FSHR

Detailed Information

Human FSHR is a member of the G-protein coupled receptor (GPCR) superfamily, which consists of 695 amino acid residues.FSHR can be divided into three parts: extracellular domain (ECD), transmembrane domain (TMD) and intracellular domain (ICD) (Figure 1). The FSHR gene is located on chromosome 2p21-p16 and contains 10 exons and 9 introns, the first 9 exons are responsible for encoding the ECD of FSHR, including most of the hinge region, and exon 10 mainly encodes the TMD and the ICD, where the ECD is composed of 10 leucine-rich repeats. consists of 10 leucine rich repeat (LRR) sequences and consecutive β-strands and α-helices, which are involved in ligand selection and specific protein-protein interactions, facilitating recognition and binding to specific receptors. The hinge region, also located in the ECD, is responsible for linking the LRR sequence-rich ECD to the seven-transmembrane-domain (7TMD) α-helices, which are involved in not only ligand high affinity but also receptor activation and intracellular molecular signaling. There are also four potential glycosylation sites at positions 191, 199, 293, and 318 (sequence NXS/T, X being any amino acid except proline).N-linked glycosylation (and disulfide bond formation) is a common feature of biosynthesis that facilitates the folding of protein precursors, and plays a critically important role in intracellular translocation of the receptor.7TMD is a polypeptide chain that traverses the lipid bilayer of the plasma membrane seven times to form a characteristic transmembrane-domain (TMD). The TMD is a polypeptide chain that crosses the plasma membrane lipid bilayer seven times, forms a characteristic transmembrane α-helix, and is interconnected by alternating extracellular loops (EL) and intracellular loops (IL), which have a homology of up to 90% in different species. ICD, on the other hand, has a lower homology and is mainly involved in intracellular transport and signaling of the receptor. Compared with other GPCRs, the large ECD is the most important feature of FSHR, which is responsible for the recognition and binding of homologous ligands and receptor activation, and then the TMD transmits the activation information to the ICD, which couples with the downstream effector proteins, and interacts with the bridging proteins, which further triggers intracellular signaling to the downstream.

Figure 1. Schematic of FSHR showing its amino acid sequence and functional domains for agonist binding, activation, and signal transduction. (Ulloa-Aguirre A, et al., 2018)

Functional realization of FSHR

In females, FSHR is mainly expressed in granulosa cells of developing follicles and is required for promoting follicular growth and development and estradiol synthesis. Its life cycle includes its synthesis, proper folding, post-translational modification, and formation of higher poly/oligomers in the endoplasmic reticulum and Golgi apparatus, followed by translocation to the surface of the cell membrane for binding to ligands and subsequent intracellular signal transduction. When its LRR-rich ECD specifically binds to FSH, it activates stimulatory G proteins, which in turn activates adenylate cyclase (AC), promotes intracytoplasmic cyclic adenosine monophosphate (cAMP) formation, and further activates protein kinases A (PKA). protein kinases A (PKA), causing protein phosphorylation, signal transduction, and phosphorylation of extracellular signal-regulated protein kinase (ERK), which can regulate cAMP-response element binding protein (CREB). element binding protein (CREB), such as aromatase transcription, as well as phosphorylation of mitogen-activated protein kinase (MAPK), a downstream pathway essential for ovulation and luteinogenesis. The FSHR primary sequence also contains specific functions involved in the transport of receptors from the endoplasmic reticulum to the cell membrane, ligand transport, and ligand binding. Mutations or single nucleotide polymorphisms in the FSHR gene can cause alterations in the primary sequence, resulting in abnormal receptor protein function and ultimately disease. The functional analysis of FSHR includes three main aspects: (1) binding ability to FSH; (2) specificity to FSH, i.e., affinity; and (3) signaling ability, i.e., the amount of cAMP production. The research process generally includes: 1. obtaining the mutation site of the pioneer or its family members' receptor by DNA sequencing; 2. constructing wild-type and mutant FSHR expression vectors; 3. transient transfection of cell lines with the target vectors; 4. measurement of receptor cell membrane expression; 5. measurement of FSHR binding ability to FSH; and 6. measurement of cAMP.

FSHR gene mutations and functional inactivation

Mutations can be categorized into two main groups, inactivating and activating mutations, based on their effect on receptor function. Since the first report of pathogenic phenotypes caused by FSHR point mutations by Finnish scholars in 1995, 22 cases of FSHR inactivating mutant loci have been reported so far. Because of the differences in mutant bases and mutation sites, the degree of impairment of ligand binding and signal transduction ability of FSHR is therefore different. The following mainly summarizes the reported mechanisms of the effects of FSHR inactivating mutations leading to female ovarian hypoplasia.1. ECD inactivating mutations: the ECD peptide chain is the longest, and there are 10 reported ECD inactivating mutations involving the signal peptide coding region, LRR-rich motifs, and the hinge region. c.44G>A is the only signal peptide coding region mutation identified so far, and this was reported from a primary infertile compound This report was from a patient with a compound heterozygous mutation (c.44G>A and exons 1 and 2 deletions) in primary infertility, who presented with secondary amenorrhea and vaginal ultrasound: normal-sized ovaries bilaterally, with four follicles (>6 mm in diameter) detectable in both, and an AMH consistent with actual age. Gene sequencing showed that c.44G>A was a missense mutation, resulting in an amino acid substitution in the FSHR signaling peptide, i.e., p.Gly15Asp. SignalP4.1 software suggested that the substitution of Asp15 for Gly significantly reduced the cleavage of the signaling peptide, which in turn caused the mislocalization of receptor subcellularity, and the results of in vitro experiments showed that the downstream production of cAMP was significantly decreased. The LRR-rich motif is the region where more inactivating mutation sites have been reported, most of which are associated with blocked FSHR membrane transport. p.Ala189Val, the first FSHR mutation reported in 1995, is located in the ECD. Desai et al. Genetic analyses of six Finnish ovarian dysplasia family lines localized the disease-associated genetic locus to chromosome 2p, a segment corresponding to the FSHR and LHR loci. Since androgen levels were not affected in males in the families, FSHR was selected for sequencing. A point mutation (c.567C>T) was detected in exon 7, which replaced the amino acid Ala with Val at position 189. All patients with ovarian dysgenesis in the families had a pure mutation. Therefore, this mutant was transfected into a mouse mesenchymal stem cell-1 (MSC-1) line for in vitro functional experiments, and it was found that the binding of FSHR to ligand was significantly reduced, and the cAMP production was significantly decreased, but the affinity of such FSHR to ligand was normal. It was later shown that the sequence of amino acids 189 to 193 (AFNGT) is highly conserved in FSHR, with an N-linked glycosylation site, which is essential for the normal folding of the protein, and that mutations in this sequence can affect the conformational integrity of the receptor. p.Ala189Val may make FSHR structurally abnormal by affecting glycosylation, and impaired transport to the cellular membrane, leading to a significant reduction in its Another mutation in the conserved sequence of AFNGT (c.573A>T, p.Asn191Ile) was reported in 1996, and in vitro experiments confirmed that the mutant produced significantly less cAMP than the wild type upon FSH stimulation, and it was hypothesized that this defect may also be due to the lack of glycosylation that affects protein folding resulting in decreased expression on the membrane surface of FSHR. Since patients carrying this heterozygous mutation were reported to be able to bear children normally, this suggests that the overall function of FSHR is less affected by the heterozygous state.