Assessing Protein Interactions in Cells by Fluorescence

DESCRIPTION (provided by applicant): Interleukin-10 (IL-10) is a dominant molecule in anti-inflammatory and immunosuppressive pathways. The receptor for IL-10 consists of two chains, IL-10R1 and IL-10R2. Despite a good deal of knowledge about the receptor components and signal transduction, no direct physical evidence has provided insight into the structure of the receptor on the cell surface in the presence and absence of ligand. By fusing blue and green fluorescent proteins (BFP, GFP and/or YFP/CFP) to the receptor chains and expressing them in cells, these fluorophores could be used to measure directly the interactions of the receptor chains on the cell surface in response to IL-10 by fluorescence resonance energy transfer (FRET). The specific aims are: 1) determine if the receptor chain pairs (specifically the R1 and R2 chains of the Interlerkin-10 receptor) are pre-associated in the absence of ligand; 2) determine changes in receptor structure after engagement of ligand; 3) determine whether homotypic receptor chains are pre-assembled in the presence and absence of heterotypic chain and ligand, and 4) examine the potential for cross-talk between the IL-10 and IFN-gamma receptor complexes. The efficient accomplishment of these aims will not only provide a detailed understanding the Interleukin-10 receptor complex, but also will have ramifications for delineating the structure and dynamics of other receptors and for analyzing protein-protein interactions, ligand-receptor interactions, signal transduction and many other cellular processes in live cells. Furthermore, questions relevant to protein interactions in cells that have been addressed indirectly should be able to be answered definitively with direct measurements by FRET and the time course of the events determined. In addition, applications to high throughput screening could be extensive not only for ligand-receptor interactions, but also for many cellular processes. This work has particularly strong potential to provide unique insights into the structural determinants that specifically underlie IL-10 receptor assembly, ligand binding, and signal transduction. IL-10 and the IL-10 receptor comprise a critical control point for a variety of anti-inflammatory and immunosuppressive responses, the importance of which is underscored by the existence of IL-10 homologous sequences in certain viral genomes. The basic knowledge provided by this investigation coupled with the development of this FRET technology into a high-throughput screening approach for IL-10 receptor ligands may permit the discovery of novel peptide and non-peptide therapies for septic shock, graft survival, allergic inflammation, inflammatory bowel disease, psoriasis, and rheumatoid arthritis, among others.