Developing an APEX Assay to Investigate Signaling and Protein Interaction of the Human Follicle Stimulating Hormone Receptor

Infertility affects millions of people in the United States. In addition to being unable to conceive, patients can face a variety of challenges ranging from mental health issues to increased economic costs. One cause of infertility is a defect in follicle stimulating hormone (hFSH). hFSH is a peptide hormone that is crucial for fertility in both males and females. In males, hFSH is involved in spermatogenesis. In females, hFSH is involved in estrogen production and ovarian follicle development. To better understand the role hFSH plays in fertility, our lab is interested in investigating the function of the hFSH receptor (hFSHR). hFSHR is a G protein-coupled receptor (GPCR); signaling from the receptor is of particular interest to us, because if we know how hFSHR signals, we can identify potential molecules to investigate that may be targets to improve fertility. However, investigating the signaling of GPCRs such as hFSHR is challenging. Ideal assays to investigate hGPCR signaling should be sensitive, simple to use, high throughput, and non-radioactive. Although assays for investigating GPCR signaling have historically been used, they have significant drawbacks. Assays using radioactive ligands are sensitive and high throughput, but are not simple to use and generate radioactive waste. Other assays such as G protein dependent functional assays are sensitive, but are only able to screen for the activity of one molecule. The APEX assay is a new assay for investigating GPCR signaling that has significant advantages over other assays. When cells expressing a GPCR-APEX fusion protein are incubated in culture media containing biotin-phenol, APEX tags signaling molecules and other proteins in a 20 nm radius with biotin-phenoxy radicals. Furthermore, this reaction can be quenched. Thus, the APEX assay can be used to investigate GPCR signaling with high spatial and temporal resolution. To develop the APEX assay to investigate hFSHR signaling, it was necessary to generate a plasmid containing the hFSHR-APEX fusion protein. Creation of this plasmid will allow us to have a high-throughput, time dependent assay to develop new therapeutic targets for infertility.