Single Molecule Analyses of Fluorescent Protein Tagged FSHR Reveals Homodimerization and Specific Heterodimerization with LHR

Abstract

Gonadotropin hormones (Follicle stimulating hormone/FSH, Luteinizing Hormone/LH) play essential roles in ovarian follicle maturation. FSH induces granulosa cell proliferation and differentiation including expression of LH receptor (LHR). Since FSH receptor (FSHR) and LHR are concurrently expressed in granulosa cells, we attempted to monitor trafficking of FSHR and possible heterodimerization with LHR in HEK293 cells. We constructed an FSHR‐fluorescent protein (FSHR‐FP) but found that FSHR‐FP did not traffic to the plasma membrane. The FSHR C‐tail was swapped with the C‐tail of LHR (LHRcT) to create a chimera to which was added a fluorescent protein (FP) [GFP, YFP, or mCherry]. The FSHR‐LHRcT‐FP bound FSH and transduced FSH binding to produce cAMP. Quantitative FRET analysis of FSHR‐LHRcT‐FP showed an average FRET E% of 12.9± 5.7. Fluorescence Correlation Spectroscopy coupled with Photon Counting Histogram analyses demonstrated that the FSHR‐LHRcT‐FP exists as a freely diffusing homodimer on the cell surface. FSHR‐LHRcT‐FP co‐expressed with LHR‐FP showed an average FRET E% of 14.4±7.5, demonstrating the formation of FSHR and LHR heterodimers/hetero‐oligomers. These data provide a structural imprimatur to understand the consequences of FSHR/LHR heteromerization in granulosa cells as a possible additional level of FSHR signal control and modulation during the process of differentiation. Supported in part by HD18407 (JAD), R21MH086796 (KHD, JEM).