G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

Overview

Journal J Vis Exp

Specialties Biology
General Medicine

Date 2016 Sep 30

PMID 27684955

Citations 4

Authors

Ansley Semack

Rabia U Malik

Sivaraj Sivaramakrishnan

Affiliations

Soon will be listed here.

Abstract

Fӧrster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between Gα subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the β2-adrenergic receptor and the Gαs C-terminus peptide upon treatment with 100 µM isoproterenol hydrochloride as previously characterized(1). Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a Gα C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Citing Articles

Bayesian network models identify co-operative GPCR:G protein interactions that contribute to G protein coupling.

Mukhaleva E, Ma N, van der Velden W, Gogoshin G, Branciamore S, Bhattacharya S bioRxiv. 2023; .

PMID: 37873104 PMC: 10592737. DOI: 10.1101/2023.10.09.561618.

Dynamic spatiotemporal determinants modulate GPCR:G protein coupling selectivity and promiscuity.

Sandhu M, Cho A, Ma N, Mukhaleva E, Namkung Y, Lee S Nat Commun. 2022; 13(1):7428.

PMID: 36460632 PMC: 9718833. DOI: 10.1038/s41467-022-34055-5.

Conformational plasticity of the intracellular cavity of GPCR-G-protein complexes leads to G-protein promiscuity and selectivity.

Sandhu M, Touma A, Dysthe M, Sadler F, Sivaramakrishnan S, Vaidehi N Proc Natl Acad Sci U S A. 2019; 116(24):11956-11965.

PMID: 31138704 PMC: 6575595. DOI: 10.1073/pnas.1820944116.

ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells.

Malik R, Dysthe M, Ritt M, Sunahara R, Sivaramakrishnan S Sci Rep. 2017; 7(1):7749.

PMID: 28798477 PMC: 5552854. DOI: 10.1038/s41598-017-08029-3.

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