A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases

Abstract

Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-β2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-β2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases.

FIGURE 1.

CXCR2 preferentially interacts with NHERF1 in neutrophils. A, HA-tagged human CXCR2 (overexpressed in HEK293 cells) was pulled down by PDZ scaffold proteins (NHERF1, NHERF2, and PDZK1). The membrane was blotted with anti-HA monoclonal antibody. B, His-S-tagged murine CXCR2 (overexpressed in HEK293 cells) was pulled down by the indicated PDZ scaffold proteins. The membrane was blotted with anti-mouse CXCR2 monoclonal antibody. C, NHERF1 and NHERF2 bound to endogenous CXCR2 from human neutrophils. The membrane was immunoblotted with anti-human CXCR2 monoclonal antibody. D, NHERF1 bound to endogenous CXCR2 from murine bone marrow neutrophils. Cell lysates of HEK293 cells that overexpressed 3HA-murine CXCR2 were loaded as a positive control. The membrane was immunoblotted with anti-murine CXCR2 monoclonal antibody. E, NHERF1 and NHERF2 bound to endogenous CXCR2 from neutrophil-like cells, dHL-60 cells. The membrane was immunoblotted with anti-human CXCR2 monoclonal antibody.

FIGURE 2.

PLC-β isoforms physically interact with PDZ scaffold proteins, whereas PLC-β2 in neutrophils preferentially interacts with NHERF1. A–C, HEK293 cells were overexpressed with FLAG-tagged PLC-β1 (A), PLC-β2 (B), and PLC-β3 (C), and the GST pulldown assays were performed with PDZ scaffold proteins as described under “Experimental Procedures.” The membranes were immunoblotted with anti-PLC-β1 (A), PLC-β2 (B), and PLC-β3 (C) monoclonal antibodies, respectively. Purified His-S-PLC-β1 (20 ng) was loaded as positive control for the PLC-β1 antibody (A). D and E, endogenous PLC-β2 from neutrophils freshly isolated from mouse bone marrow (D), or from neutrophil-like cells, dHL-60 cells (E), was pulled down by PDZ scaffold proteins. The membranes were immunoblotted with anti-PLC-β2 monoclonal antibody. Purified His-S-PLC-β2 was loaded as positive control for the PLC-β2 antibody.

FIGURE 3.

The interaction between NHERF1 and CXCR2 or PLC-β2 is PDZ motif-dependent. A, pairwise binding between GST-NHERF1 and His-S-tagged human CXCR2 C-tail (containing the last 45 amino acids) WT, PDZ motif deletion (ΔTTL), or PDZ motif mutants (ATA and AAA). The complex was pulled down by S-protein-agarose and immunoblotted with anti-NHERF1 IgG. Purified GST or GST-NHERF1 (20 ng) were loaded as negative or positive control for the anti-NHERF1 antibody, respectively. B, pairwise binding between GST-NHERF1 and His-S-tagged mouse CXCR2 C-tail (containing last 45 amino acids) with (WT) or without PDZ motif (ΔTTL). The complex was pulled down by the S-protein-agarose and immunoblotted with anti-NHERF1 IgG. C, pairwise binding between GST-NHERF1 and His-S-tagged PLC-β2 C-tail (containing last 100 amino acids) WT, PDZ motif deletion (ΔSRL), or mutation (ARA and AAA). The complex was pulled down by S-protein-agarose and immunoblotted with anti-NHERF1 IgG.

FIGURE 4.

CXCR2, NHERF1, and PLC-β2 form a macromolecular complex in vitro and in neutrophils. A, schematic representation of in vitro macromolecular complex assembly (upper panel, refer to “Experimental Procedures” for details). Macromolecular complex of PLC-β2 C-tail, PDZ scaffold proteins, and human full-length CXCR2 (lower panel) are shown. B, macromolecular complex of PLC-β2 full-length, PDZ scaffold proteins, and mouse CXCR2 C-tail. C, dose-dependent (GST-NHERF1) macromolecular complex formation of His-S-tagged PLC-β2 C-tail, GST-NHERF1, and HA-tagged human CXCR2. D, endogenous PLC-β2 and NHERF1 were co-precipitated with CXCR2 from murine bone marrow neutrophils.

FIGURE 5.

A CXCR2 C-tail-specific peptide disrupts the physical interaction between NHERF1 and CXCR2. A, binding of His-S-NHERF1 to 3HA-human CXCR2 in the presence of human CXCR2 C-tail WT peptide on a Far Western blot. The membrane was immunoblotted with HRP-conjugated S-protein, which detects S-tag within His-S-NHERF1. B, GST-NHERF1 binding to His-S-tagged mouse CXCR2 C-tail (WT) in the presence of mouse CXCR2 C-tail WT peptide in pairwise binding as described in the legend to Fig. 3B.

FIGURE 6.

Disrupting the CXCR2 macromolecular complex inhibits CXCR2 ligand-induced calcium mobilization in neutrophils. Intracellular calcium mobilization induced by MIP-2 (100 ng/ml) in mouse bone marrow-derived neutrophils pre-delivered with mouse CXCR2 WT or ΔTTL peptides. **, p < 0.01 compared with the control (no peptide).

FIGURE 7.

Disrupting the CXCR2 macromolecular complex attenuates the CXCR2 ligand-induced neutrophil chemotaxic migration. A, chemotaxic migration of mouse PMNs in response to CXCR2 ligands (MIP-2 and IL-8; both 50 ng/ml) pretreated with or without various CXCR2 C-tail peptides. *, p < 0.05; **, p < 0.01, compared with the control (no peptide). B, migration of dHL-60 cells (human PMN-like granulocytes) pre-delivered with various CXCR2 C-tail peptides when the cells were stimulated by IL-8 (CXCL8; 50 ng/ml), GROα (CXCL1; 50 ng/ml), or fMLP (1 μm). **, p < 0.01, compared with the control (no peptide).

FIGURE 8.

Disrupting the CXCR2 macromolecular complex inhibits CXCR2 ligand-induced neutrophil transepithelial migration. A, IL-8-induced dHL-60 cell transepithelial migration across HT-29 cell monolayers. dHL-60 cells (3 × 10⁶) were pre-delivered with various human CXCR2 C-tail peptides as indicated, and the cells that migrated into the lower chamber at 2 h were quantified by a colorimetric MPO assay. **, p < 0.01 compared with control (no peptide). B, transepithelial migration of dHL-60 cells across HT-29 monolayers induced by fMLP. dHL-60 cells (3 × 10⁶) were pre-delivered with various human CXCR2 C-tail peptides as indicated, and the transmigration was quantified by MPO activity assay.

FIGURE 9.

Proposed mechanism of the NHERF1-mediated coupling of CXCR2 to PLC-β2 signaling in neutrophils. Both CXCR2 and PLC-β2 directly interact with the PDZ scaffold protein NHERF1. NHERF1 clusters CXCR2 and PLC-β2 in close proximity via a PDZ-based interaction, thereby forming spatially compact signaling complexes beneath the plasma membrane. Consequently, the macromolecular signaling complex (CXCR2·NHERF1·PLC-β2) enables CXCR2 to transduce its signal to PLC-β2 with efficiency and specificity.