Intersubunit disulfides of the follitropin receptor.
The electrophoretic mobility of radioiodinated follitropin (FSH) alpha and beta subunits as well as the alpha beta dimer changed markedly depending on the concentration of reducing agents such as dithiothreitol. The changes were more dramatic in the beta subunit than in the alpha subunit. 125I-FSH, complexed to the receptor on porcine granulosa cells or in Triton X-100 extracts, was cross-linked with a cleavable (nondisulfide) homobifunctional reagent, solubilized in sodium dodecyl sulfate without reducing agents, and electrophoresed. The cross-linked sample revealed three bands of high molecular mass, in addition to the hormone subunit and dimer bands. The band of lightest mass, 110 kDa, was the major band and the other two of 76 and 62 kDa were barely noticeable. Upon reduction with dithiothreitol, the 110-kDa band decreased while the 76- and 62-kDa bands increased, indicating the existence of disulfides between components of the 110-kDa complex. Formation of the disulfide-linked complexes requires 125I-FSH, specifically bound to the hormone receptor and cross-linking, and can be prevented with an excess of native FSH but not human choriogonadotropin. Complex formation was independent of blocking free sulfhydryl groups with N-ethylmaleimide. When the cross-linked complexes were reduced in the gel matrix and analyzed on fresh gels, the 76- and 62-kDa complexes were generated from the 110-kDa band, indicating the loss of two components. The lost components were estimated to be at 14 and 34 kDa. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22-, 14-, and 34-kDa components to the FSH alpha beta dimer. The results of reduction of the cross-linked complexes demonstrate the existence of disulfide linkage between the three components.
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D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism*; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D006107:Granulosa Cells / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000378:metabolism; D010908:Pituitary Hormones, Anterior / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D011962:Receptors, FSH; D013388:Succinimides / Q000494:pharmacology; D013552:Swine
D003432:Cross-Linking Reagents; D004220:Disulfides; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010908:Pituitary Hormones, Anterior; D011956:Receptors, Cell Surface; D011962:Receptors, FSH; D013388:Succinimides; C011683:bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone; D005640:Follicle Stimulating Hormone; D004229:Dithiothreitol