Single-molecule analyses of fully functional fluorescent protein-tagged follitropin receptor reveal homodimerization and specific heterodimerization with lutropin receptor

Abstract

We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, follitropin) receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 ± 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcT-FP fusion protein exists as a freely diffusing homodimer in the plasma membrane. A central question is whether LHR could oligomerize with FSHR, because both receptors are coexpressed in differentiated granulosa cells. Indeed, FRET analysis revealed an average FRET efficiency of 14.4 ± 7.5 when the FSHR-LHR cT-mCherry was coexpressed with LHR-YFP. In contrast, coexpression of a 5-HT2cVSV-YFP with FSHR-LHR cT-mCherry showed only 5.6 ± 3.2 average FRET efficiency, a value indistinguishable from the detection limit using intensity-based FRET methods. These data demonstrate that coexpression of FSHR and LHR can lead to heterodimerization, and we hypothesize that it is possible for this to occur during granulosa cell differentiation.

Keywords: follicle-stimulating hormone (FSH/FSH receptor); gonadotropins; granulosa cells; mechanisms of hormone action; oocyte maturation.

FIG. 1

Expression of hFSHR-GFP and hFSHR-rLHR-cT-FP in HEK293 cells. A) Image of a live cell expressing hFSHR-GFP merged with DIC image. The hFSHR-GFP is not present on the cell surface and is retained in the endoplasmic reticulum. B) The same cell as in A was fixed and incubated with DiI to stain the plasma membrane and DAPI to label the nucleus. The absence of trafficking of the hFSHR-GFP is clearly demonstrated in the triple-labeled image. C and D) Trafficking of the hFSHR-rLHR-FPs to the plasma membrane of HEK293 cells is clearly evident. C) hFSHR-rLHR-cT-GFP. D) hFSHR-rLHR-cT-RFP.

FIG. 2

HEK293 cell expressing hFSHR-rLHR-cT-RFP and incubated with mAb directed against hFSHR_EDC. A) hFSHR-rLHR-cT-RFP (red channel). B) Monoclonal antibody 105.106-Alexa 647 (pseudocolored green). C) Merged image demonstrating colocalization of the two fluorescent proteins on the plasma membrane of the cell.

FIG. 3

FSHR chimeric receptors form homodimers on the cell surface. Representative images are shown of HEK293 cells that were cotransfected with FSHR-rLHR-cT-YFP and FSHR-rLHR-cT-mCherry. A) Acceptor excitation/acceptor channel shows acceptor fluorescence intensities. B) Donor excitation/donor channel shows the qD fluorescence intensities. C) Uncorrected FRET represents donor excitation/acceptor channel, which includes energy transfer levels plus the two contaminants in the FRET signal: donor cross talk and acceptor bleed-through. D) PFRET image. This image represents the actual energy transfer levels and was processed by the correction algorithm, which removes donor cross talk and acceptor bleed-through. E) The PFRET image overlaid with the automatically selected ROIs produced by the software. The images were modified with ImageJ using the same settings to enhance contrast for better visualization.

FIG. 4

Fluorescence correlation spectroscopy recording from the plasma membrane of an HEK293 cell expressing FSHR-rLHR-cT-GFP. A) Fluorescence intensity trace for one 10-sec observation period. B) Autocorrelation analysis of the fluorescence intensity trace. The red line represents the autocorrelation of the observed fluorescence signal, and the green line represents the fit to a two-component model: a fast component (240 μsec) related to the photophysical properties of the fluorescent probe, and a slower component (70 msec) representing the translational diffusion of FSH receptors in the plasma membrane. Dividing the average photon count rate (kHz), determined from the fluorescence intensity trace (A), by the number of fluorescent molecules, determined from the amplitude of the autocorrelation curve, predicts the average molecular brightness of the sample. C) Photon-counting histogram of the corresponding FCS recording. To generate a histogram, the 10-sec fluorescence intensity trace (A) was broken down into 1 million 10-μsec intervals or bins (PCH bin time = 10 μsec). 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-sec observation period. D) The residuals of the PCH curve fit 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 less than two standard deviations 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. 5

FSHR-rLHR-cT chimeric receptors form homodimers (A) and form heterodimers with rLHR (B). For both pairs, E% is independent of acceptor levels at a D:A ≈ 1. In addition, the E% values do not trend to zero with decreasing A levels, and correlation coefficients show R² = 0.0096 and slope s-value = 0.0027 for the FSHR-rLHR-cT homodimer, and R² = 0.0566 and s-value = 0.089 for the FSHR-rLHR-cT/rLHR heterodimer. This behavior is indicative of the case where molecules, here hFSHR-rLHR-cT/hFSHR-rLHR-cT or FSHR-rLHR-cT/LHR, are present as clusters either as a dimer or as a higher-order oligomer.

FIG. 6

Biochemical characterization of FSHR-LHR C-tail chimera-fluorescent fusion proteins. A) Western blot of hFSHR fusion proteins. 1, hFSHR-rLHR-cT chimera; 2, hFSHR-rLHR-cT chimera-RFP; 3, mock transfection; only nonspecific second antibody staining; 4, hFSHR; and 5, hFSHR-RFP. B) Signal transduction by hFSHR fusion proteins. C) FSH-binding activity of ¹²⁵I-FSHR with fusion proteins. Open circles, hFSHR; black boxes, hFSHR-RFP; gray triangles, hFSHR/rLHR-cT; and red diamonds, hFSHR/rLHR cT-RFP.