Fluorescence correlation spectroscopy analysis of serotonin, adrenergic, muscarinic, and dopamine receptor dimerization: the oligomer number puzzle

Abstract

The issue of G protein-coupled receptor (GPCR) oligomer status has not been resolved. Although many studies have provided evidence in favor of receptor-receptor interactions, there is no consensus as to the exact oligomer size of class A GPCRs. Previous studies have reported monomers, dimers, tetramers, and higher-order oligomers. In the present study, this issue was examined using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH) analysis, a sensitive method for monitoring diffusion and oligomer size of plasma membrane proteins. Six different class A GPCRs were selected from the serotonin (5-HT2A), adrenergic (α1b-AR and β2-AR), muscarinic (M1 and M2), and dopamine (D1) receptor families. Each GPCR was C-terminally labeled with green fluorescent protein (GFP) or yellow fluorescent protein (YFP) and expressed in human embryonic kidney 293 cells. FCS provided plasma membrane diffusion coefficients on the order of 7.5 × 10(-9) cm(2)/s. PCH molecular brightness analysis was used to determine the GPCR oligomer size. Known monomeric (CD-86) and dimeric (CD-28) receptors with GFP and YFP tags were used as controls to determine the molecular brightness of monomers and dimers. PCH analysis of fluorescence-tagged GPCRs revealed molecular brightness values that were twice the monomeric controls and similar to the dimeric controls. Reduced χ(2) analyses of the PCH data best fit a model for a homogeneous population of homodimers, without tetramers or higher-order oligomers. The homodimer configuration was unaltered by agonist treatment and was stable over a 10-fold range of receptor expression level. The results of this study demonstrate that biogenic amine receptors freely diffusing within the plasma membrane are predominantly homodimers.

Fig. 1.

Confocal microscopy of transfected HEK293 cells showing plasma membrane localization of YFP-tagged proteins. (A) β₂-AR/YFP. (B) D₁/YFP. (C) Dimeric CD-28/YFP. (D) The upper plasma membrane of an HEK293 cell expressing β₂-AR/YFP showing the location (marked by +) where an FCS recording was made. Red scale bar, 10 μm.

Fig. 2.

FCS recordings from the plasma membrane of HEK293 cells expressing YFP-tagged GPCRs. (A) Fluorescence intensity traces for one 10-second observation period. (B) Autocorrelation analysis of the fluorescence intensity traces. The red line represents the autocorrelation of the observed fluorescence signal and the green line represents the fit to a two-component model. 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 translational diffusion of fluorescence-tagged receptors in the plasma membrane. 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 Eq. 4) predicts the average molecular brightness of the sample expressed as CPSM.

Fig. 3.

PCHs of the corresponding FCS recordings shown in Fig. 2. To generate the histograms, each 10-second fluorescence intensity trace (shown in Fig. 2A) was broken down into 1 million 10-µs intervals or bins (PCH bin time = 10 μs). The number of bins is plotted on the y-axis and photon counts on the x-axis. The resulting histogram depicts the number of bins that registered 1,2,3…n photon counts during one 10-second observation period. 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 data were fit to a one-component model for a single homogenous population of homodimers. The residuals of the curve fit are <2 S.D. and are randomly distributed about zero, indicating that the data are a good fit for the selected model, with reduced χ² equal to unity.

Fig. 4.

FCS recordings from the plasma membrane of HEK293 cells expressing CD-86/GFP and CD-86/GFP-GFP. (A) Fluorescence intensity traces for one 10-second observation period. (B) Autocorrelation analysis of the fluorescence intensity traces. The red line represents the autocorrelation of the observed fluorescence signal and the green line represents the fit to a two-component model. 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 translational diffusion of the fluorescence-tagged receptors in the plasma membrane. (C) PCHs of the corresponding FCS recordings yield molecular brightness values of 9693 CPSM for CD-86/GFP and 18,194 CPSM for CD-86/GFP-GFP. (D) Residuals of the curve fit. The data were fit to a one-component model for a single homogenous population of fluorescence-tagged receptors.

Fig. 5.

BiFC. The N- and C-terminal halves of YFP were attached to the C-terminal end of β₂-AR or A_2a-adenosine receptors. (Top) HEK293 cells coexpressing β₂-AR/N-YFP with β₂-AR/C-YFP show plasma membrane YFP fluorescence 20 hour post-transfection (left). The middle panel shows the differential interference contrast image and the right panel shows the merged image. (Middle) HEK293 cells coexpressing β₂-AR/N-YFP and A_2a-adenosine/C-YFP show minimal fluorescence complementation. (Bottom) Restoration of plasma membrane fluorescence complementation in HEK293 cells coexpressing A_2a-adenosine/N-YFP and A_2a-adenosine/C-YFP. Red scale bar, 10 μm.

Fig. 6.

Single-component and multicomponent analyses for HEK293 cells expressing β₂-AR/GFP, 5-HT_2A/YFP, or D₁/YFP. PCHs and residuals of the curve fit for the following: (A) a one-component fit of the data for a single population of fluorescence-tagged receptors; (B) a mixture of monomers/dimers/tetramers in a fixed 20%/70%/10% ratio; and (C) a mixture of monomers/dimers/tetramers in a fixed 40%/40%/20% ratio. Reduced χ² values for the one-component and the multicomponent 20%70%/10% and 40%/40%/20% ratios were 0.8, 4.7, and 18, respectively, for β₂-AR/GFP; 1.1, 4.3, and 17, respectively, for 5-HT_2A/YFP; and 1.0, 4.6, and 18, respectively, for D₁/YFP.

Fig. 7.

Single-component and multicomponent analyses for HEK293 cells expressing M₁/GFP, M₂/YFP or α_1b-AR/YFP. PCH and residuals of the curve fit for the following: (A) a one-component fit of the data for a single population of fluorescence-tagged receptors; (B) a mixture of monomers/dimers/tetramers in a fixed 20%/70%/10% ratio; and (C) a mixture of monomers/dimers/tetramers in a fixed 40%/40%/20% ratio. Reduced χ² values for the one-component and the multicomponent 20%/70%/10% and 40%/40%/20% ratios were 0.7, 7.0 and 23, respectively, for M₁/GFP; 1.1, 3.2 and 16, respectively, for M₂/YFP; and 1.3, 6.0 and 23, respectively, for α_1b-AR/YFP.