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 DeltaS9-S18, DeltaK19-N30 and DeltaS9-N30 failed to bind 125I-hFSH. Alanine substitution in the mutants 2HHRI(5)/2AAAA(5), 7HCSNR(11)/7ACAAA(11), 16QES(18)/16AAA(18) and 19KVT(21)/19AAA(21) increased the affinity of hFSHR for hFSH with equilibrium dissociation constants two to fivefold lower than wild type (wt) values. Signal transduction in 2HHRI(5)/2AAAA(5) and 19KVT(21)/19AAA(21) was similar to wt values, whereas 7HCSNR(11)/7ACAAA(11) and 16QES(18)/16AAA(18) showed a twofold lower accumulation of cAMP in response to hFSH than wt. These results indicate that these regions play a role in hormone binding and signal transduction. In contrast, cells infected with mutants 12VFL(14)/12AAA(14), 22EIPS(25)/22AAPA(25) and 26DLPRN(30)/26AAPAA(30) were incapable of binding 125I-hFSH even when solubilized with nonionic detergent. Flow cytometry indicated that hFSHR in 12VFL(14)/12AAA(14), 22EIPS(25)/22AAPA(25) and 26DLPRN(30)/26AAPAA(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.