Exploring the Effects of Mutations in the Caveolin Binding Motif on Downstream Signaling of Human Follicle Stimulating Hormone Receptor

Infertility affects approximately one in six individuals worldwide. Current infertility treatments, including medication, surgery, intrauterine insemination, and in vitro fertilization, are often costly and inaccessible to many patients. Improving understanding of the molecular mechanisms underlying follicle-stimulating hormone (FSH) signaling may contribute to the development of more effective and accessible therapies. FSH is produced by the anterior pituitary and plays a critical role in human reproduction by stimulating ovarian follicle development in females and spermatogenesis in males. FSH acts through a g protein-coupled receptor (GPCR) known as the human follicle stimulating hormone receptor (hFSHR). Previous research in our lab has shown that hFSHR localizes to lipid rafts; rigid microdomains of the cell membrane rich in cholesterol and sphingolipids. Here, hFSHR makes direct protein-protein interactions with a protein commonly found in lipid rafts known as caveolin through a conserved caveolin binding motif (CBM). The CBM consensus sequence (ɸXɸXXXXɸXXɸ; where ɸ is an aromatic amino acid and X is any amino acid) is found in hFSHR transmembrane helix 4 between amino acids 479-489 (FAFAAALFPIF). Site-directed mutagenesis was used to substitute phenylalanine residues with leucine at positions F479, F481, F486, and F489 (assigned letters A-D for clarity) to disrupt the CBM. hFSHR intracellular signaling has been demonstrated to activate cyclic AMP production and phosphorylation of the p44/42 MAP kinase. The effects of these mutations on receptor signaling were evaluated by measuring p44/42 MAP kinase activation using Western blot analysis and cyclic AMP production using luciferase reporter assays. Preliminary studies of signaling of the mutant receptors found that the F479L mutation negatively impacted cAMP signaling but increased activation of the p44/42 MAP kinase pathway. These studies aim to clarify how CBM-mediated interactions regulate hFSHR signaling and may provide insight into mechanisms underlying fertility and potential therapeutic targets for infertility.