Photoaffinity labeling of the follitropin receptor.
A photoactivatable derivative of human follitropin was used to identify the follitropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycine, and radioiodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself, but with a slightly lower Ka, 1.12 X 10(10) M-1 compared with 1.4 X 10(10) M-1 for the 125I-hormone. The binding could be blocked with untreated hormone. Its alpha and beta subunits could be cross-linked to produce alpha beta dimer by photolysis. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, two new bands (106 and 61 kDa) of lower electrophoretic mobility appeared in addition to the alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis, and the 125I-hormone derivative specifically bound to cells bearing the receptor. Binding could be blocked by excess untreated follitropin but not with human choriogonadotropin and thyrotropin. Under nonreducing conditions, one major band (104 kDa) of cross-linked complexes appeared. Upon reduction with dithiothreitol and second-dimensional electrophoresis, the 104-kDa band produced two smaller complexes of 75 and 61 kDa, indicating the loss of two components and the existence of intercomponent disulfides. Successful production of the 104-kDa complex requires blocking of free sulfhydryl groups with N-ethylmaleimide. It is, however, independent of various protease inhibitors or the temperature and the time period of hormone incubation with cells or the plasma membrane fraction. The mass estimates and the interaction with the hormone of the photoaffinity-labeled components are discussed.
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D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents; D004220:Disulfides; D005260:Female; D005640:Follicle Stimulating Hormone / Q000031:analogs & derivatives* / Q000378:metabolism; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D006107:Granulosa Cells / Q000378:metabolism*; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D010446:Peptide Fragments; D010777:Photochemistry; D010908:Pituitary Hormones, Anterior; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011962:Receptors, FSH; D013552:Swine
C046992:4-azidobenzoylglycyl-FSH; D000345:Affinity Labels; D001386:Azides; 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; C046991:N-hydroxysuccinimidyl-4-azidobenzoyl glycine; D005640:Follicle Stimulating Hormone