CXCR4, a chemokine GPCR, is essential for migration of neuronal and hematopoietic cells during embryonic development, and for breast cancer cells during cancer metastasis whereby CXCR4 dysregulation promotes migration and invasion. Despite the knowledge of the role of CXCR4 in breast cancer organ-specific metastasis, effective treatments for metastatic breast cancer are still lacking, especially for patients with triple negative breast cancer. A better understanding of how CXCR4 promotes metastasis is necessary for therapeutic discovery and targeting. It is known that following activation and signaling upon SDF ligand binding, CXCR4 is rapidly phosphorylated on serine and threonine residues in the C-terminal tail, which initiates β-arrestin recruitment, receptor desensitization, and trafficking to endocytic sites. In stark contrast to this classically rapid GPCR downregulation, we have shown that stimulation with gradient SDF significantly delays receptor phosphorylation, previously, and inhibits receptor trafficking, herein, which leads to robust sustained signaling to a novel CXCR4-SHP2-ERK pathway. It remained unknown however, how CXCR4 relayed this signaling to SHP2. Here we identified a novel ITIM sequence ( immunoreceptor tyrosine-based inhibition motif) in CXCR4 that regulates both SHP2 binding and sustained signaling, and is critical for CXCR4-mediated migration. ITIMs are a hallmark of inhibitory immune receptors which recruit SHP2 to inhibit downstream signaling pathways, however to date, evidence for ITIMs in GPCRs is limited. In Aim I, we determined whether the CXCR4 ITIM is functional by evaluating phosphorylation of the central tyrosine (Y302) through development of a novel phospho-specific CXCR4 antibody, and by determining whether it binds and activates SHP2 utilizing mutagenesis and co-immunoprecipitation studies. In Aim II, we analyzed the importance of the CXCR4 ITIM in downstream signaling, internalization, and migratory function with immunoblot, ELISA, and transwell assays. Our results suggest that the CXCR4 ITIM is phosphorylated as demonstrated through the development and validation of a p-Y302-CXCR4 antibody. By utilizing an ITIM mutant we demonstrate that the ITIM is necessary for SHP2 binding and activation as demonstrated by co- immunoprecipitation and gradient signaling assays. We also show that Y302 of the ITIM is dispensable for gradient sensing and normal trafficking, but that it is required for migration of breast cancer cells. Our data support a working model that CXCR4 contains a functional ITIM sequence capable of promoting breast cancer cell migration through the recruitment and activation of the proto-oncogene SHP2. These data have implications on metastatic breast cancer cells known to have aberrant CXCR4 and SHP2 expression.
|Advisor:||Moore, Catherine C.|
|Commitee:||Chen, Bin, Janetopoulos, Christopher, Wang, Zhihong, Weeraratna, Ashani|
|School:||University of the Sciences in Philadelphia|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 79/04(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Pharmacology, Oncology|
|Keywords:||CXC chemokine receptor 4, CXCR4, GPCR migration, ITIM, Immunoreceptor tyrosine-based inhibition motif, Metastasis, SDF, SH2 domain-containing non-transmembrane protein tyrosine phosphatase, SHP2 signaling, Stromal cellderived factor 1alpha, CXCL12|
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