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 TS H receptors (LHR and TSHR). This family of receptors belong to the G-p rotein-coupled class of membrane-bound receptors. A very large extrace llular domain suggests that interaction of ligand with receptor is lik ely 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 t he hypothesis that FSHR R265-S296 is accessible in FSHR and plays a ro le in hormone binding. Rat FSHR (rFSHR) was expressed in insect cells and used as a source of receptor for binding studies. Recombinant rece ptor 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 an alysis. Functional coupling of the rat FSHR to adenylate cyclase in in sect cells was demonstrated. Antipeptide antibodies against FSHR R265- S296 inhibited binding of radiolabeled hFSH to insect cell rat FSHR. I n contrast, neither nonimmune rabbit serum nor antipeptide antibodies against FSHR G150-L183 inhibited the binding of radiolabeled hFSH to r at FSHR in insect cells. Similar results were obtained with recombinan t 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 compellin g evidence that portions of the peptide sequence FSHR R265-S296 are ac cessible to the antipeptide antibodies and may be involved in hormone binding.