Abstract
G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-third of 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 tasks, and also in receptor regulation processes, such as desensitization and internalization. The direction of protein-protein interactions and multi-protein complexes formation is crucial in understanding protein function and their implication in pathological events. Although several methods have been already developed to assay protein complexes, 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 de-orphanized 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 with proteins involved in the typical GPCR regulation, such as desensitization and internalization machinery, was investigated. The obtained results were validated by co-immunoprecipitation experiments, confirming this new method as a rapid and quantitative assay to study protein-protein interactions.
Highlights
G-Protein-coupled receptors (GPCRs) are members of a family of transmembrane proteins that mediate the transmission of a wide range of extracellular signals to the intracellular space, and initiate signalling mechanisms that regulate multiple cellular processes [1]
GPCRs translate extracellular stimuli into intracellular signals, and the intensity and duration of these are determined by complex regulation mechanisms [3], which imply the physical interaction of GPCRs with regulatory proteins such as G protein-coupled receptor kinases (GRKs) and β-arrestins [4,5,6]
In order to optimize and validate the ELISA assay, the recently deorphanized GPCR, GPR17 [12], was chosen a GPCR prototype. Both a transfected cell line and primary oligodendrocyte precursor cells (OPCs) which natively express the receptor at high levels [13], were used: in these cell models, we investigated GPR17 interactions with proteins involved in the typical GPCR desensitization and internalization machinery, upon receptor stimulation with its putative ligands [12,14,15]
Summary
G-Protein-coupled receptors (GPCRs) are members of a family of transmembrane proteins that mediate the transmission of a wide range of extracellular signals to the intracellular space, and initiate signalling mechanisms that regulate multiple cellular processes [1]. Estimates of 40%–80% false negatives and 30%–60% false positives have been assigned to high-throughput studies that have used two-hybrid techniques, affinity based techniques or computational approaches [9,10,11] Some of these methods are quite laborious, expensive, and do not generate fully quantitative results. We describe the development of a immunoenzymatic assay to detect GPCR interactions with its regulatory proteins This new method allows to skip the time-consuming steps of co-immunoprecipitation assay, and to obtain a quantitative measure of the protein-protein interaction. ELISA assay can be applied to every GPCR, in the study of the interaction with signaling proteins, as well as with other GPCRs (heterodimers)
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