FSHR Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of FSHR
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

The development of ovulatory follicles involves differentiation of the granulosa cell component from a mitotically active, estrogen-secreting type (immature cells) into a non-dividing, luteinized, progesterone-secreting type (mature cells). Granulosa cell differentiation is regulated by two pituitary hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH), as well as intra-ovarian steroids and growth factors.The follitropin, lutropin, and thyrotropin receptors (FSHR, LHR, and TSHR) belong to the family of GPCRs. They form a small sub-family of GPCRs, collectively known as the leucine-rich repeat containing GPCRs, that is characterized by the presence of relatively large extracellular domains composed of leucine-rich repeats. FSH-induced activation of the FSHR results in the internalization of the FSH-FSHR complex via clathrin-coated pits by a pathway that requires dynamin. As FSHR couples to Gαs, FSH stimulation of FSHR leads to activation adenylate cyclase which in turn catalyzes the formation of cAMP from ATP.

Figure 1. Internalization of rFSHR-EGFP stimulated with FSH. Cells were untreated (DMSO control, left panel) or treated with 20 U/ml recombinant human FSH for 2 hr (right panel). Arrows indicate the rFSHR-EGFP internalization that is detected by the image analysis algorithm.

Figure 2. FSH concentration response in the FSHR Redistribution® assay. Concentration response was measured in 9 point half log dilution series (n=8). Cells were incubated with recFSH for 2 hr. Cells were then fixed and receptor internalization was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (20 U/ml recFSH) and negative control (0.25% DMSO). The EC₅₀ of recFSH is ~2.5 U/ml.