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Leveraging a Novel ITIM Motif in GPCRs for Targeted Antibody Design
Author(s) -
Belcastro Lili T.,
Jancina Anastasia,
Adams Christina,
Paulukinas Ryan D.,
Moore Catherine C.
Publication year - 2017
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.31.1_supplement.664.13
Subject(s) - phosphorylation , g protein coupled receptor , mapk/erk pathway , microbiology and biotechnology , internalization , signal transduction , biology , endocytic cycle , signal transducing adaptor protein , receptor , tyrosine phosphorylation , chemokine receptor , cancer research , chemokine , endocytosis , biochemistry
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.