Date of Award
Doctor of Philosophy
Tim E. Sparer
Jeffrey Becker, Elias Fernandez, Todd Reynolds and Thandi Onami
Chemokines are structurally and functionally related 8-10 kDa proteins defined by four conserved cysteine residues. They consist of a superfamily of proinflammatory mediators that promote the recruitment of various kinds of leukocytes and other cell types through binding to their respective chemokine receptor, a member of the GPCR family. Abnormal control of this system results in various diseases including tumorigenesis and cancer metastasis. Deregulation can occur when constitutively active mutant (CAM) chemokine receptors are locked in the “on” position. This can lead to cellular transformation/tumorigenesis. A viral CAM receptor, ORF74, that can cause tumors in humans, also has homology to human CXC chemokine receptor 2 (CXCR2), which is a G-protein-coupled receptor (GPCR) expressed on neutrophils, some monocytes, endothelial cells, and some epithelial cells. CXCR2 activation with ELR+ CXC chemokines induces leukocyte migration, trafficking, cellular differentiation, angiogenesis and cellular transformation. Using a high throughput yeast screen we identified a novel point mutation, D9H, in CXCR2, which leads to constitutive activation (CA). Generation of positively charged substitutions, D9K and D9R, and D143V as a positive control resulted in CA CXCR2 with differential levels of cellular transformation. To further investigate how D9 mutations lead to differential CA, we used inhibitors of known signal transduction pathways. Pertusiss toxin (PTX) sensitivity in foci formation assays demonstrated that D9R uses the Gi subunit like WTCXCR2 and D143V, while D9H and D9K do not. All CA receptors use the JAK pathway based on sensitivity to the inhibitor, AG490. Phosphorylation of PLC-beta 3 and sensitivity to the PLC-beta 3 inhibitor, U73122, implicates that mutant receptors such as D143V, D9H, D9K, and D9R utilize the Gq/11 subunit. Interestingly, D9R use both Gi and Gq/11 subunits. All of the CA receptors induced phosphorylation of the epidermal growth factor receptor (EGFR) indicating a transactivation between CXCR2 and EGFR. These data describe two novel and important findings. First, N-terminal CXCR2 controls activation and signaling using multiple G protein subunits to elicit downstream signaling. Second, our work supports the “functional selectivity” model for GPCR activation. That is, mimicking agonist activation, CA CXCR2 receptors have multiple conformational states that lead to differential activation.
Park, Giljun, "NOVEL CONSTITUTIVELY ACTIVE POINT MUTATIONS IN THE NH2 DOMAIN OF CXCR2 CAPTURE THE RECEPTOR IN DIFFERENT ACTIVATION STATES. " PhD diss., University of Tennessee, 2010.