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. 2012 Jul 6;287(28):23604-14.
doi: 10.1074/jbc.M112.350249. Epub 2012 May 16.

Oligomer size of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor revealed by fluorescence correlation spectroscopy with photon counting histogram analysis: evidence for homodimers without monomers or tetramers

Katharine Herrick-Davis  1 , Ellinor Grinde, Tara Lindsley, Ann Cowan, Joseph E Mazurkiewicz
Affiliations

Oligomer size of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor revealed by fluorescence correlation spectroscopy with photon counting histogram analysis: evidence for homodimers without monomers or tetramers

Katharine Herrick-Davis et al. J Biol Chem. .

Abstract

Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) are techniques with single molecule sensitivity that are well suited for examining the biophysical properties of protein complexes in living cells. In the present study, FCS and PCH were applied to determine the diffusion coefficient and oligomeric size of G-protein-coupled receptors. FCS was used to record fluctuations in fluorescence intensity arising from fluorescence-tagged 5-hydroxytryptamine 2C (5-HT(2C)) receptors diffusing within the plasma membrane of HEK293 cells and rat hippocampal neurons. Autocorrelation analysis yielded diffusion coefficients ranging from 0.8 to 1.2 μm(2)/s for fluorescence-tagged receptors. Because the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins traveling together within a protein complex, it can be used to determine the oligomeric size of the protein complex. FCS and PCH analysis of fluorescence-tagged 5-HT(2C) receptors provided molecular brightness values that were twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5-HT. Bimolecular fluorescence complementation of the N- and C-terminal halves of YFP attached to 5-HT(2C) receptors was observed in endoplasmic reticulum/Golgi and plasma membranes with a brightness equal to monomeric YFP. When GFP-tagged 5-HT(2C) receptors were co-expressed with a large excess of untagged, non-fluorescent 5-HT(2C) receptors, the molecular brightness was reduced by half. PCH analysis of the FCS data were best described by a one-component dimer model without monomers or tetramers. Therefore, it is concluded that 5-HT(2C) receptors freely diffusing within the plasma membrane are dimeric.

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Figures

FIGURE 1.
FIGURE 1.
Confocal microscopy of HEK293 cells transfected with BiFC constructs. A, the N- and C-terminal halves of YFP were attached to the C-terminal end of 5-HT2C receptors and co-transfected (5-HT2C/N-YFP + 5-HT2C/C-YFP) into HEK293 cells. Plasma membrane YFP fluorescence, 20 h post-transfection, is shown in the first panel. The second panel shows the differential interference contrast microscopy image, and the third panel shows the fluorescence overlay on this image. Red scale bar, 10 μm. B, HEK 293 cells co-transfected with 5-HT2C/N-YFP + β2-adrenergic/C-YFP, imaged 20 h post-transfection. C, HEK293 cells co-transfected with β2-adrenergic/N-YFP + β2-adrenergic/C-YFP, imaged 20 h post-transfection. D, HEK293 cells co-transfected with 5-HT2C/N-YFP + 5-HT2C/C-YFP. Sixteen hours post-transfection, YFP fluorescence is observed in the endoplasmic reticulum/Golgi.
FIGURE 2.
FIGURE 2.
Confocal microscopy of an isolated rat hippocampal neuron transfected with 5-HT2C/YFP and maintained in culture for 1 day. The inset shows the upper plasma membrane of the cell body and the location (+) where an FCS recording was made. Red scale bar, 10 μm.
FIGURE 3.
FIGURE 3.
FCS recordings from the plasma membrane of hippocampal neurons expressing 5-HT2C/YFP and HEK293 cells expressing 5-HT2C/GFP or 5-HT2C/mCherry. A, fluorescence intensity traces for one 10-s observation period. B, biphasic FCS autocorrelation curves, generated from the intensity traces shown above. The blue line represents the autocorrelation of the observed fluorescence, and the green line represents the fit to a two-component model (for 5-HT2C/mCherry the colors are red and green, respectively). The fast component (measured in microseconds) is related to the photophysical properties of the fluorescent probe, whereas the slower component (measured in milliseconds) represents the diffusion of fluorescence-tagged receptors in the plasma membrane. The diffusion rates are calculated from the midpoint of the decay of the autocorrelation curve. C, residuals of the curve fit from the autocorrelation analysis. Dividing the average photon count rate (kHz) determined from the fluorescence intensity trace shown in A by the number of fluorescent molecules determined from the autocorrelation curve shown in B (calculated as in Equation 3, where G(0) = the y intercept at the 1 μs time point) predicts the average molecular brightness of the sample expressed as CPSM.
FIGURE 4.
FIGURE 4.
Photon counting histograms from FCS recordings made in the cytosol of HEK293 cells expressing a tandem, GFP-GFP dimeric construct (A) and the plasma membrane of HEK293 cells expressing 5-HT2C/GFP receptors (B). The inset shows the PCH on a linear scale. To generate a histogram, each 10-s fluorescence intensity trace (as illustrated in Fig. 3A) was broken down into 1 million 10-μs intervals or bins (PCH bin time = 10 μs). Histograms were constructed (using the PCH module in the Zeiss Aim 4.2 software) in which the number of 10-μs bins was plotted on the y axis, and photon counts were plotted on the x axis. The resulting histogram depicts the number of bins that registered 1,2,3 … n photon counts during one 10-s observation period. The histograms show the average number of photon counts per 10-μs bin time to be 1.24, equivalent to 124,000 counts/s. Dividing by the average number of molecules in the observation volume (7) yields an average molecular brightness of 17,714 CPSM. The residuals of the curve fit (shown in the lower panels) plot the number of bins on the y axis and photon counts on the x axis. The residuals show the deviation of the fit of the data to the selected model, providing a measure of how well the data fit the model. In this case, the data were fit to a one-component model for a single homogenous population of fluorescence-tagged receptors (i.e. dimers). The residuals of the curve fit are less than 2 S.D. values and are randomly distributed about 0, indicating that the data are a good fit for the selected model, with reduced χ2 equal to unity.
FIGURE 5.
FIGURE 5.
Live cell time course of β-arrestin recruitment to the plasma membrane following activation of 5-HT2C receptors. HEK293 cells were co-transfected with β-arrestin2/CFP (shown in green) and 5-HT2C/YFP (shown in red). Images were recorded prior to treatment with 5-HT (t = 0) and 30 s (t = 30) and 90 s (t = 90) following application of 50 nm 5-HT. β-Arrestin is entirely cytosolic prior to 5-HT and appears co-localized with 5-HT2C receptors on the plasma membrane 30 s and 90 s after 5-HT treatment (yellow plasma membrane in the merged image).
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