Investigating Human Follicle Stimulating Hormone Receptor and its Partners Using the APEX Assay

Follicle stimulating hormone (FSH) and its receptor (FSHR) play important roles in reproduction and fertility in both men and women. Fertility problems arise when FSH, FSHR, or any downstream signaling components don't function properly. FSHR is a G-protein coupled receptor (GPCR) found on the cell surface of granulosa cells in women and Sertoli cells in men. When activated by FSH, FSHR initiates a signaling cascade that can result in downstream results such as ovarian follicular development and estrogen production in women and sperm production in men. To better understand how the receptor functions, the interactions between FSHR and effector proteins involved in signaling must be identified and understood. We plan to study the interactions between FSHR and its effector proteins using the APEX Assay. This assay utilizes an engineered ascorbate peroxidase (APEX) which is attached to the C-terminus of FSHR. APEX modifies proteins using biotin phenol and hydrogen peroxide, which creates a biotin-radical, thus labeling proteins within a small radius of FSHR because of the short lived nature of the radical. Using mass spectrometry, the biotinylated proteins can be quantitatively analyzed to give a better understanding of the proteins associated with FSHR and the changes in the proteins over time after hormone treatment. Using HEK293 cells, we created a stable cell line expressing an FSHR-APEX construct. We are currently using the FSHR-APEX expressing cells in the APEX Assay to analyze the proteins that are involved in FSHR signaling. This research will give us a better insight as to what effector proteins interact with FSHR and how these interactions change following hormone stimulation. This will provide us with a better understanding as to how the receptor signals and initiates downstream signaling and how this might affect fertility in humans.