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.
Nechamen
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CA
Wadsworth Center, David Axelrod Institute for Public Health, New York State Department of Health, 120 New Scotland Avenue, 12201-0509, Albany, NY, USA.
Dias
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JA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011962:Receptors, FSH / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D018411:Spodoptera
D011962:Receptors, FSH; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone