Macromolecular photoaffinity labeling of the lutropin receptor on granulosa cells.

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Abstract
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 .times. 109 M-1 compared with 5.1 .times. 109 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. It .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 .mu.M, 37.3% at 100 .mu.M, and 67.2% at 150 .mu.M. When the 125I-hormone derivative bound the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels under reducing conditions, 3 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 3 bands correspond to MW 96,000 .+-. 6700, 66,000 .+-. 46000, and 63,000 .+-. 4400. Because the hormone has a high carbohydrate content, and such glycoproteins are known to exhibit anomalous electrophoretic mobilities, these estimates must be tentative.