Lipid Raft Disruption Alters Human Follicle Stimulating Hormone Receptor Signaling

Abstract

Over 6.7 million people struggle with infertility each year. Studying signaling by reproductive hormones in fertility can allow us to gain a better understanding of the signaling pathways that must function correctly for proper fertility. Some infertility is due to incorrect human follicle stimulating hormone receptor (hFSHR) function. When follicle stimulating hormone (FSH) binds to hFSHR this begins a signaling cascade where the end product is the maturation of sperm by Sertoli cells in men, and egg development and production of estrogen through stimulation of granulosa cells in women. It has been determined that hFSHR is localized to microdomains of the cell membrane called lipid rafts which are characterized by higher concentrations of sphingolipids and cholesterol. This composition and structure makes them less fluid than non‐raft membranes, and it is believed that lipid rafts regulate signaling of many proteins that reside in the rafts, including hFSHR. It was hypothesized that if the lipid rafts in cell membranes were removed then hFSHR signaling would be altered. To remove lipid rafts HEK293 cells stably expressing hFSHR were treated with sphingolipid synthesis inhibitors (Myriocin or Fumonisin B1) or a cholesterol withdrawing drug (Methyl‐β‐cyclodextrin). Removing either the sphingolipids or cholesterol results in disruption of the lipid rafts. hFSHR signaling from the cells with disrupted lipid rafts were compared to wild type hFSHR signaling by western blot. Two pathways were investigated: cAMP production and the activation of p44/42 MAP kinase (ERK1/2). cAMP production was measured indirectly by detecting phosphorylated cAMP Response Element Binding protein (p‐CREB) and p44/42 MAPK signaling was measured by directly detecting the phosphorylation of the kinase (p‐p44/42). It was found that treatment with the lipid raft disrupting agents resulted in increased basal cAMP production (as measured by pCREB activation). However, time‐dependent hFSH stimulation was decreased compared to cells with intact lipid rafts. In contrast, p‐p44 signaling in the drug treated cells was not altered compared to untreated cells. These results suggest that G protein mediated activation of adenylyl cyclase to produce cAMP is lipid raft dependent while p44/42 MAPK activation is not. Further understanding of how lipid raft residency allows for functional hFSHR signaling would allow for the development of new treatments and pharmaceuticals for men and women struggling with infertility.