Premium
Investigating Human Follicle Stimulating Hormone Receptor and its Partners Using the APEX Assay
Author(s) -
Temple Alexandra Elizabeth,
Cohen Brian D.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.03589
Subject(s) - follicle stimulating hormone receptor , lipid raft , microbiology and biotechnology , receptor , hormone receptor , biology , follicle stimulating hormone , signal transduction , medicine , chemistry , endocrinology , biochemistry , hormone , luteinizing hormone , genetics , cancer , breast cancer
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 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 different downstream results such as ovarian follicular development and estrogen production in women and sperm production and regulation in men. To better understand how the receptor functions, the interactions between FSH, FSHR and other effector proteins involved in signaling must be identified and understood. One hypothesized mechanism of signaling is through the receptor localization in lipid rafts. The lipid rafts act as a signaling complex where FSHR and other effector proteins colocalize to make for efficient signaling of FSHR. This colocalization in lipid rafts is believed to play an important role and we plan to study the interactions between FSHR, effector proteins, and lipid rafts using the APEX Assay. The APEX Assay has been developed to study these interactions using different GPCRs. 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, 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, as well as lipid raft localization. Using HEK293 cells, we are working on creating a stable cell line expressing an FSHR‐APEX construct. Once the cell line is created, the FSHR‐APEX receptor can be used 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 FSHR interacts with, when these interactions occur, and if they occur in lipid rafts. 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.