Screening for novel constitutively active CXCR2 mutants and their cellular effects.
Chemokines play an important role in inflammatory, developmental, and homeostatic processes. Deregulation 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. The CXC chemokine receptor 2 (CXCR2) is a G-protein-coupled receptor (GPCR) expressed on neutrophils, some monocytes, endothelial cells, and some epithelial cells. CXCR2 activation with CXC chemokines induces leukocyte migration, trafficking, leukocyte degranulation, cellular differentiation, and angiogenesis. Activation of CXCR2 can lead to cellular transformation. We hypothesized that CAM CXCR2s may play a role in cancer development. In order to identify CXCR2 CAMs, potential mutant CXCR2 receptors were screened using a modified Saccharomyces cerevisiae high-throughput system. S. cerevisiae has been used successfully to identify GPCR/G-protein interactions and autocrine selection for peptide agonists. The CXCR2 CAMs identified from this screen were characterized in mammalian cells. Their ability to transform cells in vitro was shown using foci formation, soft-agar growth, impedance measurement assays, and in vivo tumor growth following hind flank inoculation into mice. Signaling pathways contributing to cellular transformation were identified using luciferase reporter assays. Studying constitutively active GPCRs is an approach to "capturing" pluridimensional GPCRs in a "locked" activation state. In order to address the residues necessary for CXCR2 activation, we used S. cerevisiae for screening novel CAMs and characterized them using mammalian reporter assays.
The University of Tennessee, Department of Microbiology, Knoxville, Tennessee, USA.
Giljun
G
Park
Tom
T
Masi
Chang K
CK
Choi
Heejung
H
Kim
Jeffrey M
JM
Becker
Tim E
TE
Sparer
D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D017097:Electric Impedance; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D016296:Mutagenesis; D009154:Mutation; D041681:NIH 3T3 Cells; D009369:Neoplasms / Q000473:pathology; D023063:Receptors, Interleukin-8B / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection / Q000379:methods
D023063:Receptors, Interleukin-8B; D008156:Luciferases