Macromolecular photoaffinity labeling of the lutropin receptor on granulosa cells.
Lutropin, a pituitary hormone, and human choriogonadotropin bind to the same receptors in the ovary and elicit identical responses. A photoactivable derivative of human choriogonadotropin was used to identify the lutropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycylglycine, and iodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself did but with a slightly lower Ka, 2.98 X 10(9) M-1 compared with 5.1 X 10(9) M-1 for 125I-hormone. The binding could be blocked with untreated hormone or lutropin but not with follitropin, prolactin, insulin, or bovine serum albumin. Its alpha and beta subunits could be crosslinked to produce alpha beta dimer by photolysis, the extent of crosslinking being dependent upon the reagent concentration used for the derivatization: 22.8% at 50 microM, 37.3% at 100 microM, and 67.2% at 150 microM. 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, three new bands of lower electrophoretic mobility appeared in addition to alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis and the presence of both cells bearing the receptor and the 125I-hormone derivative. It could be blocked by excess untreated hormone. The three bands correspond to molecular weights, 96,000 +/- 6,700, 66,000 +/- 4,600, and 63,000 +/- 4,400. Because the hormone has a high carbohydrate content and such glycoproteins are known to exhibit anomalous electrophoretic mobilities, these estimates must be tentative.
D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D006063:Chorionic Gonadotropin / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism*; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011974:Receptors, LH; D013552:Swine
D000345:Affinity Labels; D006063:Chorionic Gonadotropin; D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D011974:Receptors, LH