Composition of cross-linked 125I-follitropin-receptor complexes.
Both of the alpha and beta subunits of intact human follitropin (FSH) were radioiodinated with 125I-sodium iodide and chloramine-T and could be resolved on sodium dodecyl sulfate-polyacrylamide gels. Radioiodinated FSH was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to its membrane receptor on the porcine granulosa cell surface as well as to a Triton X-100-solubilized form of the receptor. Cross-linked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65, 83, and 117 kDa, in addition to the hormone bands. The hormone alpha beta dimer band corresponded to 43 kDa. Formation of the three bands requires the 125I-hormone to bind specifically to the receptor with subsequent cross-linking. Binding was prevented by an excess of the native hormone but not by other hormones. A monofunctional analog of the cross-linking reagent failed to produce the three bands. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of cross-linked complexes were treated to cleave covalent cross-links and then electrophoresed in a second dimension, 18-, 22-, and 34-kDa components were released, in addition to the alpha and beta subunits of the hormone.
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D000818:Animals; D003432:Cross-Linking Reagents / 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; D046911:Macromolecular Substances; D008970:Molecular Weight; D017830:Octoxynol; D010446:Peptide Fragments / Q000378:metabolism; D010908:Pituitary Hormones, Anterior / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D011962:Receptors, FSH; D012995:Solubility; D013388:Succinimides / Q000494:pharmacology; D013552:Swine
D003432:Cross-Linking Reagents; 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; D011092:Polyethylene Glycols; C011683:bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone; D005640:Follicle Stimulating Hormone; D017830:Octoxynol