Abstract
The present study was intended to examine the structure of the rat Leydig cell gonadotropin receptor. Leydig cell suspensions were prepared by either collagenase digestion or mechanical disruption of the testes. The cells were incubated with 125I-human chorionic gonadotropin (hCG) following which the bound 125I-hCG was covalently cross-linked to the cell surface receptor using a cleavable (dithiobis(succinimidyl propionate] and a noncleavable (disuccinimidyl suberate) cross-linking reagent. The extracted cross-linked membrane proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and subjected to autoradiographic analysis. Under nonreducing conditions, three radiolabeled bands, in addition to intact hCG and its alpha-subunit, were detected with apparent molecular weights of 184,000, 136,000, and 103,000. However, under reducing conditions, three radiolabeled bands migrated on the gel corresponding to molecular weights of 144,000, 106,000, and 75,000. The binding of 125I-hCG to the receptor was inhibited by hCG and luteinizing hormone, but not by a number of other peptides or proteins. The radiolabeled bands were not detectable in hCG down-regulated Leydig cells. Furthermore, a similar autoradiographic pattern of 125I-hCG-linked complexes was seen when the 125I-linked receptor complex was subjected to immunoprecipitation with anti-hCG antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, evidence was obtained indicating that these three labeled bands were derived from the same molecular species. The data suggests that the hCG receptor in Leydig cell is probably an oligomeric complex with a molecular weight of about 250,000, which is composed of three polypeptide chains of molecular weights 121,000, 83,000, and 52,000 held together through noncovalent forces. Additionally, collagenase treatment of Leydig cells does not appear to alter the autoradiographic pattern of the 125I-hCG-linked receptor.
Highlights
Underreducing conditions, three radiola- one may purify a component of.the receptor instead of the beled bands migrated on the gel corresponding tomo- whole receptor species, and thepurified component might still lecular weightsof 144,000,106,000,and 75,000
The present study shows that the affinity-labeled Human choriogonadotropin (hCG)
Receptor complex extracted with Triton X-100 has an apparent molecular weightof about 300,000 basedon the gel filtration data
Summary
The existence of lZ5I-hCG-linkedreceptors was demonstrated by competition experiments in which Leydig cells were cross-linkedto IZ5I-hCGin the absence or presence of various FIG. 1. The existence of lZ5I-hCG-linkedreceptors was demonstrated by competition experiments in which Leydig cells were cross-linkedto IZ5I-hCGin the absence or presence of various FIG. 1. The specificityof cross-linking '*"I-hCGto ratLeyunlabeled proteins in the incubation medium. Cells the autoradiographic profile of '2511-hCG-linkepdroteins sep- were incubated with '"I-hCG at 37 "C for 1.5 h in the presence of 1.5 arated on SDS-PAGE under reducing conditions. In addition to lZ5I-hCGand thea subunit of lZ5I-. P~ of the indicated unlabeled proteins or peptides. LZ'I-hCG-equilibrated Leydig cells were cross-linked with 1 m M DSS. The migration distance of the molecular markers is shown to the left of hCG, appeared in the control ( l a n e 1 ). GnRH, gonadotropin-releasing hormone; CT, cholera toxin, insulin, gonadotropin-releasing hormone, or cholera toxin; BSA, bovine serum albumin
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