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. 2015 Nov 3;109(9):1798-806.
doi: 10.1016/j.bpj.2015.09.004.

Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States

Sarah L Latty  1 , James H Felce  2 , Laura Weimann  1 , Steven F Lee  1 , Simon J Davis  3 , David Klenerman  4
Affiliations

Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States

Sarah L Latty et al. Biophys J. .

Abstract

The extent to which Rhodopsin family G-protein-coupled receptors (GPCRs) form invariant oligomers is contentious. Recent single-molecule fluorescence imaging studies mostly argue against the existence of constitutive receptor dimers and instead suggest that GPCRs only dimerize transiently, if at all. However, whether or not even transient dimers exist is not always clear due to difficulties in unambiguously distinguishing genuine interactions from chance colocalizations, particularly with respect to short-lived events. Previous single-molecule studies have depended critically on calculations of chance colocalization rates and/or comparison with unfixed control proteins whose diffusional behavior may or may not differ from that of the test receptor. Here, we describe a single-molecule imaging assay that 1) utilizes comparisons with well-characterized control proteins, i.e., the monomer CD86 and the homodimer CD28, and 2) relies on cell fixation to limit artifacts arising from differences in the distribution and diffusion of test proteins versus these controls. The improved assay reliably reports the stoichiometry of the Glutamate-family GPCR dimer, γ-amino butyric acid receptor b2, whereas two Rhodopsin-family GPCRs, β2-adrenergic receptor and mCannR2, exhibit colocalization levels comparable to those of CD86 monomers, strengthening the case against invariant GPCR oligomerization.

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Figures

Figure 1
Figure 1
Principle of referenced colocalization exemplified for CD86 and CD28 controls expressed in HEK-293T cells. (A) Representation of transfected HaloTag- (red) and SNAP-tag (green)-labeled monomeric CD86 protein. (B) Representative data obtained for CD86 in the green and red channels showing raw data (top row) and reconstructions of spot detection after application of the tracking algorithm (bottom row). Blue-boxed regions are shown magnified at far right. (C) HaloTag- (red) and SNAP-tag-labeled (green) dimeric CD28 protein. (D) Representative raw and reconstructed data obtained for CD28 showing higher levels of coincidence. Scale bars are 5 μm.
Figure 2
Figure 2
Referenced colocalization analysis differentiates between GPCR oligomerization states in HEK-293T cells. (A) Coincidence for CD28 and GABAbR2, but not mCannR2 or β2AR, is significantly larger (p > 0.05, two-tailed t-test) than that measured for CD86. Values are mean percentage coincidence ± SE for data from individual cells. (B) Representative images showing fluorescence of GPCRs expressed in HEK-293T cells, in the form of both raw data (top) and reconstructed data after spot detection (bottom). Equivalent images for CD86 and CD28 are given in Fig. 1. Red spots correspond to fluorescent HaloTag-labeled proteins and green spots to fluorescent SNAP-tag-labeled proteins. Scale bars are 5 μm.
Figure 3
Figure 3
Referenced colocalization analysis reveals that β2AR does not behave as a dimer in CHO-K1 cells. (A) The coincidence value for β2AR is not significantly larger than that measured for CD86. (B) Representative raw (top) and reconstructed (bottom) data obtained for HaloTag- (red) and SNAP-tag-labeled (green) CD86, CD28, and β2AR expressed in CHO-K1 cells. Scale bars are 5 μm.
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