A rapid and efficient immunoenzymatic assay to detect receptor protein interactions : G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-thofd the current therapeutic drugs on the market. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs), which take part in receptor proper folding, targeting to the appropriate subcellular compartments and in receptor signaling task and also in receptor regulation process,esuch as desensitization and internalizoni. The direction of protein-protein interactions and multi-protein complexes formation is crucial in understanding protein function and their implication in pathological. Although several methods have been already developed to assay protein compl s, some of them are quite laborious, expensive, and, more important, they do not generate fully quantitative results. Herein, we show a rapid immunoenzymatic assay to quantify GPCR interactionswith its signaling proteins. The recently -odrphanized GPCR, GPR17, was chosen as a GPCR prototype to optimize the assay. In a GPR17 transfected cell line and primary oligodendrocyte precursor cells, GPR17 interaction withp roteins involved in the typical GPCR regulation, such as desensitization and internalizationmachinery, was investigated. The obtained results were validated by immunoprecipitation experiments, confirming this new method as a rapid and quantitative assay to study protein-protein interactions.