Abstract
The immune system includes abundant examples of biologically-relevant cross-regulation of signaling pathways by the T cell antigen receptor (TCR) and the G protein-coupled chemokine receptor, CXCR4. TCR ligation induces transactivation of CXCR4 and TCR-CXCR4 complex formation, permitting the TCR to signal via CXCR4 to activate a phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein (PREX1)-dependent signaling pathway that drives robust cytokine secretion by T cells. To understand this receptor heterodimer and its regulation, we characterized the molecular mechanisms required for TCR-mediated TCR-CXCR4 complex formation. We found that the cytoplasmic C-terminal domain of CXCR4 and specifically phosphorylation of Ser-339 within this region were required for TCR-CXCR4 complex formation. Interestingly, siRNA-mediated depletion of G protein-coupled receptor kinase-2 (GRK2) or inhibition by the GRK2-specific inhibitor, paroxetine, inhibited TCR-induced phosphorylation of CXCR4-Ser-339 and TCR-CXCR4 complex formation. Either GRK2 siRNA or paroxetine treatment of human T cells significantly reduced T cell cytokine production. Upstream, TCR-activated tyrosine kinases caused inducible tyrosine phosphorylation of GRK2 and were required for the GRK2-dependent events of CXCR4-Ser-339 phosphorylation and TCR-CXCR4 complex formation. Downstream of TCR-CXCR4 complex formation, we found that GRK2 and phosphatidylinositol 3-kinase γ (PI3Kγ) were required for TCR-stimulated membrane recruitment of PREX1 and for stabilization of cytokine mRNAs and robust cytokine secretion. Together, our results identify a novel role for GRK2 as a target of TCR signaling that is responsible for TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that signals via PI3Kγ/PREX1 to mediate cytokine production. Therapeutic regulation of GRK2 or PI3Kγ may therefore be useful for limiting cytokines produced by T cell malignancies or autoimmune diseases.
Highlights
The immune system includes abundant examples of biologically-relevant cross-regulation of signaling pathways by the T cell antigen receptor (TCR) and the G protein– coupled chemokine receptor, CXCR4
Our results identify a novel role for G protein– coupled receptor kinase-2 (GRK2) as a target of TCR signaling that is responsible for TCR-induced transactivation of CXCR4 and TCR–CXCR4 complex formation that signals via PI3K␥/PREX1 to mediate cytokine production
We previously reported that the association of TCR with CXCR4 in response to ligation of the TCR is required for activation of a cytokine mRNA stabilization pathway that leads to robust production of IL-2 and IL-10 [12]
Summary
Be useful for limiting cytokines produced by T cell malignancies or autoimmune diseases. Upon TCR recognition of its cognate antigen/MHC ligand, TCR receptor-associated intracellular tyrosine kinases such as Src kinases and ZAP-70 are activated and initiate multiple signaling pathways that drive T cell proliferation and cytokine production [14]. We show that GRK2 is required for the TCR-mediated transactivation of CXCR4, mediating phosphorylation of the CXCR4 residue Ser339, which is required for TCR–CXCR4 complex formation Consistent with these results, we show that either depletion or inhibition of GRK2 significantly impaired the ability of T cells to produce high levels of IL-2 and IL-10 cytokines. PREX1 localization was inhibited using the PI3K␥ inhibitor IPI-549, and either inhibition or depletion of PI3K␥ significantly reduced TCR-mediated cytokine production Together, these results significantly expand understanding of the molecular mechanisms responsible for cross-regulation of CXCR4 and the TCR in T cells
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