Regulation of CXC Chemokine Receptor Function Through Intracellular Trafficking and Novel Receptor-Interacting Proteins

Abstract

Chemokines are a family of small chemotactic cytokines that bind seven transmembrane G protein-coupled receptors. Roles for CXC chemokines and the receptors CXCR2 and CXCR4 in inflammation-mediated tumorigenesis, angiogenesis, and metastasis have established these proteins as anti-cancer therapeutic targets. Therefore, understanding the biology of chemokines and their receptors is crucial for the development of novel therapeutics. We hypothesized that the cellular responses elicited by chemokines are in part regulated through the internalization and intracellular trafficking of chemokine receptors through small GTPases and by novel protein complexes that interact with the cytoplasmic domains of the receptors. We used three approaches to investigate this hypothesis: 1) We investigated the role of the small GTPase RhoB in the intracellular trafficking of CXCR2. Experimental results indicate that the oscillation of the RhoB GTPase activity is essential for appropriate sorting decisions and for directing CXCR2 degradation and recycling, events that are required for optimal chemotaxis. 2) A proteomics approach for the identification of novel ligand-dependent CXC chemokine receptor-interacting proteins was utilized. This approach led to the identification of a number of CXCR2-interacting proteins, including the scaffolding protein IQGAP1 and the actin cytoskeletal modifying protein VASP which may link CXCR2 to major signaling pathways and the actin cytoskeleton. Our studies also describe a direct, phosphorylation-dependent interaction between CXCR2 and VASP and suggest that VASP plays a critical role in CXCR2-mediated leukocyte recruitment in vivo as VASP -/- mice exhibit impaired responses. In addition, a direct interaction of CXCR2 with the amino-terminus of IQGAP1 was identified. CXCL8 stimulation was further shown to regulate IQGAP1 association with the small GTPase Cdc42, which plays a major role in the establishment of cell polarity. 3) The role of the carboxyl-terminus of CXCR4 in breast cancer tumorigenesis was also investigated. We found that the truncated CXCR4 receptor constitutively activates signaling pathways due to enhanced ligand-independent recycling. As a result, this truncated receptor leads to enhanced motility, proliferation, and metastasis of breast cancer cells. Taken together, our data demonstrate the critical importance of the proper intracellular trafficking of CXC chemokine receptors and of its interacting proteins in mediating biological responses elicited by chemokines.