Follitropin or follicle stimulating hormone (FSH), a glycoprotein hormone secreted by the pituitary gland, is essential for male and female reproductive competency. Improper functioning of FSH and its cognate receptor, hFSHR, can have severe effects such as impaired puberty, precocious puberty, primary ovarian failure, azoospermia and others. The hFSHR receptor is a G Protein-Coupled Receptor (GPCR) found in specialized lipid rafts and caveolae. The function of other GPCRs has been shown to be influenced by interactions with scaffolding proteins like caveolin-1, which stimulates the localization and subsequent de-localization of signaling molecules and proteins within the membrane lipid raft. This begs the question as to whether caveolin-1 is involved in the FSH signaling pathway. To address this, my project focuses on determining whether hFSHR and caveolin-1 have direct physical interactions.
To determine this interaction, co-immunoprecipitation was used. Human embryonic kidney cells stably expressing hFSHR and transiently transfected with myc-tagged caveolin-1 were extracted with different buffers and detergents to optimize extract hFSHR while maintaining any protein-protein interactions that occur in the cellular environment. An antibody specific for hFSHR was allowed to interact with the cell extracts and then isolated. Western blotting was then used to detect the presence of myc-tagged caveolin-1 in the isolates. Preliminary research suggests that hFSHR and caveolin-1 co-immunoprecipitate but only weakly. Future research to optimize the interaction will utilize chemical cross-linking to bind proteins in the local environment of hFSHR followed by immunoprecipitation and immunoblotting.
It is crucial to develop improved techniques to study hFSHR interactions with other proteins in its environment to better understand the function of hFSHR in vivo and potentially develop better treatments for infertility associated with lack of FSH function.