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Originally published In Press as doi:10.1074/jbc.M408481200 on December 22, 2004

J. Biol. Chem., Vol. 280, Issue 8, 6692-6700, February 25, 2005
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Identification of the Cytoplasmic Domains of CXCR4 Involved in Jak2 and STAT3 Phosphorylation*

Barbara Ahr{ddagger}§, Mélanie Denizot{ddagger}§, Véronique Robert-Hebmann{ddagger}, Anne Brelot||, and Martine Biard-Piechaczyk{ddagger}**

From the {ddagger}Laboratoire Infections Rétrovirales et Signalisation Cellulaire, CNRS UMR 5121, Institut de Biologie, 4, Bd Henri IV, CS 89508, 34960 Montpellier Cedex 2, France and the ||Institut Cochin, U567, 22 rue Méchain, 75014 Paris, France

Received for publication, July 27, 2004 , and in revised form, December 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The chemokine SDF-1{alpha} transduces Gi-dependent and -independent signals through CXCR4. Activation of Jak2/STAT3, a Gi-independent signaling pathway, which plays a major role in survival signals, is known to be activated after SDF-1{alpha} binding to CXCR4 but the domains of CXCR4 involved in this signaling remain unexplored. Using human embryonic kidney HEK-293 cells stably expressing wild-type or mutated forms of CXCR4, we demonstrated that STAT3 phosphorylation requires the N-terminal part of the third intracellular loop (ICL3) and the tyrosine 157 present at the end of the second intracellular loop (ICL2) of CXCR4. In contrast, neither the conserved Tyr135 in the DRY motif at the N terminus of ICL2 nor the Tyr65 and Tyr76 in the first intracellular loop (ICL1) are involved in this activation. ICL3, which does not contain any tyrosine residues, is needed to activate Jak2. These results demonstrate that two separate domains of CXCR4 are involved in Jak2/STAT3 signaling. The N-terminal part of ICL3 is needed to activate Jak2 after SDF-1{alpha} binding to CXCR4, leading to phosphorylation of only one cytoplasmic Tyr, present at the C terminus of ICL2, which triggers STAT3 activation. This work has profound implications for the understanding of CXCR4-transduced signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The seven transmembrane G protein-coupled receptor (GPCR)1 CXCR4 is expressed on many cell types and binds the CXC chemokine stromal-derived factor 1 (SDF-1{alpha}), also named CXCL12 (1, 2). SDF-1{alpha}/CXCR4 plays a role in embryonic development, regulation of cell proliferation, and migration (3–11) as well as in pathological situations such as asthma, tumor invasion, arteriosclerosis, and HIV infection (12–14). After binding to CXCR4, SDF-1{alpha} activates multiple signal transduction pathways including phosphorylation of focal adhesion components, extracellular signal-regulated kinases 1 and 2 (ERK1 and 2), phospholipase C-{gamma}, protein kinase C, and phosphatidylinositol 3-kinase as well as calcium mobilization, activation of the nuclear factor {kappa}B (NF{kappa}B) and chemotaxis (15, 16). Activation of the Janus kinase (Jak)/signal transduction and activation of transcription (STAT) pathway is also triggered after SDF-1{alpha} binding to CXCR4 in T, B, monocytic, and hematopoietic progenitor cell lines (17–19).

Previous studies have shown that chemokine binding to its receptor leads to activation of Jak proteins (20). Activated Jaks in turn phosphorylate the chemokine receptor on intracellular tyrosines, providing docking sites for recruitment of STAT family proteins. After STAT phosphorylation and dimerization, these proteins translocate to the nucleus and mediate transcription of several cytokine-responsive genes (21–23). This pathway is independent of heterotrimeric Gi protein activation but G{alpha}i protein association with GPCR is dependent on Jak activation (17).

Stimulation of cells with chemokine has been shown to induce cell surface GPCR oligomerization that brings about the local aggregation of associated Jaks, resulting in their activation by transphosphorylation (20, 24, 25). However, several GPCRs are already expressed at the cell surface as dimers in the absence of ligand (26–32), indicating that receptor dimerization and activation could be separable events, even if dimerization is required for Jak/STAT signaling. In 1999, Vila-Coro et al. (18) demonstrated that SDF-1{alpha} binding to CXCR4 induces receptor dimerization, but recent data indicate that CXCR4 can also form constitutive dimers without ligand stimulation (33, 34).

Several members of Jak and STAT proteins are activated depending on the receptor/ligand pair and on the cell types. Activation of the Jak2/STAT3 pathway was described in several chemokine receptors such as CCR2b, CXCR4, CCR5, and platelet-activating factor receptor (PAFR) (18, 35–37) and Jak2 is required for SDF-1{alpha}-induced activation of PI 3-kinase, tyrosine phosphorylation of multiple focal adhesion proteins and chemotaxis of hematopoietic progenitor cells (19). Other GPCRs such as luteinizing hormone, angiotensin II, PAR-1, and serotonin 5-HT2A receptors also activate Jak2 after ligand binding (38–42) underlying the role of Jak2 in GPCR signaling. In the same way, STAT3 is ubiquitously expressed and plays an important role in cell growth regulation, inflammation and early embryonic development (43). This STAT protein is required for cell migration and was shown to be activated after SDF-1{alpha} binding to CXCR4 (17, 18).

Activation of the Jak/STAT signal transduction pathway depends on specific cytoplasmic domains of receptors. These domains have been well characterized for several cytokine receptors such as the interleukin-6 signal transducer gp130 (44, 45), the interleukin-2 receptor {beta} chain (46), the interferon receptor chain 2c (47), the granulocyte-macrophage colony-stimulating factor (GM-CSF) (48), the cMpl (49), and the erythropoietin receptor (50). Depending on the receptor, one or several tyrosine residues are phosphorylated during Jak/STAT activation (44, 47, 49, 51, 52). In contrast, nearly no information is available about the cytoplasmic domains of GPCRs needed for Jak/STAT signaling. To our knowledge, the only data on this subject focus on CCR2b and the angiotensin II AT1 receptor (36, 53) in which the DRY site (36) and the YIPP motif in the carboxyl tail (53), respectively, are essential for Jak2 activation.

In the present study, we analyzed the cytoplasmic domains and the intracellular tyrosines of CXCR4 involved in Jak2/STAT3 activation after SDF-1{alpha} binding. To address this question, we used previously constructed stable HEK-293 cells that express mutated forms of CXCR4 in which each intracellular loop (ICL) was replaced by a scrambled amino acid sequence of ICL1 that does not contain serine, threonine and tyrosine, or a CXCR4 form truncated at position 308 (54). We also constructed stable HEK-293 cells that express point mutation CXCR4 receptors in which each intracellular tyrosine, Tyr65 (ICL1), Tyr76 (ICL1), Tyr135 (ICL2), or Tyr157 (ICL2) was replaced by a nonphosphorylatable amino acid and deleted forms of ICL3. We demonstrate here that SDF-1{alpha}-mediated Jak2/STAT3 signaling is transduced through the N terminus of ICL3 and Tyr157 of CXCR4.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—SDF-1{alpha}, polyclonal rabbit anti-SDF-1{alpha} antibody (Ab), and mouse monoclonal (mAb) anti-CXCR4 (MAB173) antibody were purchased from R & D Systems (R & D Systems Europe, Abington, United Kingdom). The anti-HA and Jak2 Abs were purchased from Upstate Cell Signaling Solutions (Euromedex, Mundolsheim, France). Anti-FLAG Ab M2 was purchased from Sigma-Aldrich. Anti-phosphotyrosine mAb (PY20) and anti-phosphorylated ERK1/2 Ab were purchased from Santa Cruz Biotechnology (Tebu-bio, Le Perray en Yvelines, France). Anti-P-STAT3 (Tyr705) and anti-STAT3 Abs were purchased from Cell Signaling Technology (Ozyme, Saint Quentin Yvelines, France). Fluorescein isothiocyanate (FITC) and peroxidase-labeled Fab'2 anti-mouse and anti-rabbit immunoglobulins were from Sigma-Aldrich. Bordetella pertussis toxin (PTX) and AG490 were purchased from Calbiochem (France Biochem, Meudon, France). The pRK5-Jak2 vector was kindly provided by J. Ihle, St. Jude Children's Research Hospital, Memphis, TN.

Construction of the CXCR4 Mutants and Tagged HA CXCR4 —Point mutation mutants of CXCR4, in which tyrosines at positions 65, 76, 135, or 157 were replaced by alanine or phenylalanine, were constructed using GeneEditor in vitro site-directed mutagenesis system from Promega or QuikChange site-directed mutagenesis kit from Stratagene (Stratagene Europe, Amsterdam, The Netherlands) using the wild-type CXCR4 pcDNA3 Zeo expression vector as template according to the manufacturer's instructions. Each mutated clone (CXCR4.Y65F, CXCR4.Y76A, CXCR4.Y135F, and CXCR4.Y157A) was sequenced on an Applied Biosystems (Courtaboeuf, France) Model 373A automated sequencer, using Taq polymerase and dye terminator. CXCR4 and all the constructed mutants are tagged in the C-terminal part of the molecule with the FLAG epitope. A plasmid (pcDNA3 Zeo) encoding the HA tag epitope at the N terminus of wild-type CXCR4 (CXCR4.HA) was constructed by PCR and sequenced.

Cell Culture and Transfection—HEK-293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen, Life Technologies), 1% penicillin-streptomycin, 1% glutamax. 107 cells were transfected with 5 µg of pcDNA Zeo containing the CXCR4 mutant genes (CXCR4.Y65F, CXCR4.Y76A, CXCR4.Y135F, and CXCR4.Y157A) using the FuGENE 6 transfection reagent (Roche Diagnostics, Meylan, France) according to the manufacturer's instructions. To construct stable HEK-293 cell lines expressing CXCR4 mutants containing deletions in ICL3, named CXCR4.{Delta}ICL3-A, CXCR4.{Delta}ICL3-B, and CXCR4.{Delta}ICL3-C (55), 107 cells were cotransfected with 5 µg of pcDNA containing the CXCR4 mutant genes and 0.5 µg of pcDNA Zeo. Stable clones able to grow in the presence of 250 µg/ml zeocin were selected by flow cytometry for receptor surface expression.

Flow Cytometry—Cells (1 x 105) were incubated for 1 h at 4 °C with 50 µl of PBS or PBS supplemented with the appropriate mAb. After three washings with PBS, bound mAb was revealed by addition of 50 µl of a 1:100 dilution of fluorescein-conjugated (FITC) secondary immunoglobulin. After 1 h of staining, cells were washed with PBS, and fluorescence intensity at 543 nm was measured on a EPICS XL4-C cytofluorometer (Beckman-Coulter). To study the direct binding of SDF-1{alpha} to CXCR4 expressed on transfected HEK-293 cells, 106 cells were incubated in 30 µl of PBS for 1 h at 4 °C to prevent subsequent internalization, and 30 µl of a SDF-1{alpha} solution at 200 nM was then incubated for 20 min at 4 °C after cell centrifugation. After washing with PBS, 30 µl of an anti-SDF-1{alpha} antibody at 10 µg/ml was added for 30 min, and bound Ab was revealed as described previously (54).

Calcium Signaling—The technique used was described previously (54). Briefly, 107 cells were resuspended in Hank's solution (Invitrogen, Life Technologies, Inc.), loaded with Fluo-3-AM at 2 µM for 20 min at room temperature and stimulated with buffer alone or SDF-1{alpha} at 250 nM. Ionomycin at 10–6 M was then added to verify the capability of the cells to induce a calcium influx. Ratio of fluorescence of bound to free Fluo-3 was analyzed each 10 s on an EPICS XL4-C cytofluorometer.

SDF-1{alpha} Activation and Western Blot Analysis—Cells were starved of serum during 2 days at 37 °C, 5% CO2. After washing three times in PBS, 5 x 106 cells/ml were resuspended in 100 µl of PBS and incubated at 37 °C for 15 min. Cells were then stimulated with SDF-1{alpha} at 125 nM for the specified times at 37 °C and lysed in 50 mM Tris (pH 8), 1% Triton X-100, 100 mM NaCl, 1 mM MgCl2, 2 mM benzamidine, 2 µg/ml leupeptin, 150 µM phenylmethylsulfonyl fluoride, containing NaF, Na3VO4 and {beta}-glycerophosphate. The proteins were subjected to electrophoresis through 10% SDS-PAGE and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were then blocked in Tris-buffered saline (TBS), 0.05% Tween 20, and 10% milk for 1 h at 20 °C. Blots were incubated overnight at 4 °C with the primary antibody diluted in TBS-Tween-5% milk. After 3 washings with TBS-Tween, the blots were incubated for 1 h at 20 °C with peroxidase-coupled antiserum diluted 1:2000 in TBS-Tween-5% milk. After further washing, the immune complexes were revealed by enhanced chemiluminescence (ECL, PerkinElmer Life Sciences) and subjected to autoradiography. Quantification of protein phosphorylation was performed by using the ImageJ program after autoradiography scanning.

DNA Binding STAT3 Assay—After activation, cells were lysed in 50 mM Tris-HCl pH 7.9, 1% Nonidet P-40, 150 mM NaCl, 0.1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mg/ml aprotinin. The oligonucleotide sequence GTC-GACATTTCCCGTAAATC was used to immunopurify DNA binding STAT3. After incubation for 1 h at 4 °C of the cell lysate with 1 µg of double-stranded, 5'-biotinylated oligonucleotide coupled to 30 µl of a 50% suspension of streptavidin agarose. Complexes were washed twice in lysis buffer and affinity-purified proteins were fractionated by electrophoresis, transferred to PVDF membrane as described above. Western blot analysis was performed with anti-P-STAT3 Ab and immunoreactive bands were visualized by chemiluminescence.

In Vitro Jak2 Kinase Assay—Cells were starved of serum over 1 day at 37 °C, 5% CO2, and transfected with 2 µg of pRK5-Jak2 by using the FuGENE 6 transfection reagent. The following day, cells were resuspended in 100 µl of PBS, stimulated with SDF-1{alpha} at 100 ng/ml for 1 min at 37 °C, and lysed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 1 mM PMSF. After centrifugation, the pellet was resuspended in 500 µl of lysis buffer containing 1% Nonidet P-40 and incubated for 15 min at 4 °C. Jak2 was immunoprecipitated as described previously (54). Briefly, 2 mg of proteins were used for immunoprecipitation with anti-Jak2 Ab and protein G-conjugated magnetic beads (Miltenyi Biotech) for 3 h at 4 °C. The immune complexes were then separated on magnetic columns and incubated in the presence of 2 µCi of [{gamma}-32P]ATP in Tris buffer pH 7.4 containing 13.5 mM Mg2+ for 30 min at room temperature. After washing several times, phosphorylated products were eluted, loaded onto 10% SDS-PAGE, transferred onto PVDF membrane, and revealed by autoradiography. Phosphorylated Jak2 was identified by incubating the membrane with anti-Jak2 Ab.

Membrane Protein Extraction—107 cells were washed in PBS and treated with 100 µg/ml of the membrane permeant cross-linking reagent DSP (3,3'-dithiopropionic acid di(N-hydroxysuccinimide ester)) for 1 h on ice. The reaction was stopped by adding PBS containing 100 mM glycine and 20 mM NEM (N-ethylmaleimide). After centrifugation for 5 min at 3000 x g, the pellet was resuspended in 150 µl of a lysis buffer containing 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, and protease inhibitors, and incubated for 30 min at 4 °C. After two steps of freezing in liquid nitrogen/thawing at 37 °C and centrifugation for 30 min at 15,000 x g, the supernatant containing the membrane fraction was subjected to electrophoresis. Gels were transferred to PVDF membranes, and Western blot was performed as described previously. CXCR4 was revealed using the anti-FLAG Ab.

Immunoprecipitation—107 cells stably expressing wild-type CXCR4 or CXCR4.ICL3m molecules containing a FLAG at the C terminus were transiently transfected with 5 µg of pcDNA Zeo containing CXCR4 tagged at the N terminus with the HA epitope (CXCR4.HA) by using the FuGENE 6 transfection reagent. After membrane protein extraction and lysate preclearing by incubation with protein A Sepharose, CXCR4.HA receptors were immunoprecipitated using an anti-HA Ab and protein A Sepharose. To analyze CXCR4 tyrosine phosphorylation after SDF-1{alpha} activation, phosphorylated proteins contained in cell lysates were immunoprecipitated using an anti-phosphotyrosine Ab, and analysis was performed as described above.

Statistical Analysis—Variance analysis was performed after arc sine transformation of the data (56). *, p < .05; **, p < .01; ***, p < .001.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Construction and Expression of the CXCR4 Mutants—To investigate the role of intracellular tyrosines of CXCR4 in Jak2/STAT3 activation pathway after SDF-1{alpha} binding, CXCR4 mutants containing single amino acid substitutions in which Tyr65 and Tyr135 were replaced by a phenylalanine and Tyr76 and Tyr157 by alanine were constructed and stably transfected in HEK-293 cells. We also used previously constructed HEK-293 cells expressing CXCR4 mutants in which each entire intracellular loop was modified (CXCR4.ICL1m, CXCR4.ICL2m, and CXCR4.ICL3m) and a HEK-293 cell line expressing a truncated form of CXCR4 (CXCR4.7TM) (54). Modification of ICL1 has suppressed Tyr65, but not Tyr76 and CXCR4.ICL2m has lost Tyr135 in the DRY box but Tyr157 is still present. All these CXCR4 mutants express a FLAG at the C terminus end of the receptor. Finally, to define the domains of ICL3 involved in Jak2/STAT3 activation, stably transfected HEK-293 cells expressing CXCR4 mutants containing deletions in ICL3, named CXCR4.{Delta}ICL3-A, CXCR4.{Delta}ICL3-B, and CXCR4.{Delta}ICL3-C, described previously (55) were constructed. The amino acids 227–230, 230–233, and 233–236 are deleted in, respectively, CXCR4.{Delta}ICL3-A, -B, and -C. A schematic representation of CXCR4 mutants used for this study is described in Fig. 1. This diagram is based on that proposed by Doranz et al. (60).



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  		FIG. 1.
Schematic diagram of CXCR4 receptor and mutants. The intracellular tyrosines are numbered and indicated with a thick line. The intracellular loops that were modified in CXCR4.ICL1m, CXCR4.ICL2m, and CXCR4.ICL3m are indicated with thin lines. The limits of the deletion in ICL3 are indicated. The FLAG at the C terminus is noted in bold and italic. The lipid bilayer is depicted as gray rectangles.

 
Expression of the CXCR4 mutants was measured by flow cytometry and compared with expression of the CXCR4 wild-type molecule on HEK-293 cells (Fig. 2). Cell surface expression of CXCR4.ICL1–3m and CXCR4.7TM was described previously (54). CXCR4.ICL1m and CXCR4.ICL2m are functional but weakly expressed. This led us to construct CXCR4 mutants in which each cytoplasmic tyrosine, including tyrosines already absent in CXCR4.ICL1m and CXCR4.ICL2m, was modified. CXCR4, CXCR4.Y65F, CXCR4.Y135F, and the three mutants of ICL3 are very well expressed, at an almost identical level at the cell surface. CXCR4.Y76A and CXCR4.Y157A are well expressed on HEK-293 cells, but at a slightly lower level than that of CXCR4.



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  		FIG. 2.
Expression of wild-type and mutated CXCR4 molecules at the surface of the HEK-293 cell line. Cells were incubated with medium containing the anti-CXCR4 mAb at 10 µg/ml. Bound mAb was detected with a FITC-labeled anti-mouse Ig. The white histogram represents binding of anti-CXCR4 mAb to HEK-293 cells, and black histograms to the different CXCR4 molecules analyzed. The fluorescence intensity was recorded in log mode on an EPICS XL4 cytofluorometer. Representative data from one to five independent experiments are shown.

 
CXCR4 Mutants Expressed on HEK-293 Cells Are Functional—To confirm that the mutated forms of CXCR4 are still able to bind SDF-1{alpha} when expressed on HEK-293 cells, SDF-1{alpha} at 200 nM was incubated with the stably transfected cell lines expressing these mutants. SDF-1{alpha} binding to these cells was revealed by an anti-SDF-1{alpha} Ab. As shown in Fig. 3A, SDF-1{alpha} binds to all the CXCR4 mutants. CXCR4.Y65F, CXCR4.Y135F, CXCR4.Y157A and CXCR4.{Delta}ICL3-A, -B, and -C bind SDF-1{alpha} with the same efficacy as wild-type CXCR4. CXCR4.Y76A is still able to bind SDF-1{alpha}, but at a lower level.



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  		FIG. 3.
Functional cell surface expression of wild-type and mutated CXCR4 receptors after stable transfection of the HEK-293 cell line. A, cells were incubated with PBS alone (white histograms) or a solution of SDF-1{alpha} at 200 nM (black histograms) in PBS at 4 °C. After incubation of the anti-SDF-1{alpha} Ab at 10 µg/ml, bound Ab was detected with a FITC-labeled anti-rabbit Ig. Representative data from one to five experiments are described. B, stably transfected HEK-293 cells expressing wild-type and mutated CXCR4 receptors were loaded with Fluo-3-AM. SDF-1{alpha}-induced calcium flux was measured as described under "Experimental Procedures." Representative curves of [Ca2+]i changes after stimulation with SDF-1 {alpha} and ionomycin (I) from one to four experiments are described. C, serum-starved transfected cells were stimulated for 2 min with SDF-1{alpha} at 125 nM and lysed as described under "Experimental Procedures." Protein samples were run on an SDS-polyacrylamide gel and Western blotted with an anti-phospho-ERK1/2 Ab. Protein loading was controlled using a total anti-ERK2 Ab. Results shown are from 3 to 5 independent experiments.

 
The CXCR4 mutants in which the tyrosine residues are mutated (Y65F, Y76A, Y135F, and Y157A) contain a native form of ICL3, known to transduce Gi-dependent signals such as calcium mobilization and ERK phosphorylation. To confirm that these CXCR4 mutants are still able to transduce a signal after SDF-1{alpha} binding when expressed on HEK-293 cells, Gi-dependent signal activation was studied. SDF-1{alpha} binding to the four mutants induces calcium mobilization (Fig. 3B) and ERK phosphorylation (Fig. 3C), confirming their ability to transduce signaling.

The Third Intracellular Loop and the Tyrosine 157 of CXCR4 Are Required for STAT3 Phosphorylation—To investigate which sites within the cytoplasmic domains of CXCR4 are responsible for STAT3 activation, we used different stable cell lines expressing CXCR4 mutants in which the entire cytoplasmic domain or punctual mutations are performed. Tyrosines, residues that are phosphorylated during this activation pathway, are present in ICL1 (Tyr65 and Tyr76) and ICL2 (Tyr135 and Tyr157). Neither ICL3 nor the C-terminal domain of CXCR4 contains any tyrosine. We first analyzed the capability of CXCR4 mutants in which each entire cytoplasmic domain was modified to phosphorylate STAT3 after SDF-1{alpha} binding. After SDF-1{alpha} stimulation, STAT3 phosphorylation occurred in the HEK/CXCR4, HEK/CXCR4.ICL1m, HEK/CXCR4.ICL2m, and HEK/CXCR4.7TM cells, whereas no activation of this kinase was found in HEK-293 and HEK/CXCR4.ICL3m cells (Fig. 4, A and B, left). To pursue this study, we analyzed the role of each intracellular tyrosine in STAT3 phosphorylation after SDF-1{alpha} binding using HEK-293 cells stably expressing CXCR4 receptors mutated on each intracellular tyrosine. Only Tyr157 is involved in STAT3 phosphorylation (Fig. 4, A and B, right). Tyr135, present in the DRY motif, is not involved in STAT3 signaling, confirming the results obtained with the CXCR4.ICL2m mutant. This signal was not inhibited by PTX, in contrast to ERK phosphorylation, confirming that this signal is independent on Gi proteins. At the opposite, STAT3 activation was totally inhibited by AG490, a specific inhibitor of Jak2 kinase (Fig. 4C). AG9, a control peptide, did not block this signaling (data not shown). To further analyze the role of Tyr157 in this pathway, DNA binding of STAT3 was studied after SDF-1{alpha} stimulation in HEK/CXCR4 and HEK/CXCR4.Y157A cells. Biotinylated oligonucleotides comprising high affinity binding sites for STAT3 were used as an affinity matrix to purify DNA binding STAT3 complexes from cell lysates of HEK/CXCR4 and HEK/CXCR4.Y157A cells after SDF-1{alpha} stimulation. Fig. 4D shows that SDF-1{alpha} binding to wild-type CXCR4, but not to CXCR4.Y157A triggered STAT3 transcriptional activity. At last, to define the crucial role of Tyr157 in CXCR4 phosphorylation, HEK, HEK/CXCR4, and HEK/CXCR4.Y157A cells were serum-starved and stimulated with SDF-1{alpha} for 2 min. Cells lysates were immunoprecipitated with anti-phosphotyrosine Ab, and the immunoprecipitates were analyzed by Western blotting with an anti-FLAG Ab. Only wild-type CXCR4 is phosphorylated after stimulation by SDF-1{alpha}, indicating that Tyr157 is involved in CXCR4 phosphorylation after SDF-1{alpha} binding (Fig. 4E).



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  		FIG. 4.
ICL3 and Tyr157 are involved in STAT3 activation. A, serum-starved HEK-293 cells stably expressing CXCR4 mutants of the intracellular domains (left) or CXCR4 mutated on each intracellular tyrosine (right) were stimulated for 2 min with SDF-1{alpha} at 125 nM and lysed as described under "Experimental Procedures." Protein samples were run on an SDS-polyacrylamide gel and Western blotted with an anti-phospho-STAT3 Ab. Protein loading was controlled using a total anti-STAT3 Ab. B, densitometric analysis of phosphorylated STAT3 expression. Data are presented as fold increase of STAT3 phosphorylation, where the amount of STAT3 phosphorylation in cells that do not express CXCR4 is assigned a value of 1.0 after SDF-1{alpha} activation. All the relative intensities were calculated by normalizing the intensity of phosphorylated STAT3 protein to its STAT3 protein loading control. Results shown are from 3–5 independent experiments; error bars reflect S.D. Statistical analysis was performed as described under "Experimental Procedures." *, p < 0.05; **, p < 0.01; ***, p < .001. C, STAT3 and ERK1/2 phosphorylation induced by SDF-1{alpha} in HEK/CXCR4 in the presence or absence of PTX and AG490. D, after SDF-1 stimulation and lysis of the cells, affinity purification of DNA-binding proteins using oligonucleotides specific for STAT3 was performed. Purified proteins were resolved on SDS-polyacrylamide gel and Western-blotted with an anti-phospho-STAT3 Ab as described under "Experimental Procedures." Result is representative of two independent experiments. E, immunoprecipitation of Tyr phosphorylated proteins was performed with anti-phosphotyrosine Ab. Immune complexes were separated on SDS-polyacrylamide gel, transferred on PVDF membrane and Western-blotted with an anti-FLAG Ab as described under "Experimental Procedures." Result is representative of two independent experiments.

 
SDF-1{alpha}-induced Jak2 Phosphorylation Depends on the Third Intracellular Loop of CXCR4 —CXCR4.Y157A and CXCR4.ICL3m are unable to activate STAT3 after SDF-1{alpha} binding. Because Jak2 phosphorylation acts upstream STAT3 activation, we analyzed the capability of these mutants to phosphorylate Jak2. Only CXCR4.ICL3m was unable to activate Jak2 after SDF-1{alpha} binding (Fig. 5), indicating that ICL3 is the only part of CXCR4 needed for Jak2 phosphorylation.



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  		FIG. 5.
ICL3 is needed for Jak2 phosphorylation. In vitro Jak2 kinase assay was performed in cells expressing the CXCR4 mutants that do not activate STAT3, as described under "Experimental Procedures." HEK-293 cells that do or do not express wild-type CXCR4 were used as controls. Phosphorylated Jak2 was identified by incubating the membrane with anti-Jak2 Ab. Results representative of three independent experiments are shown.

 
CXCR4.ICL3m Exists as Oligomers at the Cell Surface— Receptor oligomerization is a crucial step in Jak/STAT signaling. As the domains of CXCR4 involved in dimerization are unknown and ICL3 of GPCRs (31) can form dimers, we cannot exclude a role of ICL3 in CXCR4 oligomerization. For this purpose, we first examined whether wild-type CXCR4 and/or CXCR4.ICL3m could form oligomers at the cell surface without SDF-1{alpha} stimulation. Membrane extracts of HEK-293 cells stably expressing wild-type CXCR4 or CXCR4.ICL3m were analyzed by SDS-PAGE and Western blot analysis as described under "Experimental Procedures." Both monomeric and oligomeric forms were detected (Fig. 6A), indicating that CXCR4.ICL3m is able to oligomerize as wild-type CXCR4 at the cell surface. It is worth noting that replacement of ICL3 by ICL1 in CXCR4.ICL3m decreases the molecular weight of the receptor. Similar results were obtained with the other CXCR4 mutants and after SDF-1{alpha} or RANTES stimulation (data not shown). Furthermore, CXCR4 and CXCR4.ICL3m stably expressed at the cell surface of HEK-293 cells were coimmunoprecipitated with transiently transfected CXCR4.HA without SDF-1{alpha} activation, confirming that ICL3 is not responsible for CXCR4 oligomerization (Fig. 6B). Blots containing immunoprecipitates were immunostained with anti-HA Ab to verify the efficiency of transfection (data not shown).



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  		FIG. 6.
CXCR4.ICL3m exists as oligomers at the cell surface. A, CXCR4.ICL3m and wild-type CXCR4 extracted from cell membrane were detected by Western blot analysis using the anti-FLAG Ab as described under "Experimental Procedures." Asterisks indicate the positions of monomers (*), dimers (**), and trimers (***). Data are representative of three separate experiments. B, transitory transfected CXCR4.HA in cells stably expressing wild-type CXCR4 or CXCR4.ICL3m was immunoprecipitated using an anti-HA Ab. The immunoprecipitates were then immunoblotted with the anti-FLAG. Results showed are representative of three independent experiments.

 
The Domain A of ICL3 Is Involved in Jak2/STAT3 and Gi Protein Activation—To define the domains of ICL3 critical for Jak2/STAT3 activation, stable cell lines that express CXCR4 mutants containing deletions in ICL3 were activated by SDF-1{alpha} and STAT3 and Jak2 phosphorylation were analyzed. STAT3 activation occurs after SDF-1{alpha} binding in HEK/CXCR4.{Delta}ICL3-B and HEK/CXCR4.{Delta}ICL3-C whereas no activation of this kinase was found in HEK/CXCR4.{Delta}ICL3-A (Fig. 7, A and B). In the same way, only HEK/CXCR4.{Delta}ICL3-B and HEK/CXCR4.{Delta}ICL3-C are able to phosphorylate Jak2 after SDF-1{alpha} stimulation. SDF-1{alpha} binding to HEK/CXCR4.{Delta}ICL3-A does not induce any Jak2 phosphorylation, indicating that the domain A of CXCR4, present at the N terminus part of ICL3, is critical for Jak2/STAT3 activation (Fig. 7C). ICL3 of CXCR4 was described as necessary for Gi activation, signal shown to act downstream the Jak/STAT activation pathway. ERK phosphorylation and calcium mobilization, Gi-dependent signals, occur in HEK/CXCR4.{Delta}ICL3-B and HEK/CXCR4.{Delta}ICL3-C but not in HEK/CXCR4.{Delta}ICL3-A (Fig. 7, A and D). In conclusion, the domain A of ICL3 is needed to activate Jak2/STAT3 pathway and Gi signaling.



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  		FIG. 7.
The domain A of ICL3 is involved in Jak2/STAT3 and Gi protein activation. A, serum-starved HEK-293 cells stably expressing ICL3 mutants of CXCR4 were stimulated for 2 min with SDF-1{alpha} at 125 nM and lysed as described under "Experimental Procedures." Protein samples were run on an SDS-polyacrylamide gel, transferred on PVDF membrane, and Western-blotted with an anti-phospho-STAT3 Ab. Analysis of SDF-1-induced ERK1/2 phosphorylation was also performed as described in Fig. 3. Results showed are representative of three independent experiments. Protein loading was controlled using actin Ab. B, densitometric analysis of phosphorylated STAT3 expression was performed as in Fig. 4. Results shown are from three independent experiments. C, in vitro Jak2 kinase assay was performed in cells expressing ICL3 mutants of CXCR4 as presented in Fig. 5. D, representative curves of [Ca2+]i changes after stimulation of cells expressing CXCR4 mutated on ICL3 with SDF-1{alpha} and ionomycin. One to three experiments are described.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study was aimed at delineating the intracellular sites of CXCR4 necessary for Jak2/STAT3 activation. Although SDF-1-mediated activation of other members of the Jak/STAT family has also been described (17–19), we focused our work on the Jak2/STAT3 pathway because of its major role in GPCR signal transduction (18, 35–37) and survival signals (57). GPCRs contain four cytoplasmic domains, three loops (ICL1–3) and a C terminus part, that are potentially able to recruit signaling molecules leading to activation of a wide variety of cellular responses after ligand binding. However, the three-dimensional structure of GPCRs at the atomic level is unknown, except for the light receptor rhodopsin. Thus, the precise location of the intracellular loops of CXCR4 remains fuzzy, which led us to analyze all the tyrosine residues exposed toward the cytosol.

Although SDF-1-induced signaling is well known (3, 4, 15, 16, 18, 58), very little data are available on the cytoplasmic CXCR4 domains involved in signal activation. To date, ICL3 has been shown to be responsible for Gi protein (54, 55) and {beta}-arrestin (59) interaction, and the C terminus part links to {beta}-arrestin (59) and triggers CXCR4 internalization (54). ICL2, ICL3, and the C-terminal part of CXCR4 are needed for SDF-1{alpha}-induced cell migration, a complex event that requires activation of several signaling pathways (54). It is well established that Jaks bind specifically and constitutively to intracellular domains of receptors and catalyze ligand-induced phosphorylation of themselves and of cytoplasmic tyrosine residues on the receptor, creating docking sites for STATs, but the cascade of events by which GPCRs activate the Jak/STAT pathway remains poorly understood, and is completely unknown for CXCR4.

To determine the CXCR4 domains involved in Jak2/STAT3 activation, we first analyzed the capability of previously constructed CXCR4 mutants to activate STAT3 after SDF-1{alpha} binding. In these mutants, each intracellular domain, determined from the schematic representation of CXCR4 described by Doranz et al. (60), has been modified. Surprisingly, only CXCR4.ICL3m is unable to phosphorylate STAT3 whereas this loop does not contain any Tyr residue, raising several questions. (i) ICL3 is the docking site for Jak2, and is thus needed for Jak2 transactivation. Phosphorylated Jak2 could then phosphorylate one or several intracellular tyrosines within CXCR4 which has (have) not been removed in CXCR4.ICL1m and CXCR4.ICL2m and which is (are) able to recruit and activate STAT3. (ii) ICL3 may also be important for CXCR4 oligomerization, a step known to be essential for Jak/STAT activation. (iii) Activation of Gi-dependent events, known to be exclusively triggered by ICL3 of CXCR4, is necessary for SDF-1{alpha}-induced Jak/STAT activation. This last hypothesis can be excluded because SDF-1{alpha}-mediated STAT3 activation occurs in the presence of PTX, according to our result and data from the literature (17, 18, 36, 61–63).

A growing number of data indicates that GPCRs, including CXCR4, form multimeric complexes at the cell surface in the absence of ligand (26, 31, 33, 34, 64). However, few data are available on the GPCR domains involved in receptor oligomerization. The hydrophobic transmembranes (30, 65–67) and ICL3 (31) could be involved in GPCR oligomerization, depending on the receptor. Recently, Trettel et al. (64) demonstrated by Western blot analysis and immunoprecipitation studies that CXCR2 expressed on HEK-293 is able to form oligomers independently of receptor activation by agonist. In the same way, to analyze the putative role of ICL3 in CXCR4 oligomerization, we studied the capability of the wild-type CXCR4 molecule and CXCR4.ICL3m to homomultimerize and demonstrated that CXCR4.ICL3m, as well as wild-type CXCR4, are present at the cell membrane as preformed multimers. Although the present work does not provide insight into the domains of CXCR4 involved, this result indicates that ICL3 of CXCR4 is not needed to form stable oligomers at the cell membrane.

The domains involved in Jak/STAT activation pathway were extensively studied in class I cytokine receptors. They possess two short conserved motifs in the membrane-proximal intracellular region (box 1 and box 2) that mediate binding of Jaks. The box 1 region contains a proline-rich motif of eight amino acids. One proline-rich motif, YIPP, present in the C-terminal part of the AT1 receptor, is involved in physical association of Jak2 kinase (38), but this motif is not conserved among the GPCRs, and is not present in CXCR4. The box 2 region possesses a cluster of hydrophobic amino acid residues, often followed by charged amino acids (68, 69). Three hydrophobic amino acids, Leu253, Ile257, and Trp258, conserved among the cytokine receptors, are part of an essential, precisely oriented, hydrophobic motif in the juxtamembrane cytosolic domain of the erythropoietin receptor, and are critical for Jak2 signaling (70, 71). Constantinescu et al. (70, 71) proposed a model explaining Jak2 transphosphorylation, in which the juxtamembrane hydrophobic patch forms a single continuous {alpha}-helix with the transmembrane domain able to transmit a conformation specifically required to activate Jak2. These amino acids are not found in ICL3 of CXCR4, but this loop contains many hydrophobic residues and its predicted structure, using SOPMA, MLRC, DSC, and PHD programs, indicates the presence of cytoplasmic {alpha}-helices continuous to the two transmembrane {alpha}-helices at each extremity of the loop. Further investigation on the specific residues involved in Jak2 binding and activation is needed. However, based on the data from Constantinescu et al. (70), we hypothesize that the specific conformation induced after SDF-1{alpha} binding to CXCR4 could be transmitted by transmembrane domains, in particular TM5, to the juxtamembrane cytoplasmic residues at the N terminus of ICL3 that contain hydrophobic amino acids included in an {alpha}-helix functionally continuous to the TM5 {alpha}-helix, allowing Jak2 transphosphorylation. This hypothesis is reinforced by the fact that the motif SHSK, named domain A of ICL3 is needed for Jak2 phosphorylation and STAT3 activation. It is worth noting that G{alpha}i association with chemokine receptors is known to be dependent on Jak activation (61), explaining that CXCR4.{Delta}ICL3-A is unable to phosphorylate ERK and induces a highly reduced calcium mobilization after SDF-1{alpha} binding. ICL3 of CXCR4, which also contains in its C-terminal part the RKALK motif described as a structural determinant for Gi-stimulating function (55, 72), is thus sequentially involved in Jak2/STAT3 and Gi protein activation pathways. It is worth noting that this RKALK motif (BBXXB) is present in CXCR4.{Delta}ICL3-A and -B, and HALK (BXXB) is still present in CXCR4.{Delta}ICL3-C. CXCR4.{Delta}ICL3-C can also transduce Gi-dependent signals after SDF-1 binding, indicating that the HALK sequence may be sufficient to activate Gi proteins.

One or several tyrosine residues in CXCR4 would thus be phosphorylated after Jak2 activation. The absence of precise data about the Tyr of CXCR4 exposed to the cytoplasm led us to construct mutants in which the Tyr65, Tyr76, Tyr135, and Tyr157 are replaced by a nonphosphorylatable amino acid. For many cytokine receptors, the motif YXX(L/I/V) serves as a docking site for different SH2 domain-containing proteins such as ZAP-70, SHP-2, and Stat5 (50, 73, 74). This motif (YLAI) is present in ICL2 of CXCR4, in which Tyr135 is included in the conserved DRY site. Furthermore, this DRY motif has been shown to be critical for Jak2/STAT3 activation through CCR2b, another chemokine receptor (36, 75). Indeed, Mellado et al. (36) demonstrated that MCP-1 binding to wild-type CCR2b expressed on HEK-293 cells, but not to CCR2b in which the Tyr139 present in the conserved DRY site has been replaced by Phe (CCR2b.Y139F), triggers activation of the Jak2/STAT3 pathway. Surprisingly, CXCR4.ICL2m, in which the entire ICL2 was replaced, and CXCR4.Y135F, the homologous mutant of CCR2b.Y139F, are still able to phosphorylate STAT3 after SDF-1{alpha} binding, indicating that Tyr135 is dispensable for STAT3 activation. Thus, the role of the DRY motif in GPCRs, even if highly conserved, depends on the receptors.

The C terminus part of CXCR4 that does not contain any tyrosine and ICL1 in which two Tyr are present at each extremity of the loop play no role in STAT3 activation. At the opposite, the Tyr residue 157 of CXCR4, present at the far end of ICL2, is specifically involved in STAT3 activation. Indeed, Tyr157 is crucial for phosphorylation and DNA binding of STAT3 after SDF-1{alpha} binding to CXCR4. Thus, a single tyrosine module is sufficient for STAT activation, as already described by Behrmann et al. (52), even if multiple tyrosine residues in the cytosolic domains can also promote Jak/STAT activation (49–51,76). This Tyr157 is present in an original motif, EKVVYVGVW, different from the two main structural motifs, YXXQ and GXGYM, previously identified (48, 77–84) but which is a predicted tyrosine phosphorylation site (score 0.822) using the NetPhos 2.0 server. It is worth noting that Tyr157 is the only Tyr residue in CXCR4 located toward the cell cytoplasm present in a consensus motif of phosphorylation. We confirm here its capability to be phosphorylated after SDF-1{alpha} binding to CXCR4. These data strongly support the concept that activation of STAT3 is determined by a single phosphotyrosine module in GPCRs.

In summary, this study is the first that delineates the domains of CXCR4 involved in Jak2/STAT3 activation. Our data demonstrate that very specific, nonredundant domains of CXCR4 trigger Jak2/STAT3 activation. ICL3 is needed for Jak2 phosphorylation, and the very membrane proximal Tyr157, sequentially the closest Tyr residue to ICL3, is involved in STAT3 activation. Interestingly, Tyr from the DRY box of CXCR4 is not involved in Jak2/STAT3 activation. These data will aid in understanding the physiological and pathological role of CXCR4/SDF-1 pair.


   FOOTNOTES
 
* This work was supported by institutional funds from the Centre National de la Recherche Scientifique (CNRS) and the University (UM1), and grants from Ensemble contre le SIDA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

Recipient of a fellowship from the Agence Nationale de Recherches sur le SIDA (ANRS). Back

** To whom correspondence should be addressed: Laboratoire Infections Rétrovirales et Signalisation Cellulaire, CNRS UMR 5121, Institut de Biologie, 4, Bd Henri IV, CS 89508, 34960 Montpellier Cedex 2, France. Tel.: 33-4-67-60-86-60; Fax: 33-4-67-60-44-20; E-mail: martine.biard{at}univ-montp1.fr.

1 The abbreviations used are: GPCR, G protein-coupled receptor; SDF, stromal-derived factor 1; Ab, antibody; mAb, monoclonal antibody; HA, hemagglutinin; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; Jak, Janus kinase; ERK, extracellular signal-regulated kinase; FITC, fluorescein isothiocyanate; HIV, human immunodeficiency virus; ICL, intracellular loop; STAT, signal transduction and activation of transcription. Back


   ACKNOWLEDGMENTS
 
We thank A. Ferrand and V. Lafond for Jak2 expression and DNA binding experiments, respectively, and for helpful discussions; B. Hemonnot for excellent technical assistance; L. Meunier for helpful scientific discussions and S. Thebault for critical reading of the manuscript. The pRK5-Jak2 vector and the anti-STAT3 Ab were kindly provided from J. Ihle, St. Jude Children's Research Hospital, Memphis, TN and M. Jourdan, INSERM U475, Montpellier, France, respectively.



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