Human follicle stimulating hormone receptor trafficking and hormone binding sites in the amino terminus

Previous studies of the rat follicle-stimulating hormone receptor (rFSHR) demonstrated that the amino terminus is important in FSH binding and signal transduction. To define the structure function correlates of this region, we prepared deletion and alanine scanning mutants of amino acids (a.a.) 9-30 in human FSHR (hFSHR). The deletion mutants Delta S9-S18, Delta K19-N30 and Delta S9-N30 failed to bind I-125-hFSH. Alanine substitution in thr mutants (HHRI5)-H-2/(2)AAA(5), (HCSNR11)-H-7/(7)ACAAA(11), (16)QES(18)/(16)AAA(18) and (KVT21)-K-19/(19)AAA(21) increased the affinity of hFSHR for hFSH with equilibrium dissociation constants two to fivefold lower than wild type (wt) values. Signal transduction in (HHRI5)-H-2/(2)AAAA(5) and (KVT21)-K-19/(19)AAA(21) was similar to wt values, whereas (HCSNR11)-H-7/(7)ACAAA(11) and (16)QES(18)/(16)AAA(18) showed a twofold lower accumulation of cAMP in response to hFSH than wt. These result indicate that these regions play a role in hormone binding and signal transduction. In contrast, cells infected with mutants (VFL14)-V-12/(12)AAA(14), (EIPS25)-E-22/(22)AAPA(25) and (DLPRN30)-D-26/(26)AAPAA(30) were incapable of binding I-125-hFSH even when solubilized with nonionic detergent. Flow cytometry indicated that hFSHR in (VFL14)-V-12/(12)AAA(14), (EIPS25)-E-22/(22)AAPA(25) and (DLPRN30)-D-26/(26)AAPAA(30) was not present on the cell surface although the protein was expressed at high levels as determined by Western blotting. These results suggest that a discontinuous epitope in the N-terminus, likely stabilized by disulfide bonds and outside of the leucine-rich repeat domains, constitutes a hormone binding site, membrane localization signal or both. (C) 2000 Elsevier science Ireland Ltd. All rights reserved,