CXCR4, a chemokine GPCR, is essential for migration of neuronal, hematopoietic, and breast cancer cells during metastasis whereby CXCR4 dysregulation promotes migration and invasion. Following SDF stimulation, CXCR4 is phosphorylated on Ser/Thr residues which initiates adaptor recruitment, receptor desensitization, and trafficking to endocytic sites. Here we show that stimulation with gradient SDF, delays receptor phosphorylation and trafficking, leading to sustained signaling to a novel CXCR4‐SHP2‐ERK pathway. SHP2 is a tyrosine phosphatase implicated in HER2(+) and triple‐negative breast cancers, whereby it transduces mitogenic and migratory signals driving hyperproliferation and invasion. SHP2 is recruited to tyrosine phosphorylated ITIM motifs ( i mmmunoreceptor t yrosine‐based i nhibitory consensus m otifs), a hallmark found in inhibitory immune receptors with little evidence in GPCRs. Here we identify an ITIM motif in CXCR4 that regulates both SHP2 binding and signaling. Specifically, we assessed if gradient SDF stimulation of CXCR4 1) delays receptor phosphorylation and trafficking, 2) sustains signaling to SHP2‐ERK, 3) induces SHP2‐dependent migration; and if CXCR4 Tyr mutation within the ITIM motif 4) maintains SDF gradient sensing ability, and 5) disrupts interaction with and signaling to SHP2. Our data demonstrate that gradient SDF delays receptor Ser/Thr phosphorylation and internalization thereby sustaining signaling to SHP2‐ERK and driving SHP2‐dependent migration. Furthermore, the ITIM mutant maintains SDF gradient sensing ability, but disrupts interaction with and signaling to SHP2. Our data support a working model that CXCR4 contains a functional ITIM motif which we are currently leveraging for targeted antibody design for use in migration studies of aggressive breast cancer cells with dysregulated CXCR4. Support or Funding Information These studies were supported by NIH grant GM‐097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund.
CXCR4, a chemokine G protein‐coupled receptor (GPCR), is essential for migration of neuronal and hematopoietic cells, as well as breast cancer cells during metastasis whereby CXCR4 dysregulation promotes a migratory and invasive phenotype. Normally following activation, CXCR4 is rapidly phosphorylated on Ser/Thr residues by GRKs, a key step in initiating adaptor recruitment, which promotes receptor desensitization and receptor trafficking to endocytic sites. Previously we identified that CXCR4 Ser/Thr phosphorylation is delayed upon SDF gradient sensing, and correlates with sustained signaling to a novel CXCR4‐SHP2‐ERK pathway. SHP2 is a protein tyrosine phosphatase (PTP) implicated in HER2(+) and triple‐negative breast cancers, whereby SHP2 transduces both mitogenic and migratory signals, driving hyperproliferation and invasion. SHP2 is also known to be recruited to tyrosine phosphorylated ITIM motifs ( i mmmunoreceptor t yrosine‐based i nhibitory consensus m otifs), a hallmark found in inhibitory immune receptors with little to no evidence for its presence and function in GPCRs. We have identified a putative ITIM motif in CXCR4 that regulates SHP2 binding and signaling. Specifically, we assessed 1) if CXCR4 is tyrosine phosphorylated within the putative ITIM motif, using a rSHP2 phosphatase assay, pAb development, and mutagenesis, 2) if SHP2 interaction with CXCR4 is disrupted upon ITIM Tyr mutation, by co‐immunoprecipitation, 3) if CXCR4 signaling to SHP2 is disrupted upon ITIM Tyr mutation, and 4) if differences in signaling are due to improper localization and/or function of the mutant, by ELISA. Our results demonstrate that rSHP2 phosphatase is active on CXCR4, suggesting pTyr of CXCR4. Specific pTyr antibodies are under development to confirm these results. Additionally, Tyr mutation within the ITIM alters CXCR4 phosphorylation, suggesting pTyr of CXCR4 occurs within this motif. Furthermore, Tyr mutation disrupts CXCR4 immunocomplex formation with SHP2. Finally, sustained signaling to the SHP2‐ERK pathway is attenuated upon tyrosine mutation within the ITIM motif, and this effect is not due to improper receptor localization or function as demonstrated by cell surface expression and agonist‐dependent internalization. To date, our data support a working model that CXCR4 contains a functional ITIM motif capable of binding SHP2 and transducing sustained signaling to SHP2 upon SDF gradient sensing. These data have implications on aggressive breast cancers with dysregulated CXCR4 and SHP2. Support or Funding Information Supported by NIH GM‐097718 and PDH SAP4100057688 grants.
Abstract CXCR4, a chemokine GPCR, is essential for migration of neuronal, hematopoietic, and breast cancer cells during metastasis whereby CXCR4 dysregulation promotes migration and invasion. Following SDF stimulation, CXCR4 is phosphorylated on Ser/Thr residues which initiates adaptor recruitment, receptor desensitization, and trafficking to endocytic sites. Here we show that stimulation with gradient SDF, delays receptor phosphorylation and trafficking, leading to sustained signaling to a novel CXCR4-SHP2-ERK pathway. SHP2 is a tyrosine phosphatase implicated in HER2(+) and triple-negative breast cancers, whereby it transduces mitogenic and migratory signals driving hyperproliferation and invasion. SHP2 is recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine-based inhibitory consensus motifs), a hallmark found in inhibitory immune receptors with little evidence in GPCRs. Here we identify an ITIM motif in CXCR4 that regulates both SHP2 binding and signaling. Specifically, we assessed if gradient SDF stimulation of CXCR4 1) delays receptor phosphorylation and trafficking, 2) sustains signaling to SHP2-ERK, 3) induces SHP2-dependent migration; and if CXCR4 Tyr mutation within the ITIM motif 4) maintains SDF gradient sensing ability, and 5) disrupts interaction with and signaling to SHP2. Our data demonstrate that gradient SDF delays receptor Ser/Thr phosphorylation and internalization thereby sustaining signaling to SHP2-ERK and driving SHP2-dependent migration. Furthermore, the ITIM mutant maintains SDF gradient sensing ability, but disrupts interaction with and signaling to SHP2. Our data support a working model that CXCR4 contains a functional ITIM motif which we are currently leveraging for targeted antibody design for use in migration studies of aggressive breast cancer cells with dysregulated CXCR4. These studies were supported by NIH grant GM-097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund. Citation Format: Lili T. Belcastro, Anastasia Jancina, Christina Adams, Ryan D. Paulukinas, Catherine C. Moore. Leveraging a novel ITIM motif in GPCRs for targeted antibody design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 360. doi:10.1158/1538-7445.AM2017-360
Aberrant expression of CXCR4, a chemokine G protein‐coupled receptor (GPCR), promotes a migratory and invasive phenotype during breast cancer metastasis, however the mechanism is unknown. Here we investigate the role of CXCR4 tyrosine phosphorylation within a novel ITIM motif. Following activation, CXCR4 is normally phosphorylated on Ser/Thr residues, which promotes receptor desensitization and internalization. We found previously that Ser/Thr phosphorylation of CXCR4 is delayed upon SDF gradient sensing, and correlates with sustained signaling to SHP2, a protein that drives hyperproliferation and invasion of breast cancer. Since SHP2 is known to be recruited to tyrosine phosphorylated ITIM motifs ( i mmmunoreceptor tyrosine‐based inhibitory consensus mo tifs), we investigated if CXCR4 contains an ITIM motif, and if tyrosine phosphorylation within this motif regulates SHP2 binding, signaling, and migration. Specifically, we assessed 1) if CXCR4 is tyrosine phosphorylated as measured by recombinant phosphatase assay, phospho‐Ab development, and tyrosine mutagenesis (YF); 2) if tyrosine mutagenesis alters CXCR4 membrane localization, internalization, and SDF gradient sensing, by ELISA; 3) if tyrosine mutagenesis disrupts CXCR4 interaction with SHP2, by co‐IP; 4) if tyrosine mutagenesis alters CXCR4 sustained signaling to SHP2, by Western; and 5) if tyrosine mutation (YF) within the novel ITIM motif in CXCR4 alters migration of metastatic breast cancer cells, by transwell motility assays. Our results demonstrate that CXCR4 phosphorylation is sensitive to recombinant tyrosine phosphatase treatment and tyrosine mutagenesis, suggesting that CXCR4 is indeed tyrosine phosphorylated within the ITIM motif. We determined that while tyrosine phosphorylation within the ITIM motif is not required for CXCR4 membrane localization, internalization, or SDF gradient sensing, it does regulate CXCR4 binding and signaling to SHP2. Investigating the role in CXCR4 mediated migration is on going. To date, our data support a working model that tyrosine phosphorylation of CXCR4 within an ITIM motif is critical for binding to SHP2 and transducing sustained signaling to SHP2. These data have implications on aggressive breast cancers with dysregulated CXCR4 and SHP2. Support or Funding Information These studies were supported by NIH grant GM‐097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund.
Abstract Aberrant expression of CXCR4, a chemokine G protein-coupled receptor (GPCR), promotes a migratory and invasive phenotype during breast cancer metastasis, however the mechanism is unknown. Here we investigate the role of CXCR4 tyrosine phosphorylation within a novel ITIM motif. Following activation, CXCR4 is normally phosphorylated on Ser/Thr residues, which promotes receptor desensitization and internalization. We found previously that Ser/Thr phosphorylation of CXCR4 is delayed upon SDF gradient sensing, and correlates with sustained signaling to SHP2, a protein that drives hyperproliferation and invasion of breast cancer. Since SHP2 is known to be recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine-based inhibitory consensus motifs), we investigated if CXCR4 contains an ITIM motif, and if tyrosine phosphorylation within this motif regulates SHP2 binding, signaling, and migration. Specifically, we assessed 1) if CXCR4 is tyrosine phosphorylated as measured by recombinant phosphatase assay, phospho-Ab development, and tyrosine mutagenesis (YF); 2) if tyrosine mutagenesis alters CXCR4 membrane localization, internalization, and SDF gradient sensing, by ELISA; 3) if tyrosine mutagenesis disrupts CXCR4 interaction with SHP2, by co-IP; 4) if tyrosine mutagenesis alters CXCR4 sustained signaling to SHP2, by Western; and 5) if tyrosine mutation (YF) within the novel ITIM motif in CXCR4 alters migration of metastatic breast cancer cells, by transwell motility assays. Our results demonstrate that CXCR4 phosphorylation is sensitive to recombinant tyrosine phosphatase treatment and tyrosine mutagenesis, suggesting that CXCR4 is indeed tyrosine phosphorylated within the ITIM motif. We determined that while tyrosine phosphorylation within the ITIM motif is not required for CXCR4 membrane localization, internalization, or SDF gradient sensing, it does regulate CXCR4 binding and signaling to SHP2. Investigating the role in CXCR4 mediated migration is on going. To date, our data support a working model that tyrosine phosphorylation of CXCR4 within an ITIM motif is critical for binding to SHP2 and transducing sustained signaling to SHP2. These data have implications on aggressive breast cancers with dysregulated CXCR4 and SHP2. These studies were supported by NIH grant GM-097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund. Citation Format: Ryan D. Paulukinas, Lili T. Belcastro, Catherine C. Moore. Investigating a novel GPCR ITIM motif in regulating Shp2 binding, signaling, and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 341. doi:10.1158/1538-7445.AM2017-341
Abstract ID 95428Poster Board 248 CXCR4, a chemokine GPCR, is essential for migration of neuronal and hematopoietic cells during embryonic development, and for migration of breast cancer cells during metastasis whereby CXCR4 dysregulation promotes cell motility and invasion. Following uniform SDF stimulation, CXCR4 is rapidly phosphorylated on serine and threonine residues in the C-terminal tail, which initiates b-arrestin recruitment, receptor desensitization, and trafficking to endocytic sites. Here we show that stimulation with gradient SDF however, significantly delays this receptor phosphorylation and trafficking, leading to sustained signaling to a novel CXCR4-SHP2-ERK pathway. SHP2 is a tyrosine phosphatase implicated in HER2+ and triple-negative breast cancers (TNBC), whereby it transduces mitogenic and migratory signals driving hyper-proliferation and invasion. SHP2 is recruited to tyrosine phosphorylated ITIM motifs (immunoreceptor tyrosine-based inhibitory consensus motifs), a hallmark of inhibitory immune receptors with little evidence for their presence in GPCRs. Here we identify a novel ITIM motif in the chemokine GPCR CXCR4, that drives gradient signaling to SHP2 and directional motility. Specifically, first we show that gradient SDF stimulation of the CXCR4 receptor 1) delays receptor phosphorylation and trafficking, and 2) sustains signaling to a novel CXCR4-SHP2-ERK pathway. Next, we identify an ITIM motif in the CXCR4 receptor that drives gradient SDF mediated 3) sustained signaling to SHP2, and 4) directional migration in TNBC cells. Additionally, we demonstrate that this novel ITIM motif can be 5) leveraged for targeted antibody design, and 6) peptide inhibitor development. Overall our data demonstrate that gradient SDF delays receptor Ser/Thr phosphorylation and internalization thereby sustaining signaling to SHP2-ERK and driving SHP2-dependent migration. Furthermore, a CXCR4 ITIM motif is critical for transducing this SDF gradient sensing to SHP2 binding and signaling, and directional motility. Coupled with our ITIM targeted antibody and peptide inhibitor development, these data have therapeutic implications for metastatic diseases driven by aberrant CXCR4 and SHP2 expression.
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