FSH receptor-specific residues L501 and I505 in extracellular loop 2 are essential for its function.

The extracellular loop 2 (EL2) of FSH receptor (FSHR) plays a pivotal role in various events downstream of FSH stimulation. Because swapping the six FSHR-specific residues in EL2 (chimeric EL2M) with those from LH/choriogonadotropin receptor resulted in impaired internalization of FSH-FSHR complex and low FSH-induced cAMP production, six substitution mutants of EL2 were generated to ascertain the contribution of individual amino acids to the effects shown by chimeric EL2M. Results revealed that L(501)F mainly and I(505)V to a lesser extent contribute to the diminished receptor function in chimeric EL2M. HEK293 cells stably expressing WT and chimeric EL2M FSHR were generated to track the fate of the receptors post FSH induction. The chimeric EL2M FSHR stable clone showed weak internalization and cAMP response similar to transiently transfected cells. Furthermore, reduced FSH-induced ERK phosphorylation was also observed. The interaction of activated chimeric EL2M and L(501)F FSHR with ß-arrestins was weak compared with WT FSHR, thus explaining the impaired internalization of chimeric EL2M and corroborating the indispensable role of EL2 in receptor function.

Extracellular loop 2 in the FSH receptor is crucial for ligand mediated receptor activation.

The present study aims to determine the role of the specific residues of the extracellular loops (ELs) of the FSH receptor (FSHR) in hormone binding and receptor activation. By substituting the sequences of each of the ELs of human FSHR with those of the luteinizing hormone/choriogonadotropin receptor (LH/CGR), we generated three mutant constructs where the three ELs were individually replaced. A fourth construct had all the three substituted ELs. The receptor expression and hormone binding ability of the mutants were comparable to that of the wild type. Hormone-induced signaling and internalization were lower in the EL2 substitution mutant (EL2M). In this mutant, the EL2 of FSHR was substituted with the corresponding loop of LH/CGR. Interestingly, homology modeling revealed a change in the orientation of EL2 in the mutant receptor. Thus, disruption of EL2 affected overall receptor function, suggesting the role of FSHR specific residues of the loop in ligand mediated signaling.

Accessibility of the extracellular loops of follicle stimulating hormone receptor and their role in hormone-receptor interaction.

The follicle stimulating hormone receptor (FSHR), a member of the G-protein coupled receptor family, has a large extracellular domain (ECD) which is responsible for hormone binding specificity. Whether the extracellular loops (ELs) of FSHR which are outward projections of its transmembrane domain have any role in receptor function is not yet well understood. Here, we use antipeptide antibodies to peptides corresponding to the FSHR-ELs to check the surface accessibility of the loops. These antibodies were further used to understand the involvement of the loops in hormone binding and signaling. The results demonstrate that EL1 and EL3 are surface accessible on the mature receptor in spite of the presence of a large ECD. It is observed that the EL1 and EL3 serve as secondary binding sites and they possibly interact with the ECD-bound hormone's alpha subunit which is common to the gonadotropins.

Characterization of peptide 20-30 of follicle stimulating hormone receptor as an antagonist of receptor activity: significance of charged residues.

We had previously reported the region (20-30) from follicle stimulating hormone receptor as being an immunodominant epitope and the smallest reported peptide capable of inhibiting hormone binding. We now report it to be an effective antagonist of ligand-induced cAMP signalling as well. The region (20-30) of follicle stimulating hormone receptor has three charged residues, namely, E(22), D(26) and R(29) that are specific to follicle stimulating hormone receptor and are conserved in mammals. This study aimed to verify whether the charged residues contribute to the activity of the follicle stimulating hormone receptor peptide (20-30). This was done using analogs of follicle stimulating hormone receptor peptide (20-30), each having an alanine substitution for a corresponding charged residue. The analog peptides displayed a loss of activity and could not inhibit hormone binding or the subsequent signal transduction. The ability of follicle stimulating hormone receptor peptide (20-30) to bind antipeptide antibodies against follicle stimulating hormone receptor peptide (9-30) was either decreased or abolished with the alanine substituted analog peptides of follicle stimulating hormone receptor peptide (20-30). The loss of function led us to verify whether there was a conformational change as well. CD spectral analysis did not reveal a significant change. These observations indicate that the charged aminoacids present in follicle stimulating hormone receptor peptide (20-30) are crucial for the observed follicle stimulating hormone antagonistic activity. This information could form the basis for the design of novel compounds capable of functioning as follicle stimulating hormone antagonists.