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complementary dna mutant cxcr4 cdna  (R&D Systems)


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    R&D Systems complementary dna mutant cxcr4 cdna
    VLA-4 phosphorylation at S988 is dependent on shed Sdc1-mediated VEGFR2 activation. (A) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without small interfering RNA (si) against human (h)Sdc1 and <t>cDNA</t> constructs for mouse (m)Sdc1 or mSdc1ΔPVD. After 72 h, the cells were plated on 100 µg/ml IIICS for 2.5 h and lysates were analyzed by immunoblotting with an anti-α4-pS988, anti-total α4 integrin, anti-hSdc1 or anti-mSdc1 antibodies. (B) Cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM OGT2115 (HPSE inhibitor), 30 µM SSTNVEGFR2, 10 µM vandetanib (VEGFR2 inhibitor), or 10 µM H-89 (PKA inhibitor). The whole-cell lysates were analyzed by immunoblotting with anti-α4-pS988 and anti-total α4 integrin antibody. (C–E) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without siRNA against α4 integrin and siRNA-resistant cDNA constructs for HA-tagged WT, S988A or S988D α4 integrin for 48 h. Cells were allowed to migrate towards 100 µg/ml IIICS in the absence or presence of 30 µM SSTNVEGFR2, 10 µM vandetanib, or 10 µM H-89 for 16 h. (C) Cells accumulated on the bottom side of the filter were imaged at 20× magnification. Scale bars: 50 µm. (D,E) Migrated cells were quantified from five random images for each condition and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments.
    Complementary Dna Mutant Cxcr4 Cdna, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/complementary+dna+mutant+cxcr4+cdna/pmc06826009-373-0-15?v=R%26D+Systems
    Average 90 stars, based on 1 article reviews
    complementary dna mutant cxcr4 cdna - by Bioz Stars, 2026-06
    90/100 stars

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    1) Product Images from "VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1"

    Article Title: VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.232645

    VLA-4 phosphorylation at S988 is dependent on shed Sdc1-mediated VEGFR2 activation. (A) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without small interfering RNA (si) against human (h)Sdc1 and cDNA constructs for mouse (m)Sdc1 or mSdc1ΔPVD. After 72 h, the cells were plated on 100 µg/ml IIICS for 2.5 h and lysates were analyzed by immunoblotting with an anti-α4-pS988, anti-total α4 integrin, anti-hSdc1 or anti-mSdc1 antibodies. (B) Cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM OGT2115 (HPSE inhibitor), 30 µM SSTNVEGFR2, 10 µM vandetanib (VEGFR2 inhibitor), or 10 µM H-89 (PKA inhibitor). The whole-cell lysates were analyzed by immunoblotting with anti-α4-pS988 and anti-total α4 integrin antibody. (C–E) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without siRNA against α4 integrin and siRNA-resistant cDNA constructs for HA-tagged WT, S988A or S988D α4 integrin for 48 h. Cells were allowed to migrate towards 100 µg/ml IIICS in the absence or presence of 30 µM SSTNVEGFR2, 10 µM vandetanib, or 10 µM H-89 for 16 h. (C) Cells accumulated on the bottom side of the filter were imaged at 20× magnification. Scale bars: 50 µm. (D,E) Migrated cells were quantified from five random images for each condition and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments.
    Figure Legend Snippet: VLA-4 phosphorylation at S988 is dependent on shed Sdc1-mediated VEGFR2 activation. (A) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without small interfering RNA (si) against human (h)Sdc1 and cDNA constructs for mouse (m)Sdc1 or mSdc1ΔPVD. After 72 h, the cells were plated on 100 µg/ml IIICS for 2.5 h and lysates were analyzed by immunoblotting with an anti-α4-pS988, anti-total α4 integrin, anti-hSdc1 or anti-mSdc1 antibodies. (B) Cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM OGT2115 (HPSE inhibitor), 30 µM SSTNVEGFR2, 10 µM vandetanib (VEGFR2 inhibitor), or 10 µM H-89 (PKA inhibitor). The whole-cell lysates were analyzed by immunoblotting with anti-α4-pS988 and anti-total α4 integrin antibody. (C–E) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without siRNA against α4 integrin and siRNA-resistant cDNA constructs for HA-tagged WT, S988A or S988D α4 integrin for 48 h. Cells were allowed to migrate towards 100 µg/ml IIICS in the absence or presence of 30 µM SSTNVEGFR2, 10 µM vandetanib, or 10 µM H-89 for 16 h. (C) Cells accumulated on the bottom side of the filter were imaged at 20× magnification. Scale bars: 50 µm. (D,E) Migrated cells were quantified from five random images for each condition and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments.

    Techniques Used: Phospho-proteomics, Activation Assay, Transfection, Small Interfering RNA, Construct, Western Blot, One-tailed Test

    CXCR4 is required for VLA-4-dependent cell migration in a ligand-independent manner. (A) CAGHPSE, HMEC-1 or M14 cells were transfected with two different CXCR4 siRNAs for 48 h prior to 16 h transfilter migration assays towards 100 µg/ml IIICS. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells. (B) CAGHPSE, HMEC-1 or M14 cells transfected with CXCR4 siRNA for 48 h were analyzed by immunoblotting for integrin α4-pS988, PKA pT197, CXCR4 or β-actin. (C) 16 h transfilter migration assays towards IIICS were performed with CAGHPSE and M14 cells treated with or without 10 µg/ml CXCR4-blocking antibody or 10 µM AMD3100 in the absence or presence of 20 ng/ml SDF-1. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments. (D) CAGHPSE or M14 cells were transfected with or without VEGFR2 siRNA for 48 h. 16 h transfilter migration assays towards 100 µg/ml IIICS were performed with or without 30 µM SSTNVEGFR2 or 10 µM vandetanib. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (E) HMEC-1 cells were plated on 100 µg/ml IIICS with 30 µM SSTNVEGFR2 for 2.5 h. Cell lysates were subjected to immunoprecipitation with rabbit anti-VEGFR2 antibody. The VEGFR2-associated complexes were analyzed by immunoblotting for VEGFR2 (with mouse anti-VEGFR2), CXCR4, AC7, PKA, hSdc1 or α4 integrin. (F) HMEC-1 or M14 cells were transfected with two different Sdc1 siRNA for 48 h and plated on 100 µg/ml IIICS for 2 h. Cell lysates were subjected to immunoprecipitation with rabbit anti-VEGFR2. The associated complexes were probed with mouse anti-VEGFR2, anti-CXCR4, anti-α4 integrin, anti-AC7, anti-hSdc1 or anti-PKA antibodies. Silencing of Sdc1 expression was confirmed by immunoprecipitation with rabbit polyclonal anti-Sdc1 and probed with mouse anti-human Sdc1 antibody. (G) CAGHPSE or M14 cells were transfected with VEGFR2 siRNAs for 48 h and plated on 100 µg/ml IIICS for 2 h. Cell lysates were subjected to immunoprecipitation with rabbit polyclonal anti-Sdc1 antibody. The associated complexes were analyzed by immunoblotting for AC7, PKA, CXCR4, Sdc1 or α4 integrin. The whole-cell lysates were analyzed by immunoblotting for VEGFR2. (H) HMEC-1 cells were transfected with two different CXCR4 siRNAs for 48 h and then cell lysates were subjected to immunoprecipitation with anti-VEGFR2 antibody. The associated complexes were analyzed by immunoblotting for AC7, PKA, VEGFR2 or Gβγ. The whole-cell lysates were analyzed by immunoblotting for CXCR4.
    Figure Legend Snippet: CXCR4 is required for VLA-4-dependent cell migration in a ligand-independent manner. (A) CAGHPSE, HMEC-1 or M14 cells were transfected with two different CXCR4 siRNAs for 48 h prior to 16 h transfilter migration assays towards 100 µg/ml IIICS. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells. (B) CAGHPSE, HMEC-1 or M14 cells transfected with CXCR4 siRNA for 48 h were analyzed by immunoblotting for integrin α4-pS988, PKA pT197, CXCR4 or β-actin. (C) 16 h transfilter migration assays towards IIICS were performed with CAGHPSE and M14 cells treated with or without 10 µg/ml CXCR4-blocking antibody or 10 µM AMD3100 in the absence or presence of 20 ng/ml SDF-1. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments. (D) CAGHPSE or M14 cells were transfected with or without VEGFR2 siRNA for 48 h. 16 h transfilter migration assays towards 100 µg/ml IIICS were performed with or without 30 µM SSTNVEGFR2 or 10 µM vandetanib. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (E) HMEC-1 cells were plated on 100 µg/ml IIICS with 30 µM SSTNVEGFR2 for 2.5 h. Cell lysates were subjected to immunoprecipitation with rabbit anti-VEGFR2 antibody. The VEGFR2-associated complexes were analyzed by immunoblotting for VEGFR2 (with mouse anti-VEGFR2), CXCR4, AC7, PKA, hSdc1 or α4 integrin. (F) HMEC-1 or M14 cells were transfected with two different Sdc1 siRNA for 48 h and plated on 100 µg/ml IIICS for 2 h. Cell lysates were subjected to immunoprecipitation with rabbit anti-VEGFR2. The associated complexes were probed with mouse anti-VEGFR2, anti-CXCR4, anti-α4 integrin, anti-AC7, anti-hSdc1 or anti-PKA antibodies. Silencing of Sdc1 expression was confirmed by immunoprecipitation with rabbit polyclonal anti-Sdc1 and probed with mouse anti-human Sdc1 antibody. (G) CAGHPSE or M14 cells were transfected with VEGFR2 siRNAs for 48 h and plated on 100 µg/ml IIICS for 2 h. Cell lysates were subjected to immunoprecipitation with rabbit polyclonal anti-Sdc1 antibody. The associated complexes were analyzed by immunoblotting for AC7, PKA, CXCR4, Sdc1 or α4 integrin. The whole-cell lysates were analyzed by immunoblotting for VEGFR2. (H) HMEC-1 cells were transfected with two different CXCR4 siRNAs for 48 h and then cell lysates were subjected to immunoprecipitation with anti-VEGFR2 antibody. The associated complexes were analyzed by immunoblotting for AC7, PKA, VEGFR2 or Gβγ. The whole-cell lysates were analyzed by immunoblotting for CXCR4.

    Techniques Used: Migration, Transfection, One-tailed Test, Western Blot, Blocking Assay, Immunoprecipitation, Expressing

    VEGFR2 activates CXCR4 by phosphorylating Y135 in its DRY motif. (A) CAGHPSE, HMEC-1 or M14 cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM vandetanib or 30 µM SSTNVEGFR2 for 2.5 h. CXCR4 was immunoprecipitated with rabbit anti-CXCR4 antibody and the CXCR4-containing complexes were analyzed by immunoblotting with anti-phosphotyrosine or rat anti-CXCR4 antibody. (B–F) CAGHPSE, HMEC-1 or M14 cells were co-transfected with CXCR4 3′UTR siRNA and CXCR4 cDNA lacking the 3′UTR (GFP-tagged WT and Y→F mutants or GFP alone) for 48 h. The cells were then analyzed by flow cytometry, immunoprecipitation and migration assays. (B) GFP-tagged WT or Y135F CXCR4 mutant HMEC-1 cells were incubated with PBS alone or 200 nM SDF-1 in PBS at 4°C. After incubation with 10 µg/ml anti-SDF-1 antibody versus an isotype-matched control mouse IgG1 for 1 h, cells were fixed and blocked with 3% BSA in PBS. The bound antibody was detected with a RPE-labeled anti-mouse IgG and analyzed by flow cytometry for SDF-1-bound CXCR4. (C) CAGHPSE cell lysates were subjected to immunoprecipitation with anti-GFP antibody and then analyzed with anti-VEGFR2, anti-α4 integrin, anti-hSdc1, anti-AC7, anti-PKA or anti-CXCR4 antibodies. (D) M14 cell lysates were subjected to immunoprecipitation with anti-GFP antibody and then analyzed with anti-phosphotyrosine (pY20) or anti-CXCR4 antibodies. (E) Quantification of 16 h transfilter migration of CAGHPSE cells towards 100 µg/ml IIICS in the absence or presence of 20 nM SDF-1 is plotted as the mean±s.d. of three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (F) CAGHPSE cells were kept in suspension or plated on 100 µg/ml IIICS in the absence or presence of 20 nM SDF-1 with or without 10 µM vandetanib for 2 h. Cell lysates were subjected to immunoprecipitation with anti-GFP or anti-GTP-Gαi antibodies, then probed with anti-Gαi antibody. The whole-cell lysates were analyzed by immunoblotting for integrin α4-pS988 and β-actin.
    Figure Legend Snippet: VEGFR2 activates CXCR4 by phosphorylating Y135 in its DRY motif. (A) CAGHPSE, HMEC-1 or M14 cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM vandetanib or 30 µM SSTNVEGFR2 for 2.5 h. CXCR4 was immunoprecipitated with rabbit anti-CXCR4 antibody and the CXCR4-containing complexes were analyzed by immunoblotting with anti-phosphotyrosine or rat anti-CXCR4 antibody. (B–F) CAGHPSE, HMEC-1 or M14 cells were co-transfected with CXCR4 3′UTR siRNA and CXCR4 cDNA lacking the 3′UTR (GFP-tagged WT and Y→F mutants or GFP alone) for 48 h. The cells were then analyzed by flow cytometry, immunoprecipitation and migration assays. (B) GFP-tagged WT or Y135F CXCR4 mutant HMEC-1 cells were incubated with PBS alone or 200 nM SDF-1 in PBS at 4°C. After incubation with 10 µg/ml anti-SDF-1 antibody versus an isotype-matched control mouse IgG1 for 1 h, cells were fixed and blocked with 3% BSA in PBS. The bound antibody was detected with a RPE-labeled anti-mouse IgG and analyzed by flow cytometry for SDF-1-bound CXCR4. (C) CAGHPSE cell lysates were subjected to immunoprecipitation with anti-GFP antibody and then analyzed with anti-VEGFR2, anti-α4 integrin, anti-hSdc1, anti-AC7, anti-PKA or anti-CXCR4 antibodies. (D) M14 cell lysates were subjected to immunoprecipitation with anti-GFP antibody and then analyzed with anti-phosphotyrosine (pY20) or anti-CXCR4 antibodies. (E) Quantification of 16 h transfilter migration of CAGHPSE cells towards 100 µg/ml IIICS in the absence or presence of 20 nM SDF-1 is plotted as the mean±s.d. of three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (F) CAGHPSE cells were kept in suspension or plated on 100 µg/ml IIICS in the absence or presence of 20 nM SDF-1 with or without 10 µM vandetanib for 2 h. Cell lysates were subjected to immunoprecipitation with anti-GFP or anti-GTP-Gαi antibodies, then probed with anti-Gαi antibody. The whole-cell lysates were analyzed by immunoblotting for integrin α4-pS988 and β-actin.

    Techniques Used: Immunoprecipitation, Western Blot, Transfection, Flow Cytometry, Migration, Mutagenesis, Incubation, Control, Labeling, One-tailed Test, Suspension

    The CXCR4 Y135D phosphomimetic mutant rescues the effects of VEGFR2 inhibition. (A,B) CAGHPSE, HMEC-1 or M14 cells were transfected with GFP alone, GFP–CXCR4 WT, or GFP–CXCR4 Y135D together with siRNA targeting 3′UTR of endogenous CXCR4 for 48 h. (A) 16 h transfilter cell migration assay towards 100 µg/ml IIICS was performed in the absence or presence of 10 µM vandetanib. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01, n.s., not significant. (B) M14 cell lysates were subjected to immunoprecipitation with anti-GTP-Gαi and probed for Gαi. Whole-cell lysates were analyzed by immunoblotting for Gαi or α4 integrin (total or pS988). (C) Model depicting how paxillin bound to VLA-4 causes inherent inhibition of Rac GTPase at this site due to its binding of ArfGAP (Nishiya et al., 2005). VEGFR2 forms a complex with CXCR4, AC7 and PKA, but this complex is inactive unless Sdc1 is shed. Trimming of the HS chains on Sdc1 by HPSE facilitates its shedding by MMP9 (Purushothaman et al., 2010; Yang et al., 2007). Sdc1 freed of its membrane anchorage couples VEGFR2 and its integrin phosphorylation machinery to VLA-4 clusters. VEGFR2 activated by this clustering event phosphorylates CXCR4 at Y135, activating its heterotrimeric G-protein. Gβγ freed of Gαi activates AC7, generating cAMP that activates PKA. PKA phosphorylation of the α4 integrin cytoplasmic domain at S988 dissociates paxillin–ArfGAP from VLA-4, allowing local lamellipodium formation and directed cell migration.
    Figure Legend Snippet: The CXCR4 Y135D phosphomimetic mutant rescues the effects of VEGFR2 inhibition. (A,B) CAGHPSE, HMEC-1 or M14 cells were transfected with GFP alone, GFP–CXCR4 WT, or GFP–CXCR4 Y135D together with siRNA targeting 3′UTR of endogenous CXCR4 for 48 h. (A) 16 h transfilter cell migration assay towards 100 µg/ml IIICS was performed in the absence or presence of 10 µM vandetanib. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01, n.s., not significant. (B) M14 cell lysates were subjected to immunoprecipitation with anti-GTP-Gαi and probed for Gαi. Whole-cell lysates were analyzed by immunoblotting for Gαi or α4 integrin (total or pS988). (C) Model depicting how paxillin bound to VLA-4 causes inherent inhibition of Rac GTPase at this site due to its binding of ArfGAP (Nishiya et al., 2005). VEGFR2 forms a complex with CXCR4, AC7 and PKA, but this complex is inactive unless Sdc1 is shed. Trimming of the HS chains on Sdc1 by HPSE facilitates its shedding by MMP9 (Purushothaman et al., 2010; Yang et al., 2007). Sdc1 freed of its membrane anchorage couples VEGFR2 and its integrin phosphorylation machinery to VLA-4 clusters. VEGFR2 activated by this clustering event phosphorylates CXCR4 at Y135, activating its heterotrimeric G-protein. Gβγ freed of Gαi activates AC7, generating cAMP that activates PKA. PKA phosphorylation of the α4 integrin cytoplasmic domain at S988 dissociates paxillin–ArfGAP from VLA-4, allowing local lamellipodium formation and directed cell migration.

    Techniques Used: Mutagenesis, Inhibition, Transfection, Cell Migration Assay, One-tailed Test, Immunoprecipitation, Western Blot, Binding Assay, Membrane, Phospho-proteomics, Migration

    sSdc1-mediated VEGFR2 activation suppresses cytotoxic T cell and NK cell migration by displacing VLA-4 from LFA-1. (A) Lysates from CAGHPSE cells, NK cells and T cells were analyzed by immunoblotting for VEGFR2, VLA-4, LFA-1, Sdc1, PKA, CXCR4, AC7 and β-actin (loading control). (B) NK cells and T cells were plated on 10 µg/ml VCAM-1 in the absence or presence of 30 µM SSTNVEGFR2 for 2.5 h. VEGFR2 was immunoprecipitated and the VEGFR2-containing complexes analyzed by immunoblotting for VEGFR2, AC7, Sdc1, PKA, CXCR4, α4 integrin [total (VLA-4) and pS988], LFA-1 and VLA-5. (C) NK cells and T cells were plated on either ICAM-1 or VCAM-1 in the absence or presence of SDF-1 with or without anti-VLA-4 or anti-LFA-1 antibodies or CXCR4 inhibitor (AMD3100). Attached NK cells and T cells in five random images for each experiment were quantified and graphed as a percent of cells treated with SDF-1 alone (set to 100%). All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (D) 16 h transfilter cell migration assays towards 10 µg/ml VCAM-1 alone, 10 µg/ml ICAM-1 alone, or a mixture of 10 µg/ml ICAM-1 with 10 µg/ml VCAM-1, analysis after treatment with 30 µM SSTNVEGFR2, 10 µg/ml anti-VLA-4, or 10 µg/ml anti-LFA-1 in the absence or presence of SDF-1. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells on each ligand; **P<0.01, n.s., not significant between treatments. (E) 16 h transfilter cell migration assays towards 10 µg/ml VCAM-1 alone or mixture of 10 µg/ml ICAM-1 plus 10 µg/ml VCAM-1 was performed as in D in the absence or presence of 30 µM SSTNVEGFR2, 1 µM vandetanib or 10 µM AMD3100. All data were compared using the unpaired one-tailed t-test. **P<0.01, n.s., not significant. (F) NK cells and T cells were plated on 10 µg/ml VCAM-1 alone or mixture of 10 µg/ml ICAM-1 with 10 µg/ml VCAM-1 in the absence or presence of SSTNVEGFR2. Cell lysates were subjected to immunoprecipitation of VLA-4 and probed for VLA-4 or LFA-1. Whole-cell lysates were analyzed by immunoblotting for integrin α4-pS988, VEGFR2-pY1054/1059 or β-actin. (G) Model depicting how LFA-1 and VLA-4 on NK cells and T cells associate in an adhesion complex in which signaling from FAK and PYK2, localized to VLA-4 by paxillin, transregulates LFA-1 activity leading to LFA-1-dependent cell migration (Cantor et al., 2015; Rose et al., 2003). If Sdc1 is shed by the leukocytes, or high levels of shed Sdc1 accumulate in the tumor microenvironment from HPSE-overexpressing tumor cells, the shed syndecan couples VEGFR2 and its phosphorylation machinery to VLA-4. Activation of CXCR4 by SDF-1, or by VEGFR2-mediated phosphorylation of Y135 in the CXCR4 cytoplasmic loop 2, causes phosphorylation of S988 in the α4 integrin subunit, displacing paxillin and suppressing LFA-1-mediated influx of cytotoxic leukocytes to the tumor. SSTNVEGFR2, which reverses the effects of shed syndecan, relieves this immunosuppression while at the same time blocking tumor cell migration and metastasis (see Fig. 6C).
    Figure Legend Snippet: sSdc1-mediated VEGFR2 activation suppresses cytotoxic T cell and NK cell migration by displacing VLA-4 from LFA-1. (A) Lysates from CAGHPSE cells, NK cells and T cells were analyzed by immunoblotting for VEGFR2, VLA-4, LFA-1, Sdc1, PKA, CXCR4, AC7 and β-actin (loading control). (B) NK cells and T cells were plated on 10 µg/ml VCAM-1 in the absence or presence of 30 µM SSTNVEGFR2 for 2.5 h. VEGFR2 was immunoprecipitated and the VEGFR2-containing complexes analyzed by immunoblotting for VEGFR2, AC7, Sdc1, PKA, CXCR4, α4 integrin [total (VLA-4) and pS988], LFA-1 and VLA-5. (C) NK cells and T cells were plated on either ICAM-1 or VCAM-1 in the absence or presence of SDF-1 with or without anti-VLA-4 or anti-LFA-1 antibodies or CXCR4 inhibitor (AMD3100). Attached NK cells and T cells in five random images for each experiment were quantified and graphed as a percent of cells treated with SDF-1 alone (set to 100%). All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (D) 16 h transfilter cell migration assays towards 10 µg/ml VCAM-1 alone, 10 µg/ml ICAM-1 alone, or a mixture of 10 µg/ml ICAM-1 with 10 µg/ml VCAM-1, analysis after treatment with 30 µM SSTNVEGFR2, 10 µg/ml anti-VLA-4, or 10 µg/ml anti-LFA-1 in the absence or presence of SDF-1. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells on each ligand; **P<0.01, n.s., not significant between treatments. (E) 16 h transfilter cell migration assays towards 10 µg/ml VCAM-1 alone or mixture of 10 µg/ml ICAM-1 plus 10 µg/ml VCAM-1 was performed as in D in the absence or presence of 30 µM SSTNVEGFR2, 1 µM vandetanib or 10 µM AMD3100. All data were compared using the unpaired one-tailed t-test. **P<0.01, n.s., not significant. (F) NK cells and T cells were plated on 10 µg/ml VCAM-1 alone or mixture of 10 µg/ml ICAM-1 with 10 µg/ml VCAM-1 in the absence or presence of SSTNVEGFR2. Cell lysates were subjected to immunoprecipitation of VLA-4 and probed for VLA-4 or LFA-1. Whole-cell lysates were analyzed by immunoblotting for integrin α4-pS988, VEGFR2-pY1054/1059 or β-actin. (G) Model depicting how LFA-1 and VLA-4 on NK cells and T cells associate in an adhesion complex in which signaling from FAK and PYK2, localized to VLA-4 by paxillin, transregulates LFA-1 activity leading to LFA-1-dependent cell migration (Cantor et al., 2015; Rose et al., 2003). If Sdc1 is shed by the leukocytes, or high levels of shed Sdc1 accumulate in the tumor microenvironment from HPSE-overexpressing tumor cells, the shed syndecan couples VEGFR2 and its phosphorylation machinery to VLA-4. Activation of CXCR4 by SDF-1, or by VEGFR2-mediated phosphorylation of Y135 in the CXCR4 cytoplasmic loop 2, causes phosphorylation of S988 in the α4 integrin subunit, displacing paxillin and suppressing LFA-1-mediated influx of cytotoxic leukocytes to the tumor. SSTNVEGFR2, which reverses the effects of shed syndecan, relieves this immunosuppression while at the same time blocking tumor cell migration and metastasis (see Fig. 6C).

    Techniques Used: Activation Assay, Migration, Western Blot, Control, Immunoprecipitation, One-tailed Test, Activity Assay, Phospho-proteomics, Blocking Assay



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    complementary dna mutant cxcr4 cdna  (R&D Systems)
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    VLA-4 phosphorylation at S988 is dependent on shed Sdc1-mediated VEGFR2 activation. (A) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without small interfering RNA (si) against human (h)Sdc1 and <t>cDNA</t> constructs for mouse (m)Sdc1 or mSdc1ΔPVD. After 72 h, the cells were plated on 100 µg/ml IIICS for 2.5 h and lysates were analyzed by immunoblotting with an anti-α4-pS988, anti-total α4 integrin, anti-hSdc1 or anti-mSdc1 antibodies. (B) Cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM OGT2115 (HPSE inhibitor), 30 µM SSTNVEGFR2, 10 µM vandetanib (VEGFR2 inhibitor), or 10 µM H-89 (PKA inhibitor). The whole-cell lysates were analyzed by immunoblotting with anti-α4-pS988 and anti-total α4 integrin antibody. (C–E) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without siRNA against α4 integrin and siRNA-resistant cDNA constructs for HA-tagged WT, S988A or S988D α4 integrin for 48 h. Cells were allowed to migrate towards 100 µg/ml IIICS in the absence or presence of 30 µM SSTNVEGFR2, 10 µM vandetanib, or 10 µM H-89 for 16 h. (C) Cells accumulated on the bottom side of the filter were imaged at 20× magnification. Scale bars: 50 µm. (D,E) Migrated cells were quantified from five random images for each condition and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments.
    Complementary Dna Mutant Cxcr4 Cdna, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    VLA-4 phosphorylation at S988 is dependent on shed Sdc1-mediated VEGFR2 activation. (A) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without small interfering RNA (si) against human (h)Sdc1 and cDNA constructs for mouse (m)Sdc1 or mSdc1ΔPVD. After 72 h, the cells were plated on 100 µg/ml IIICS for 2.5 h and lysates were analyzed by immunoblotting with an anti-α4-pS988, anti-total α4 integrin, anti-hSdc1 or anti-mSdc1 antibodies. (B) Cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM OGT2115 (HPSE inhibitor), 30 µM SSTNVEGFR2, 10 µM vandetanib (VEGFR2 inhibitor), or 10 µM H-89 (PKA inhibitor). The whole-cell lysates were analyzed by immunoblotting with anti-α4-pS988 and anti-total α4 integrin antibody. (C–E) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without siRNA against α4 integrin and siRNA-resistant cDNA constructs for HA-tagged WT, S988A or S988D α4 integrin for 48 h. Cells were allowed to migrate towards 100 µg/ml IIICS in the absence or presence of 30 µM SSTNVEGFR2, 10 µM vandetanib, or 10 µM H-89 for 16 h. (C) Cells accumulated on the bottom side of the filter were imaged at 20× magnification. Scale bars: 50 µm. (D,E) Migrated cells were quantified from five random images for each condition and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments.

    Journal: Journal of Cell Science

    Article Title: VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1

    doi: 10.1242/jcs.232645

    Figure Lengend Snippet: VLA-4 phosphorylation at S988 is dependent on shed Sdc1-mediated VEGFR2 activation. (A) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without small interfering RNA (si) against human (h)Sdc1 and cDNA constructs for mouse (m)Sdc1 or mSdc1ΔPVD. After 72 h, the cells were plated on 100 µg/ml IIICS for 2.5 h and lysates were analyzed by immunoblotting with an anti-α4-pS988, anti-total α4 integrin, anti-hSdc1 or anti-mSdc1 antibodies. (B) Cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM OGT2115 (HPSE inhibitor), 30 µM SSTNVEGFR2, 10 µM vandetanib (VEGFR2 inhibitor), or 10 µM H-89 (PKA inhibitor). The whole-cell lysates were analyzed by immunoblotting with anti-α4-pS988 and anti-total α4 integrin antibody. (C–E) CAGHPSE, HMEC-1 or M14 cells were co-transfected with or without siRNA against α4 integrin and siRNA-resistant cDNA constructs for HA-tagged WT, S988A or S988D α4 integrin for 48 h. Cells were allowed to migrate towards 100 µg/ml IIICS in the absence or presence of 30 µM SSTNVEGFR2, 10 µM vandetanib, or 10 µM H-89 for 16 h. (C) Cells accumulated on the bottom side of the filter were imaged at 20× magnification. Scale bars: 50 µm. (D,E) Migrated cells were quantified from five random images for each condition and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments.

    Article Snippet: Complementary DNA Mutant CXCR4 cDNA was prepared by PCR using hCXCR4 VersaClone cDNA obtained through R&D Systems (RDC0032) as a template.

    Techniques: Phospho-proteomics, Activation Assay, Transfection, Small Interfering RNA, Construct, Western Blot, One-tailed Test

    CXCR4 is required for VLA-4-dependent cell migration in a ligand-independent manner. (A) CAGHPSE, HMEC-1 or M14 cells were transfected with two different CXCR4 siRNAs for 48 h prior to 16 h transfilter migration assays towards 100 µg/ml IIICS. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells. (B) CAGHPSE, HMEC-1 or M14 cells transfected with CXCR4 siRNA for 48 h were analyzed by immunoblotting for integrin α4-pS988, PKA pT197, CXCR4 or β-actin. (C) 16 h transfilter migration assays towards IIICS were performed with CAGHPSE and M14 cells treated with or without 10 µg/ml CXCR4-blocking antibody or 10 µM AMD3100 in the absence or presence of 20 ng/ml SDF-1. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments. (D) CAGHPSE or M14 cells were transfected with or without VEGFR2 siRNA for 48 h. 16 h transfilter migration assays towards 100 µg/ml IIICS were performed with or without 30 µM SSTNVEGFR2 or 10 µM vandetanib. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (E) HMEC-1 cells were plated on 100 µg/ml IIICS with 30 µM SSTNVEGFR2 for 2.5 h. Cell lysates were subjected to immunoprecipitation with rabbit anti-VEGFR2 antibody. The VEGFR2-associated complexes were analyzed by immunoblotting for VEGFR2 (with mouse anti-VEGFR2), CXCR4, AC7, PKA, hSdc1 or α4 integrin. (F) HMEC-1 or M14 cells were transfected with two different Sdc1 siRNA for 48 h and plated on 100 µg/ml IIICS for 2 h. Cell lysates were subjected to immunoprecipitation with rabbit anti-VEGFR2. The associated complexes were probed with mouse anti-VEGFR2, anti-CXCR4, anti-α4 integrin, anti-AC7, anti-hSdc1 or anti-PKA antibodies. Silencing of Sdc1 expression was confirmed by immunoprecipitation with rabbit polyclonal anti-Sdc1 and probed with mouse anti-human Sdc1 antibody. (G) CAGHPSE or M14 cells were transfected with VEGFR2 siRNAs for 48 h and plated on 100 µg/ml IIICS for 2 h. Cell lysates were subjected to immunoprecipitation with rabbit polyclonal anti-Sdc1 antibody. The associated complexes were analyzed by immunoblotting for AC7, PKA, CXCR4, Sdc1 or α4 integrin. The whole-cell lysates were analyzed by immunoblotting for VEGFR2. (H) HMEC-1 cells were transfected with two different CXCR4 siRNAs for 48 h and then cell lysates were subjected to immunoprecipitation with anti-VEGFR2 antibody. The associated complexes were analyzed by immunoblotting for AC7, PKA, VEGFR2 or Gβγ. The whole-cell lysates were analyzed by immunoblotting for CXCR4.

    Journal: Journal of Cell Science

    Article Title: VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1

    doi: 10.1242/jcs.232645

    Figure Lengend Snippet: CXCR4 is required for VLA-4-dependent cell migration in a ligand-independent manner. (A) CAGHPSE, HMEC-1 or M14 cells were transfected with two different CXCR4 siRNAs for 48 h prior to 16 h transfilter migration assays towards 100 µg/ml IIICS. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells. (B) CAGHPSE, HMEC-1 or M14 cells transfected with CXCR4 siRNA for 48 h were analyzed by immunoblotting for integrin α4-pS988, PKA pT197, CXCR4 or β-actin. (C) 16 h transfilter migration assays towards IIICS were performed with CAGHPSE and M14 cells treated with or without 10 µg/ml CXCR4-blocking antibody or 10 µM AMD3100 in the absence or presence of 20 ng/ml SDF-1. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01 between treatments. (D) CAGHPSE or M14 cells were transfected with or without VEGFR2 siRNA for 48 h. 16 h transfilter migration assays towards 100 µg/ml IIICS were performed with or without 30 µM SSTNVEGFR2 or 10 µM vandetanib. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (E) HMEC-1 cells were plated on 100 µg/ml IIICS with 30 µM SSTNVEGFR2 for 2.5 h. Cell lysates were subjected to immunoprecipitation with rabbit anti-VEGFR2 antibody. The VEGFR2-associated complexes were analyzed by immunoblotting for VEGFR2 (with mouse anti-VEGFR2), CXCR4, AC7, PKA, hSdc1 or α4 integrin. (F) HMEC-1 or M14 cells were transfected with two different Sdc1 siRNA for 48 h and plated on 100 µg/ml IIICS for 2 h. Cell lysates were subjected to immunoprecipitation with rabbit anti-VEGFR2. The associated complexes were probed with mouse anti-VEGFR2, anti-CXCR4, anti-α4 integrin, anti-AC7, anti-hSdc1 or anti-PKA antibodies. Silencing of Sdc1 expression was confirmed by immunoprecipitation with rabbit polyclonal anti-Sdc1 and probed with mouse anti-human Sdc1 antibody. (G) CAGHPSE or M14 cells were transfected with VEGFR2 siRNAs for 48 h and plated on 100 µg/ml IIICS for 2 h. Cell lysates were subjected to immunoprecipitation with rabbit polyclonal anti-Sdc1 antibody. The associated complexes were analyzed by immunoblotting for AC7, PKA, CXCR4, Sdc1 or α4 integrin. The whole-cell lysates were analyzed by immunoblotting for VEGFR2. (H) HMEC-1 cells were transfected with two different CXCR4 siRNAs for 48 h and then cell lysates were subjected to immunoprecipitation with anti-VEGFR2 antibody. The associated complexes were analyzed by immunoblotting for AC7, PKA, VEGFR2 or Gβγ. The whole-cell lysates were analyzed by immunoblotting for CXCR4.

    Article Snippet: Complementary DNA Mutant CXCR4 cDNA was prepared by PCR using hCXCR4 VersaClone cDNA obtained through R&D Systems (RDC0032) as a template.

    Techniques: Migration, Transfection, One-tailed Test, Western Blot, Blocking Assay, Immunoprecipitation, Expressing

    VEGFR2 activates CXCR4 by phosphorylating Y135 in its DRY motif. (A) CAGHPSE, HMEC-1 or M14 cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM vandetanib or 30 µM SSTNVEGFR2 for 2.5 h. CXCR4 was immunoprecipitated with rabbit anti-CXCR4 antibody and the CXCR4-containing complexes were analyzed by immunoblotting with anti-phosphotyrosine or rat anti-CXCR4 antibody. (B–F) CAGHPSE, HMEC-1 or M14 cells were co-transfected with CXCR4 3′UTR siRNA and CXCR4 cDNA lacking the 3′UTR (GFP-tagged WT and Y→F mutants or GFP alone) for 48 h. The cells were then analyzed by flow cytometry, immunoprecipitation and migration assays. (B) GFP-tagged WT or Y135F CXCR4 mutant HMEC-1 cells were incubated with PBS alone or 200 nM SDF-1 in PBS at 4°C. After incubation with 10 µg/ml anti-SDF-1 antibody versus an isotype-matched control mouse IgG1 for 1 h, cells were fixed and blocked with 3% BSA in PBS. The bound antibody was detected with a RPE-labeled anti-mouse IgG and analyzed by flow cytometry for SDF-1-bound CXCR4. (C) CAGHPSE cell lysates were subjected to immunoprecipitation with anti-GFP antibody and then analyzed with anti-VEGFR2, anti-α4 integrin, anti-hSdc1, anti-AC7, anti-PKA or anti-CXCR4 antibodies. (D) M14 cell lysates were subjected to immunoprecipitation with anti-GFP antibody and then analyzed with anti-phosphotyrosine (pY20) or anti-CXCR4 antibodies. (E) Quantification of 16 h transfilter migration of CAGHPSE cells towards 100 µg/ml IIICS in the absence or presence of 20 nM SDF-1 is plotted as the mean±s.d. of three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (F) CAGHPSE cells were kept in suspension or plated on 100 µg/ml IIICS in the absence or presence of 20 nM SDF-1 with or without 10 µM vandetanib for 2 h. Cell lysates were subjected to immunoprecipitation with anti-GFP or anti-GTP-Gαi antibodies, then probed with anti-Gαi antibody. The whole-cell lysates were analyzed by immunoblotting for integrin α4-pS988 and β-actin.

    Journal: Journal of Cell Science

    Article Title: VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1

    doi: 10.1242/jcs.232645

    Figure Lengend Snippet: VEGFR2 activates CXCR4 by phosphorylating Y135 in its DRY motif. (A) CAGHPSE, HMEC-1 or M14 cells were plated on 100 µg/ml IIICS in the absence or presence of 10 µM vandetanib or 30 µM SSTNVEGFR2 for 2.5 h. CXCR4 was immunoprecipitated with rabbit anti-CXCR4 antibody and the CXCR4-containing complexes were analyzed by immunoblotting with anti-phosphotyrosine or rat anti-CXCR4 antibody. (B–F) CAGHPSE, HMEC-1 or M14 cells were co-transfected with CXCR4 3′UTR siRNA and CXCR4 cDNA lacking the 3′UTR (GFP-tagged WT and Y→F mutants or GFP alone) for 48 h. The cells were then analyzed by flow cytometry, immunoprecipitation and migration assays. (B) GFP-tagged WT or Y135F CXCR4 mutant HMEC-1 cells were incubated with PBS alone or 200 nM SDF-1 in PBS at 4°C. After incubation with 10 µg/ml anti-SDF-1 antibody versus an isotype-matched control mouse IgG1 for 1 h, cells were fixed and blocked with 3% BSA in PBS. The bound antibody was detected with a RPE-labeled anti-mouse IgG and analyzed by flow cytometry for SDF-1-bound CXCR4. (C) CAGHPSE cell lysates were subjected to immunoprecipitation with anti-GFP antibody and then analyzed with anti-VEGFR2, anti-α4 integrin, anti-hSdc1, anti-AC7, anti-PKA or anti-CXCR4 antibodies. (D) M14 cell lysates were subjected to immunoprecipitation with anti-GFP antibody and then analyzed with anti-phosphotyrosine (pY20) or anti-CXCR4 antibodies. (E) Quantification of 16 h transfilter migration of CAGHPSE cells towards 100 µg/ml IIICS in the absence or presence of 20 nM SDF-1 is plotted as the mean±s.d. of three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (F) CAGHPSE cells were kept in suspension or plated on 100 µg/ml IIICS in the absence or presence of 20 nM SDF-1 with or without 10 µM vandetanib for 2 h. Cell lysates were subjected to immunoprecipitation with anti-GFP or anti-GTP-Gαi antibodies, then probed with anti-Gαi antibody. The whole-cell lysates were analyzed by immunoblotting for integrin α4-pS988 and β-actin.

    Article Snippet: Complementary DNA Mutant CXCR4 cDNA was prepared by PCR using hCXCR4 VersaClone cDNA obtained through R&D Systems (RDC0032) as a template.

    Techniques: Immunoprecipitation, Western Blot, Transfection, Flow Cytometry, Migration, Mutagenesis, Incubation, Control, Labeling, One-tailed Test, Suspension

    The CXCR4 Y135D phosphomimetic mutant rescues the effects of VEGFR2 inhibition. (A,B) CAGHPSE, HMEC-1 or M14 cells were transfected with GFP alone, GFP–CXCR4 WT, or GFP–CXCR4 Y135D together with siRNA targeting 3′UTR of endogenous CXCR4 for 48 h. (A) 16 h transfilter cell migration assay towards 100 µg/ml IIICS was performed in the absence or presence of 10 µM vandetanib. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01, n.s., not significant. (B) M14 cell lysates were subjected to immunoprecipitation with anti-GTP-Gαi and probed for Gαi. Whole-cell lysates were analyzed by immunoblotting for Gαi or α4 integrin (total or pS988). (C) Model depicting how paxillin bound to VLA-4 causes inherent inhibition of Rac GTPase at this site due to its binding of ArfGAP (Nishiya et al., 2005). VEGFR2 forms a complex with CXCR4, AC7 and PKA, but this complex is inactive unless Sdc1 is shed. Trimming of the HS chains on Sdc1 by HPSE facilitates its shedding by MMP9 (Purushothaman et al., 2010; Yang et al., 2007). Sdc1 freed of its membrane anchorage couples VEGFR2 and its integrin phosphorylation machinery to VLA-4 clusters. VEGFR2 activated by this clustering event phosphorylates CXCR4 at Y135, activating its heterotrimeric G-protein. Gβγ freed of Gαi activates AC7, generating cAMP that activates PKA. PKA phosphorylation of the α4 integrin cytoplasmic domain at S988 dissociates paxillin–ArfGAP from VLA-4, allowing local lamellipodium formation and directed cell migration.

    Journal: Journal of Cell Science

    Article Title: VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1

    doi: 10.1242/jcs.232645

    Figure Lengend Snippet: The CXCR4 Y135D phosphomimetic mutant rescues the effects of VEGFR2 inhibition. (A,B) CAGHPSE, HMEC-1 or M14 cells were transfected with GFP alone, GFP–CXCR4 WT, or GFP–CXCR4 Y135D together with siRNA targeting 3′UTR of endogenous CXCR4 for 48 h. (A) 16 h transfilter cell migration assay towards 100 µg/ml IIICS was performed in the absence or presence of 10 µM vandetanib. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01, n.s., not significant. (B) M14 cell lysates were subjected to immunoprecipitation with anti-GTP-Gαi and probed for Gαi. Whole-cell lysates were analyzed by immunoblotting for Gαi or α4 integrin (total or pS988). (C) Model depicting how paxillin bound to VLA-4 causes inherent inhibition of Rac GTPase at this site due to its binding of ArfGAP (Nishiya et al., 2005). VEGFR2 forms a complex with CXCR4, AC7 and PKA, but this complex is inactive unless Sdc1 is shed. Trimming of the HS chains on Sdc1 by HPSE facilitates its shedding by MMP9 (Purushothaman et al., 2010; Yang et al., 2007). Sdc1 freed of its membrane anchorage couples VEGFR2 and its integrin phosphorylation machinery to VLA-4 clusters. VEGFR2 activated by this clustering event phosphorylates CXCR4 at Y135, activating its heterotrimeric G-protein. Gβγ freed of Gαi activates AC7, generating cAMP that activates PKA. PKA phosphorylation of the α4 integrin cytoplasmic domain at S988 dissociates paxillin–ArfGAP from VLA-4, allowing local lamellipodium formation and directed cell migration.

    Article Snippet: Complementary DNA Mutant CXCR4 cDNA was prepared by PCR using hCXCR4 VersaClone cDNA obtained through R&D Systems (RDC0032) as a template.

    Techniques: Mutagenesis, Inhibition, Transfection, Cell Migration Assay, One-tailed Test, Immunoprecipitation, Western Blot, Binding Assay, Membrane, Phospho-proteomics, Migration

    sSdc1-mediated VEGFR2 activation suppresses cytotoxic T cell and NK cell migration by displacing VLA-4 from LFA-1. (A) Lysates from CAGHPSE cells, NK cells and T cells were analyzed by immunoblotting for VEGFR2, VLA-4, LFA-1, Sdc1, PKA, CXCR4, AC7 and β-actin (loading control). (B) NK cells and T cells were plated on 10 µg/ml VCAM-1 in the absence or presence of 30 µM SSTNVEGFR2 for 2.5 h. VEGFR2 was immunoprecipitated and the VEGFR2-containing complexes analyzed by immunoblotting for VEGFR2, AC7, Sdc1, PKA, CXCR4, α4 integrin [total (VLA-4) and pS988], LFA-1 and VLA-5. (C) NK cells and T cells were plated on either ICAM-1 or VCAM-1 in the absence or presence of SDF-1 with or without anti-VLA-4 or anti-LFA-1 antibodies or CXCR4 inhibitor (AMD3100). Attached NK cells and T cells in five random images for each experiment were quantified and graphed as a percent of cells treated with SDF-1 alone (set to 100%). All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (D) 16 h transfilter cell migration assays towards 10 µg/ml VCAM-1 alone, 10 µg/ml ICAM-1 alone, or a mixture of 10 µg/ml ICAM-1 with 10 µg/ml VCAM-1, analysis after treatment with 30 µM SSTNVEGFR2, 10 µg/ml anti-VLA-4, or 10 µg/ml anti-LFA-1 in the absence or presence of SDF-1. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells on each ligand; **P<0.01, n.s., not significant between treatments. (E) 16 h transfilter cell migration assays towards 10 µg/ml VCAM-1 alone or mixture of 10 µg/ml ICAM-1 plus 10 µg/ml VCAM-1 was performed as in D in the absence or presence of 30 µM SSTNVEGFR2, 1 µM vandetanib or 10 µM AMD3100. All data were compared using the unpaired one-tailed t-test. **P<0.01, n.s., not significant. (F) NK cells and T cells were plated on 10 µg/ml VCAM-1 alone or mixture of 10 µg/ml ICAM-1 with 10 µg/ml VCAM-1 in the absence or presence of SSTNVEGFR2. Cell lysates were subjected to immunoprecipitation of VLA-4 and probed for VLA-4 or LFA-1. Whole-cell lysates were analyzed by immunoblotting for integrin α4-pS988, VEGFR2-pY1054/1059 or β-actin. (G) Model depicting how LFA-1 and VLA-4 on NK cells and T cells associate in an adhesion complex in which signaling from FAK and PYK2, localized to VLA-4 by paxillin, transregulates LFA-1 activity leading to LFA-1-dependent cell migration (Cantor et al., 2015; Rose et al., 2003). If Sdc1 is shed by the leukocytes, or high levels of shed Sdc1 accumulate in the tumor microenvironment from HPSE-overexpressing tumor cells, the shed syndecan couples VEGFR2 and its phosphorylation machinery to VLA-4. Activation of CXCR4 by SDF-1, or by VEGFR2-mediated phosphorylation of Y135 in the CXCR4 cytoplasmic loop 2, causes phosphorylation of S988 in the α4 integrin subunit, displacing paxillin and suppressing LFA-1-mediated influx of cytotoxic leukocytes to the tumor. SSTNVEGFR2, which reverses the effects of shed syndecan, relieves this immunosuppression while at the same time blocking tumor cell migration and metastasis (see Fig. 6C).

    Journal: Journal of Cell Science

    Article Title: VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1

    doi: 10.1242/jcs.232645

    Figure Lengend Snippet: sSdc1-mediated VEGFR2 activation suppresses cytotoxic T cell and NK cell migration by displacing VLA-4 from LFA-1. (A) Lysates from CAGHPSE cells, NK cells and T cells were analyzed by immunoblotting for VEGFR2, VLA-4, LFA-1, Sdc1, PKA, CXCR4, AC7 and β-actin (loading control). (B) NK cells and T cells were plated on 10 µg/ml VCAM-1 in the absence or presence of 30 µM SSTNVEGFR2 for 2.5 h. VEGFR2 was immunoprecipitated and the VEGFR2-containing complexes analyzed by immunoblotting for VEGFR2, AC7, Sdc1, PKA, CXCR4, α4 integrin [total (VLA-4) and pS988], LFA-1 and VLA-5. (C) NK cells and T cells were plated on either ICAM-1 or VCAM-1 in the absence or presence of SDF-1 with or without anti-VLA-4 or anti-LFA-1 antibodies or CXCR4 inhibitor (AMD3100). Attached NK cells and T cells in five random images for each experiment were quantified and graphed as a percent of cells treated with SDF-1 alone (set to 100%). All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells; **P<0.01, n.s., not significant between treatments. (D) 16 h transfilter cell migration assays towards 10 µg/ml VCAM-1 alone, 10 µg/ml ICAM-1 alone, or a mixture of 10 µg/ml ICAM-1 with 10 µg/ml VCAM-1, analysis after treatment with 30 µM SSTNVEGFR2, 10 µg/ml anti-VLA-4, or 10 µg/ml anti-LFA-1 in the absence or presence of SDF-1. Migrated cells were quantified and graphed as the mean±s.d. from three independent experiments. All data were compared using the unpaired one-tailed t-test. *P<0.01 against untreated parental cells on each ligand; **P<0.01, n.s., not significant between treatments. (E) 16 h transfilter cell migration assays towards 10 µg/ml VCAM-1 alone or mixture of 10 µg/ml ICAM-1 plus 10 µg/ml VCAM-1 was performed as in D in the absence or presence of 30 µM SSTNVEGFR2, 1 µM vandetanib or 10 µM AMD3100. All data were compared using the unpaired one-tailed t-test. **P<0.01, n.s., not significant. (F) NK cells and T cells were plated on 10 µg/ml VCAM-1 alone or mixture of 10 µg/ml ICAM-1 with 10 µg/ml VCAM-1 in the absence or presence of SSTNVEGFR2. Cell lysates were subjected to immunoprecipitation of VLA-4 and probed for VLA-4 or LFA-1. Whole-cell lysates were analyzed by immunoblotting for integrin α4-pS988, VEGFR2-pY1054/1059 or β-actin. (G) Model depicting how LFA-1 and VLA-4 on NK cells and T cells associate in an adhesion complex in which signaling from FAK and PYK2, localized to VLA-4 by paxillin, transregulates LFA-1 activity leading to LFA-1-dependent cell migration (Cantor et al., 2015; Rose et al., 2003). If Sdc1 is shed by the leukocytes, or high levels of shed Sdc1 accumulate in the tumor microenvironment from HPSE-overexpressing tumor cells, the shed syndecan couples VEGFR2 and its phosphorylation machinery to VLA-4. Activation of CXCR4 by SDF-1, or by VEGFR2-mediated phosphorylation of Y135 in the CXCR4 cytoplasmic loop 2, causes phosphorylation of S988 in the α4 integrin subunit, displacing paxillin and suppressing LFA-1-mediated influx of cytotoxic leukocytes to the tumor. SSTNVEGFR2, which reverses the effects of shed syndecan, relieves this immunosuppression while at the same time blocking tumor cell migration and metastasis (see Fig. 6C).

    Article Snippet: Complementary DNA Mutant CXCR4 cDNA was prepared by PCR using hCXCR4 VersaClone cDNA obtained through R&D Systems (RDC0032) as a template.

    Techniques: Activation Assay, Migration, Western Blot, Control, Immunoprecipitation, One-tailed Test, Activity Assay, Phospho-proteomics, Blocking Assay