The CXC chemokine receptor 2 (CXCR2) is a G protein coupled receptor mediating interleukin-8 chemotactic signaling and plays an important role in neutrophil mobility and tumor migration. However, efficient CXCR2 signaling requires PDZ domain-mediated scaffolding of signaling complexes at the plasma membrane and functional coupling of the signaling to specific downstream signaling pathways, in which only one PDZ protein has been characterized to interact with CXCR2. Here, we identified five novel CXCR2-binding PDZ-containing proteins, among which PDZ-RhoGEF is of particular interest because this PDZ and RGS-containing guanine nucleotide exchange factor (GEF) is also involved in cell signaling and mobility. To reveal the molecular basis of the interaction, we solved the crystal structure of PDZ-RhoGEF PDZ domain in complex with the CXCR2 C-terminal PDZ binding motif. The structure reveals that the PDZ–CXCR2 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four CXCR2 residues contributing to specific interactions. Structural comparison of CXCR2-binding PDZ domains and PDZ-RhoGEF PDZ bound with different ligands reveals PDZ- and ligand-specific interactions that may underlie the ability of promiscuous CXCR2 binding by different PDZ domains and PDZ binding promiscuity. The structure also reveals an unexpected asymmetric disulfide bond-linked PDZ dimer that allows simultaneous parallel binding of CXCR2 to two PDZ domains. This study provides not only the structural basis for PDZ-mediated CXCR2–PDZ-RhoGEF interaction, but also a new mode of PDZ dimerization, which both could prove valuable in understanding signaling complex scaffolding in CXCR2 signaling and coupling to specific signaling pathways.
Eukaryotic cellular mobility is critically dependent on actin organization and remodeling that are masterfully governed through the Rho signaling pathway activated by Rho guanine exchange factors (RhoGEF). PDZ‐RhoGEF is one unique member of the RhoGEF family which possesses a PDZ domain that is known to nucleate cell surface factors to perpetuate signaling for actin organization and cell mobility. However, the role of the PDZ domain of PDZ‐RhoGEF in signaling transduction pathways for cellular mobility is still not well understood. In this study, we identified PDZ‐RhoGEF binds directly to a novel binding partner CXCR2, a G protein coupled receptor which is known to initiate the Rho signaling program for chemotaxis, haptotaxis, cellular growth and tumor invasion. In order to understand the molecular mechanism of binding, we solved the crystal structure of the chimeric complex between the PDZ domain of PDZ‐RhoGEF and the PDZ‐binding motif of CXCR2. This complex structure reveals that the CXCR2 peptide forms an antiparallel β‐sheet against PDZ‐RhoGEF with the carboxyl‐terminal leucine packaging into a deep hydrophobic pocket within the PDZ domain. Unexpectedly, we found a disulfide bond linking the PDZ domain dimer which secures a parallel binding mode of the CXCR2 ligands to the PDZ monomers. To our knowledge, this is the first PDZ dimer structure covalently linked through a disulfide bond and may be a unique feature to PDZ‐RhoGEF family. This new type of dimerization may be important for stabilizing the CXCR2 macromolecular complex and thereby provide swift signaling for cellular mobility and cancer invasion.Support or Funding InformationNational Heart, Lung, and Blood Institute
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