Biophysics Seminar

Week of Monday, September 17th 2018


Thursday, September 27th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Keehun Kim, graduate student in Sivaraj Sivaramakrishnan’s lab, Department of Genetics, Cell Biology, and Development, University of Minnesota
Subject: A cell-free FRET-based assay for profiling the intrinsic efficacy of GPCR ligands

G protein coupled receptors (GPCRs) are 7-helix transmembrane receptors which regulate diverse intracellular signaling cascades in response to extracellular stimuli. Due to their crucial role in modulating most human physiological processes, roughly 25% of the pharmaceutical in the current market target GPCRs. The intrinsic efficacy of GPCR ligands is an essential parameter in operational models that convert ligand binding to downstream response. Measurement of intrinsic efficacy of GPCR ligands has eluded researchers, given the diversity of factors that can modulate downstream signaling, including expression levels of GPCR, G protein-dependent and independent effectors, regulatory factors such GRKs, RGS, and post-translational modifications. While ligand efficacy can be directly measured from G protein activation rates, such assays either require significant amounts of purified GPCR and G protein or are limited by the heterogeneity inherent in crude membrane preparations. We have previously used FRET based SPASM sensors to measure interactions between the full-length GPCR and the C-terminus of the α5-helix of distinct Gα subunits. We have demonstrated the utility of these sensors in live cells to probe the stabilization of G protein-selective receptor conformations by distinct GPCR ligands. While the SPASM GPCR sensors robustly report ligand-dependent G protein selection, the live cell FRET measurements are limited by constraints on sensor expression levels and ligand-stimulation times. Here, we present a cell derived assay for SPASM GPCR sensors that provides highly reproducible FRET measurements with minimal sensitivity to sensor expression levels and ligand-stimulation times. Importantly, our cell-free assay provides a scalable and stable reagent for screening GPCR ligands. Our findings for the b2 adrenergic receptor show that the GPCR-Gα C-terminus interaction is sufficient to recapitulate the intrinsic efficacy as measured from G protein activation rates. Taken together, our cell-free assay provides a scalable FRET-based readout for the intrinsic efficacy of GPCR ligands, while reinforcing the structural mechanisms underlying G protein activation.

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