Expression of extracellular domain peptides of the FSH receptor and their effect on receptor-ligand interactions in vitro.
Follicle-stimulating hormone receptor (FSH-R) displays considerable homology to luteinizing hormone receptor (LH-R) in structure and amino acid sequence. Comparison of the sequences of the extracellular domains (ECD) of the receptors reveals two regions (amino acids 4-56 and 265-319 in FSH-R) that share relatively little amino acid sequence similarity. This suggests that these variable regions may be important in providing specificity of ligand binding. We have expressed overlapping ECD peptides containing one or both of these regions (RFI, amino acids 5-125; RF2, amino acids 201-319; and RF3, amino acids 5-319) as fusion proteins in E. coli using pRSET vector. The presence of polyhistidine at the N-terminal end allowed substantial purification of the expressed proteins by a single step of affinity chromatography. The purified peptides were characterized for direct binding of hormone and their ability to block the binding of FSH and LH to the receptors. None of the peptides bound labelled hormone, while all peptides inhibited the binding of FSH to its receptor in a dose-dependent manner. However, only RF2 peptide inhibited ligand binding in a hormone-specific manner. These data suggest there is a site between amino acids 201-319 of the FSH-R ECD that is involved in FSH binding.
Sharma
S C
SC
Center for Biomedical Research, Population Council, New York, NY 10021, USA.
Catterall
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D005640:Follicle Stimulating Hormone / Q000378:metabolism*; D066298:In Vitro Techniques; D008024:Ligands; D007986:Luteinizing Hormone / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011962:Receptors, FSH / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011974:Receptors, LH / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000378:metabolism
D017931:DNA Primers; D008024:Ligands; D010446:Peptide Fragments; D011962:Receptors, FSH; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone