Accessibility of rat and human follitropin receptor primary sequence (R265-S296) in situ.
The primary sequence of FSH receptor (FSHR) is homologous to LH and TSH receptors (LHR and TSHR). This family of receptors belong to the G-protein-coupled class of membrane-bound receptors. A very large extracellular domain suggests that interaction of ligand with receptor is likely to be complex. Secondary structure analysis of the FSHR R265-S296 primary sequence, which has little homology to LHR, predicted a helix-turn-helix motif. An objective of these studies was to test directly the hypothesis that FSHR R265-S296 is accessible in FSHR and plays a role in hormone binding. Rat FSHR (rFSHR) was expressed in insect cells and used as a source of receptor for binding studies. Recombinant receptor had a Kd in the picomolar range with about 200,000 receptors/cell and appeared as two forms (180 and 75 kilodaltons) by Western blot analysis. Functional coupling of the rat FSHR to adenylate cyclase in insect cells was demonstrated. Antipeptide antibodies against FSHR R265-S296 inhibited binding of radiolabeled hFSH to insect cell rat FSHR. In contrast, neither nonimmune rabbit serum nor antipeptide antibodies against FSHR G150-L183 inhibited the binding of radiolabeled hFSH to rat FSHR in insect cells. Similar results were obtained with recombinant human FSHR in Y1 cells, measuring progesterone production as an end point. Confocal microscopy using antihuman FSHR R265-S296 demonstrated that recombinant human FSHR on Chinese hamster ovary cells existed as discrete patches on the surface. In summary, the data offer compelling evidence that portions of the peptide sequence FSHR R265-S296 are accessible to the antipeptide antibodies and may be involved in hormone binding.
Liu
X
X
Wadsworth Center, New York State Department of Health, Albany 12201-0509.
DePasquale
J A
JA
Griswold
M D
MD
Dias
J A
JA
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D005640:Follicle Stimulating Hormone / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009036:Moths / Q000378:metabolism; D011374:Progesterone / Q000096:biosynthesis; D017433:Protein Structure, Secondary; D051381:Rats; D011962:Receptors, FSH / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
D000906:Antibodies; D011962:Receptors, FSH; D011994:Recombinant Proteins; D011374:Progesterone; D005640:Follicle Stimulating Hormone; D000262:Adenylyl Cyclases