Date of Award

6-2022

Document Type

Open Access

Degree Name

Bachelor of Science

Department

Biochemistry

First Advisor

Brian Cohen

Keywords

FSHR, FSH, GPCR, signaling, caveolin, caveolin binding motif, CBM, lipid raft, infertility, endocrinology

Abstract

Globally, there are about 48 million couples and 186 million individuals of reproductive age that are affected by infertility. Some cases of infertility in both men and women have been attributed to impaired follicle stimulating hormone (FSH) signaling. The lack of proper function of the cognate receptor for FSH (FSHR) could contribute to infertility since the biochemical signal generated by FSH binding to FSHR stimulates the production of a sperm-stabilizing protein in males and follicle maturation in females. It has been demonstrated that human FSHR (hFSHR) localizes to lipid rafts, which are rigid and detergent-resistant microdomains in the cell membrane. Their structure is in part due to having higher concentrations of cholesterol and sphingolipids compared to the surrounding plasma membrane. Additionally, some lipid rafts are characterized by the presence of the protein caveolin. Signaling pathways for some cell surface receptors have been demonstrated to be regulated by residency in lipid raft domains either by separating the related molecules inhibiting premature signaling or by colocalizing the related molecules to facilitate signaling. Based on evidence that FSHR forms direct protein-protein interactions with caveolin, it was hypothesized that disruption of the caveolin binding motif (CBM), a conserved peptide sequence present in proteins associated with caveolin, would affect hFSHR signaling. In hFSHR this sequence is found in transmembrane helix 4 between amino acids 479-489. The current study investigated the effect of mutations in the CBM on hFSHR signaling. The mutants were created via site-directed mutagenesis and expression vectors were transfected in HEK293 cells. Cells stably expressing the mutant FSHRs underwent western blot analysis for qualitative comparison of signaling. Mutations in three or four of the four conserved phenylalanine residues of the CBM resulted in much higher signaling compared to the wild-type and at a faster rate of activation. Elevated signaling at shorter FSH treatment times suggests that the mutated receptor might be more sensitive to FSH and may hint at the role of the caveolin interaction in regulating hFSHR signaling. Because current infertility treatments are costly, tend to have low success rates, and are not accessible to everyone, a greater understanding of FSH signaling pathways could lead to the design of more successful infertility treatment methods.

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Rights Statement

In Copyright - Educational Use Permitted.