Mutations in the Human Follicle Stimulating Hormone Receptor Caveolin Binding Motif Cause Increased Basal Activation

Over twelve percent of women aged fifteen to forty-five in America suffer with infertility, while over seven million women have used infertility services, such as intrauterine insemination and in vitro fertilization, to conceive. Dysfunctional human follicle stimulating hormone (hFSH) signaling may cause some cases of infertility. hFSH is released from the pituitary in the hypothalamic-pituitary-gonadal (HPG) axis and plays a role in spermatogenesis in males and follicle maturation and estrogen production in females. Therefore, dysfunctional signaling leads to decreased fertility in males and complete infertility in females. It has been shown that hFSHR interacts with lipid rafts, stiffer regions of the plasma membrane with higher sphingolipid and cholesterol concentrations, and with caveolin, a protein found in certain lipid rafts. This interaction may occur through a specific sequence of amino acids in the 4th transmembrane domain of hFSHR consistent with a caveolin binding motif (CBM). Previous work from our lab suggests that hFSHR signaling is regulated through residency in lipid rafts, such that recruitment into lipid rafts inhibits signaling. The current study investigated the effect of mutations of the CBM on hFSHR signaling. It was hypothesized that complex mutation of the CBM would cause increased hFSHR signaling by disrupting interactions with caveolin, preventing inhibition via residency in lipid rafts. Site-directed mutagenesis of the CBM allowed for creation of complex mutants and expression vectors were transiently transfected into HEK293 cells to qualitatively compare hFSHR signaling between mutants via western blot. Mutation of two sites in the CBM resulted in increased basal and induced hFSHR signaling, supporting the hypothesis. Mutation of three sites decreased signaling below that of the wild type receptor, suggesting that complex mutation of three sites may cause too large of a conformational change to allow for any receptor function. Further understanding of hFSHR signaling regulation would allow for development of new fertility treatments for men and women.