FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells

Abstract

The lateral organization of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R), was investigated in living cells by fluorescence resonance energy transfer (FRET) microscopy, taking advantage of the recently developed acyl carrier protein (ACP) labeling technique. The NK1R was expressed as fusion protein with ACP to which small fluorophores were then covalently bound. Our approach allowed the recording of FRET images of receptors on living cells with unprecedented high signal-to-noise ratios and a subsequent unequivocal quantification of the FRET data owing to (i) the free choice of optimal fluorophores, (ii) the labeling of NK1Rs exclusively on the cell surface, and (iii) the precise control of the donor-acceptor molar ratio. Our single-cell FRET measurements exclude the presence of constitutive or ligand-induced homodimers or oligomers of NK1Rs. The strong dependence of FRET on the receptor concentration further reveals that NK1Rs tend to concentrate in microdomains, which are found to constitute approximately 1% of the cell membrane and to be sensitive to cholesterol depletion.

Fig. 1.

Controlled double-labeling of ACP–NK1R. (A and B) Fluorescence confocal micrographs show a HEK293 cell transiently expressing ACP–NK1Rs, which were labeled simultaneously with Cy3 and Cy5 at a Cy3 mole fraction of 0.67. The cell was imaged by using particular filter sets for donor (A) and acceptor (B). (Scale bar: 10 μm.) (C) The donor mole fraction observed on the cells, x_D,cell, as a function of the donor mole fraction used for the labeling, x_D,sol. x_D,sol is identical to x_D,cell within experimental error as shown by a linear regression yielding an intercept of 0.035 ± 0.073 and a slope of 0.94 ± 0.12. Each point is the mean (±SD) of 10 cells.

Fig. 2.

Dependence of the FRET efficiency on the donor mole fraction. HEK293 cells expressing ACP–NK1Rs were labeled with a mixture of Cy3 and Cy5 fluorophores at different donor mole fractions x_D. The apparent FRET efficiency, E_app,se, is plotted vs. x_D for two populations of cells: Gray circles represent transiently expressing cells; black circles stem from stably expressing cells showing a 2.5 times lower expression. The expression level was similar for all investigated cells in a population. Each point is a mean (±SD) of 10 cells. Fitting the data with Eq. 8 plus an offset (solid curves) yields for the higher expressing cells offset = −0.022 ± 0.030 (i.e., zero within the experimental accuracy), E = 0.26 ± 0.09, n = 3.8 ± 1.3, and for the lower expressing cells with E fixed to 0.26 offset = 0.018 ± 0.047, n = 1.09 ± 0.28. The dotted curve represents a plot of E_app,se calculated by using Eq. 12 in its range of validity, with α = 81, σ_R = 256 receptors per μm², and x_D as an independent variable.

Fig. 3.

FRET dependence on cell surface receptor density. (A) HEK293 cells transiently expressing the ACP–NK1R were labeled at a donor mole fraction of x_D = 0.67. The apparent FRET efficiency, E_app,se (gray circles), is plotted as a function of the reduced acceptor concentration, c_A (bottom), and the receptor surface density, σ_R (top). Each data point represents an image containing one to three cells. The bold dotted curve at the bottom represents E_app,se calculated with Eq. 12 assuming the absence of microdomains (α = 1; coefficients A_1;2 and k_1;2 taken for a R_c/R₀ ratio of 1.1; see Theory). The fine dotted horizontal line represents the maximal apparent efficiency determined from sensitized acceptor emission: E_app,se,max = 0.626 ± 0.042 (mean of the last 17 points ± SD). The two vertical dashed lines represent the receptor surface densities for the data in Fig. 2. The solid curve is a fit over the first 47 points using Eq. 12 with only one free parameter α = 81.1 ± 0.3. The dotted curves represent E_app,se calculated from Eq. 12 with α = 81 ± 50%. (B) Modulation of the apparent FRET efficiency by cholesterol extraction. FRET efficiency of transiently expressed ACP–NK1Rs labeled with x_D = 0.6, before (0.34 ± 0.048) and after (0.24 ± 0.076) cholesterol extraction with 2% (wt/vol) methyl-β-cyclodextrin for 45 min at room temperature (mean of 9 cells ± SE; Student’s t test P ≤ 0.005).