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

Human follicle stimulating hormone (FSH) is vital to the regulation of reproductive pathways in both males and females, promoting estrogen production and follicular 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). When FSH binds, an intracellular signaling cascade is triggered promoting fertility. Issues in FSH signaling can lead to decreased spermatogenesis along with a decrease in follicular development and estrogen production, which can result in infertility in both men and women. Research in our lab demonstrated that hFSHR localizes to lipid rafts- microdomains in the plasma membrane; some lipid rafts contain caveolin which has been shown to localize GPCRs to lipid rafts. Previous research has suggested that hFSHR forms direct protein-protein interactions with caveolin which is thought to occur through the caveolin binding motif (CBM): a conserved peptide sequence present in proteins associated with caveolin. The CBM consensus sequence (ɸXPhiXXXXPhiXXPhi where Phi is an aromatic amino acid and X is any amino acid) is found in hFSHR transmembrane helix 4 between amino acids 479-489 (FAFAAALFPIF). Mutations of the four phenylalanines in the CBM amino acid sequence (for clarity these mutated sites have been labeled A-D) were created via site-directed mutagenesis and expression vectors were transfected in human embryonic kidney 293 cells (HEK-293). hFSHR intracellular signaling has been demonstrated to activate both cAMP production as well as phosphorylation of the p44/42 MAP kinase. To determine if signaling differences occurred with hFSHR CBM mutation cAMP production was monitored through luciferase reporter assays and p44/42 activation was measured by western blot. An increase in p44/42 MAP kinase activation was observed in the ABC and ABCD mutants in comparison to wild type hFSHR. In contrast, luciferase assays have shown a decrease in cAMP signaling in the ABC and ABCD mutants in comparison to the wild type. These results suggest that mutations in the CBM alter FSH signaling potentially through disruption of hFSHR interaction with caveolin. Further investigation on the interaction of the hFSHR and caveolin is key in understanding how defects in hFSHR function can result in infertility. Building a deeper foundational knowledge about this signaling pathway can lead to the development of more accessible infertility treatments.