The human follicle stimulating hormone receptor (hFSHR) is a glycoprotein hormone receptor belonging to the g protein-coupled receptor family. It is important in both male and female reproductive processes; defects in hFSHR can lead to infertility, delayed puberty, reduced muscle bulk, and osteoporosis. Work in other labs has shown that GPCRs can be localized to microdomains located within the cell membrane called lipid rafts. These regions are highly resistant to detergents because of the high concentration of sphingolipids and cholesterol. Also present within these domains is an intracellular protein, caveolin, that is essential for the function of the microdomain. Our lab has shown that hFSHR, like many other GPCRs, localizes to lipid raft domains. We hypothesize that interaction with caveolin is important in the raft residency of hFSHR through a putative caveolin binding motif (CBM). The interaction between caveolin and hFSHR in these microdomains may regulate intracellular pathways activated by the receptor.
To test our hypothesis, we have constructed CBM mutants and are in the process of creating stable cell lines expressing the mutant receptors to study the effects of mutating the key phenylalanine residues of the CBM. Interestingly, wild type hFSHR presents as a doublet on a western blot while the F479L mutant only has a single band. We hypothesize that absence of the second band for the F479L mutant is the result of altered glycosylation of the receptor. To compare the trafficking and function of the hFSHR F479L mutant we tested the activation of the protein kinase A (PKA) and p44-MAP kinase (MAPK) pathways. The mutated receptors qualitatively showed normal p44-MAPK signaling in response to hFSH, indicating that they are reaching the cell surface and are capable of binding hormone and coupling to signaling partners. We have observed a subtle difference in the phosphorylation of downstream targets of PKA between the wild type and mutant receptors suggesting that there might be a difference in the ability to activate these downstream targets. Studying the interaction between hFSHR and caveolin has the potential to develop new contraceptive antagonists and alternative agonists to regulate fertility.