To identify biologically relevant compounds in basic biology and drug discovery processes, rapid quantitative methods for elucidating protein-protein interactions have become necessary. We describe a novel optical technique for monitoring protein-protein interactions in living cells based on complementation of split luciferase fragments from click beetle (Brazilian Pyrearinus termitilluminans). A new pair of amino-terminal and carboxy-terminal fragments of the luciferase was identified using semirational library screening, demonstrating achieved markedly higher sensitivity and signal-to-background ratio. The identified fragments were applied to the study of five G-protein coupled receptors (GPCR) that interact with beta-arrestin on the plasma membrane. By generating cell lines stably expressing the GPCRs and beta-arrestin connected with the luciferase fragments, we demonstrated rapid and sensitive screening of potential chemicals that act on GPCRs using a 96-well microtiter plate format. The screening time was reduced to 5-10 min after ligand stimulation. The maximum response became more than 15-fold higher than the background signal. This luciferase complementation method also enabled accurate spatial and temporal analyses of interactions in single living cells using bioluminescence microscopy. These GPCR assays will facilitate developments of high-throughput screening systems in a multiwell plate format. Furthermore, using specific proteins of interest, the novel fragments of luciferase will provide different assay methods for the study of many intracellular signals in living cells and animals.

Previous studies have shown that oxidized products of the phospholipid PAPC (Ox-PAPC) are strong activators of aortic endothelial cells and play an important role in atherosclerosis and other inflammatory diseases. We and others have demonstrated that Ox-PAPC activates specific signaling pathways and regulates a large number of genes. Using a phosphoproteomic approach based on phosphopeptide enrichment and mass spectrometry analysis, we identified candidate changes in Ox-PAPC-induced protein phosphorylation of 228 proteins. Functional annotation of these proteins showed an enrichment of the regulation of cytoskeleton, junctional components, and tyrosine kinases, all of which may contribute to the phenotypic and molecular changes observed in endothelial cells treated with Ox-PAPC. Many changes in protein phosphorylation induced by Ox-PAPC are reported here for the first time and provide new insights into the mechanism of activation by oxidized lipids, including phosphorylation-based signal transduction.