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The Use of Spatial Intensity Distribution Analysis to Examine G Protein-Coupled Receptor Oligomerization

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G-Protein-Coupled Receptor Dimers

Part of the book series: The Receptors ((REC,volume 33))

Abstract

Spatial Intensity Distribution Analysis (SpIDA) is a new approach for detecting protein oligomerization states that can be applied not only to live cells but also fixed cells and native tissue. This approach is based on the generation of pixel-integrated fluorescence intensity histograms from laser scanning fluorescence microscopy images. These histograms are then fit with super-Poissonian distribution functions to obtain density maps and quantal brightness values of the fluorophore that are used to determine the proportions of monomer and dimers/oligomers of the fluorophore-tagged protein. In this chapter we describe SpIDA and highlight its advantages compared to other biochemical or biophysical approaches. We provide guidelines that should be useful to readers who wish to perform SpIDA measurements and describe the application of SpIDA as a post-acquisition imaging histogram analysis software tool to investigate the oligomeric state of G protein-coupled receptors (GPCRs) at the surface of mammalian cells in order to define the steady-state proportion of monomeric and dimeric/oligomeric forms and how this may be regulated by cellular challenges such as ligand treatment.

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Abbreviations

BG:

O6-BenzylGuanine

BRET:

Bioluminescence Resonance Energy Transfer

CD86:

Cluster of Differentiation Protein 86

EGF:

Epidermal Growth Factor

EGFR:

Epidermal Growth Factor Receptor

FCS:

Fluorescence Correlation Spectroscopy

FIDA:

Fluorescence Intensity Distribution Analysis

FRET:

Fluorescence Resonance Energy Transfer

GFP:

Green Fluorescent Protein

GPCR:

G-Protein Coupled Receptor

GUI:

Graphical User interface

hD2:

human Dopamine D2 receptor

hD3:

human Dopamine D3 receptor

hM1 :

human Muscarinic acetylcholine M1 receptor

hM3 :

human Muscarinic acetylcholine M3 receptor

IU:

Intensity Unit

mEGFP:

monomeric Enhanced Green Fluorescent Protein

MEU:

Monomeric Equivalent Unit

OX1 :

human Orexin 1 receptor

PCH:

Photon Counting Histogram

PM:

Parmitoylation + Myristolation

PMT:

Photo-Multiplier Tube

PSF:

Point Spread Function

QB:

Quantal Brightness

RET:

Resonance Energy Transfer

RoI:

Region of Interest

SpIDA:

Spatial Intensity Distribution Analysis

TIRF:

Total Internal Reflection Fluorescence

WN:

White Noise

5-HT2C :

5-Hydroxytryptamine 2C

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Author notes

  1. All authors contributed equally to this work

Authors and Affiliations

  1. Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK

    Richard J. Ward Ph.D., Sara Marsango Ph.D., John D. Pediani Ph.D. & Graeme Milligan Ph.D.

Authors
  1. Richard J. Ward Ph.D.
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  2. Sara Marsango Ph.D.
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  3. John D. Pediani Ph.D.
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  4. Graeme Milligan Ph.D.
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Corresponding author

Correspondence to Graeme Milligan Ph.D. .

Editor information

Editors and Affiliations

  1. Department of Neuroscience and Experimental Therapeutics, Albany Medical College, Albany, New York, USA

    Katharine Herrick-Davis

  2. Centre for Translational Pharmacology, Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom

    Graeme Milligan

  3. Department of Physiology and Biochemistry, University of Malta, Msida, Malta

    Giuseppe Di Giovanni

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Ward, R.J., Marsango, S., Pediani, J.D., Milligan, G. (2017). The Use of Spatial Intensity Distribution Analysis to Examine G Protein-Coupled Receptor Oligomerization. In: Herrick-Davis, K., Milligan, G., Di Giovanni, G. (eds) G-Protein-Coupled Receptor Dimers. The Receptors, vol 33. Humana Press, Cham. https://doi.org/10.1007/978-3-319-60174-8_2

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