Search

Fluorescence resonance energy transfer (FRET) has emerged as a powerful tool to the study of protein-protein interactions, such as receptor-ligand binding. However, the application of FRET to the study of G protein-coupled receptors (GPCRs) has been limited by the method of labeling receptor with fluorescence probes. Here we described a novel time-resolved (TR)-FRET method to study GPCR-ligand binding by using human complement 5a (C5a) receptor (C5aR) as a model system. Human C5aR was expressed in human embryonic kidney 293 cells with a hemagglutinin (HA) epitope at the N-terminus. Purified human C5a was labeled with terbium chelate and used as the fluorescence donor. Monoclonal anti-HA antibody conjugated with Alexa Fluor 488 was used as the fluorescence acceptor. Robust FRET signal was observed when the labeled ligand and C5aR membrane were mixed in the presence of the conjugated anti-HA antibody. This FRET signal was specific and saturable. C5a binding affinity to C5aR measured by the FRET assay was consistent with the data as determined by competition binding analysis using radiolabeled C5a. The FRET assay was also used to determine affinity of C5aR antagonists by competition binding analysis, and the data are similar to those from radioligand binding studies. Compared to the commonly used radioligand binding assay, this TR-FRET-based assay provides a nonradioactive, faster, and sensitive homogeneous assay format that could be easily adapted to high-throughput screening. The principle of this assay should also be applicable to other GPCRs, especially to those receptors with peptide or protein ligands.

Protein phosphorylation is one of the major regulatory mechanisms involved in signal-induced cellular events, including cell proliferation, apoptosis, and metabolism. Because many facets of biology are regulated by protein phosphorylation, aberrant kinase and/or phosphatase activity forms the basis for many different types of pathology. The disease relevance of protein kinases and phosphatases has led many pharmaceutical and biotechnology companies to expend significant resources in lead discovery programs for these two target classes. The existence of >500 kinases and phosphatases encoded by the human genome necessitates development of methodologies for the rapid screening for novel and specific compound inhibitors. We describe here a fluorescence-based, molecular assay platform that is compatible with robotic, ultra-high throughput screening systems and can be applied to virtually all tyrosine and serine/threonine protein kinases and phosphatases. The assay has a coupled-enzyme format, utilizing the differential protease sensitivity of phosphorylated versus nonphosphorylated peptide substrates. In addition to screening individual kinases, the assay can be formatted such that kinase pathways are re-created in vitro to identify compounds that specifically interact with inactive kinases. Miniaturization of this assay format to the 1-microl scale allows for the rapid and accurate compound screening of a host of kinase and phosphatase targets, thereby facilitating the hunt for new leads for these target classes.