Abstract 341: Investigating a novel GPCR ITIM motif in regulating Shp2 binding, signaling, and migration

Abstract

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