosphorylated CXCR4. DIC images of the cells are also shown (right). Shown are representative images of cells analyzed from three independent experiments. Bars, 10 μm.

Figure 10.

AIP4 localizes to the plasma membrane upon activation of CXCR4. (A) Time-lapse images were acquired in TIRF mode for HEK293 cells transiently coexpressing AIP4-CFP and HA-CXCR4-WT, S324/5A, or S324/5D before and after treatment with 100 nM CXCL12 at ∼23°C. In total, 121 images were acquired over a period of 10 min. Shown are representative images of cells before and after treatment with agonist from each transfection. Puncta represent AIP4-CFP clusters at or near the cell surface attached to the glass support (depth of evanescent field <500 nm). (B) The graph represents the average density of AIP4-CFP puncta within the evanescent field. The average puncta density was calculated from 15 time-lapse images per cell before agonist treatment from a total of eight, nine, and 10 cells examined from CXCR4, S324/5A, and S324/5D transfected cells, respectively, from three independent experiments. The data collected after agonist treatment represent the maximal average puncta density observed from 15 continuous time-lapse images per cell. Data were analyzed by a two-way ANOVA (p < 0.01), followed by a paired t test with a Bonferroni correction revealing a significant increase in puncta density after agonist treatment in cells expressing wild-type CXCR4 (*p < 0.05). Bar, 7 μm.