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The GPCR CXCR4 and Tyrosine Phosphatase SHP2 Interact and Signal via a Novel Consensus Motif
Author(s) -
Belcastro Lili T.,
Paulukinas Ryan D.,
Jancina Anastasia,
Moore Catherine C.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.710.11
Subject(s) - g protein coupled receptor , phosphorylation , biology , protein tyrosine phosphatase , chemokine receptor , microbiology and biotechnology , signal transduction , tyrosine phosphorylation , signal transducing adaptor protein , endocytic cycle , phosphatase , receptor , biochemistry , chemokine , endocytosis
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.