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

doi:10.3791/54696

. 2016 Sep 10:(115):54696.

doi: 10.3791/54696.

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

Ansley Semack¹, Rabia U Malik², Sivaraj Sivaramakrishnan³

Affiliations

¹ Genetics, Cell Biology, and Development, University of Minnesota.
² Department of Cell and Developmental Biology, University of Michigan.
³ Genetics, Cell Biology, and Development, University of Minnesota; sivaraj@umn.edu.

PMID: 27684955
PMCID: PMC5092009
DOI: 10.3791/54696

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

Ansley Semack et al. J Vis Exp. 2016.

. 2016 Sep 10:(115):54696.

doi: 10.3791/54696.

Authors

Ansley Semack¹, Rabia U Malik², Sivaraj Sivaramakrishnan³

Affiliations

¹ Genetics, Cell Biology, and Development, University of Minnesota.
² Department of Cell and Developmental Biology, University of Michigan.
³ Genetics, Cell Biology, and Development, University of Minnesota; sivaraj@umn.edu.

PMID: 27684955
PMCID: PMC5092009
DOI: 10.3791/54696

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.

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References

1. Malik RU, et al. Detection of G Protein-selective G Protein-coupled Receptor (GPCR) Conformations in Live Cells. J. Biol. Chem. 2013;288:17167–17178. - PMC - PubMed
1. Oldham WM, Hamm HE. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat. Rev. Mol. Cell Bio. 2008;9:60–71. - PubMed
1. Rasmussen SG, et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature. 2011;477:549–555. - PMC - PubMed
1. Alexander NS, et al. Energetic analysis of the rhodopsin-G-protein complex links the α5 helix to GDP release. Nat. Struct. Mol. Biol. 2014;21(1):56–63. - PMC - PubMed
1. Conklin BR, Farfel Z, Lustig KD, Julius D, Bourne HR. Substitution of three amino acids switches receptor specificity of Gqα to that of Giα. Nature. 1993;363:274–276. - PubMed

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[1] Malik RU, et al. Detection of G Protein-selective G Protein-coupled Receptor (GPCR) Conformations in Live Cells. J. Biol. Chem. 2013;288:17167–17178. - PMC - PubMed

[2] Malik RU, et al. Detection of G Protein-selective G Protein-coupled Receptor (GPCR) Conformations in Live Cells. J. Biol. Chem. 2013;288:17167–17178. - PMC - PubMed

[3] Oldham WM, Hamm HE. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat. Rev. Mol. Cell Bio. 2008;9:60–71. - PubMed

[4] Oldham WM, Hamm HE. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat. Rev. Mol. Cell Bio. 2008;9:60–71. - PubMed

[5] Rasmussen SG, et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature. 2011;477:549–555. - PMC - PubMed

[6] Rasmussen SG, et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature. 2011;477:549–555. - PMC - PubMed

[7] Alexander NS, et al. Energetic analysis of the rhodopsin-G-protein complex links the α5 helix to GDP release. Nat. Struct. Mol. Biol. 2014;21(1):56–63. - PMC - PubMed

[8] Alexander NS, et al. Energetic analysis of the rhodopsin-G-protein complex links the α5 helix to GDP release. Nat. Struct. Mol. Biol. 2014;21(1):56–63. - PMC - PubMed

[9] Conklin BR, Farfel Z, Lustig KD, Julius D, Bourne HR. Substitution of three amino acids switches receptor specificity of Gqα to that of Giα. Nature. 1993;363:274–276. - PubMed

[10] Conklin BR, Farfel Z, Lustig KD, Julius D, Bourne HR. Substitution of three amino acids switches receptor specificity of Gqα to that of Giα. Nature. 1993;363:274–276. - PubMed

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

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

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