Dissociation of hCG-receptor complex and detection of hCG receptors in human chorionic tissues.
We developed a radioreceptor assay (RRA) system for investigating occupied hCG receptors by using a rat testis membrane fraction. Treatment with dithiothreitol, neuraminidase, and low pH (3.3) buffer partially dissociated hCG from its receptor with dissociation rates of 42.8%, 35.2%, and 68.7%, respectively. Specific receptor rebinding was unaffected and showed competitive inhibition of [125I]hCG rebinding by unlabeled hCG. Scatchard plot analysis yielded a rebinding Km of 1.798-3.496/pM. High concentrations of NaCl and MgCl2 had no obvious effect on the [125I]hCG-receptor complex. Triton X-100 and high concentration MnCl2 treatments resulted in the loss of the receptor rebinding activity. We also investigated human chorionic tissue (at 8 weeks, 11 weeks, 16 weeks, 24 weeks, and term) for hCG receptors using the above method. Treatments with different agents increased [125I]hCG binding in this tissue preparation 139-226%, but Scatchard analysis failed to demonstrate any specificity. [125I]hCG uptake by the choriocarcinoma BeWo cell-line increased 151-193% after similar treatments, but this increase could not be competitively inhibited by unlabeled hCG. Treatment with dithiothreitol, neuraminidase, and low pH (3.3) buffer seems feasible for detecting hCG-occupied receptors. Our results suggest that there are no hCG receptors distributed on the surface of the syncytiotrophoblast of human placenta.
Chen
F
F
Department of Obstetrics and Gynecology, Nagoya University School of Medicine.
Goto
S
S
Furuhashi
Y
Y
Saito
M
M
Kinoshita
Y
Y
Sugiyama
M
M
Okamoto
T
T
Tomoda
Y
Y
D000328:Adult; D000818:Animals; D002823:Chorion / Q000032:analysis*; D006063:Chorionic Gonadotropin / Q000032:analysis*; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008345:Manganese / Q000494:pharmacology; D011247:Pregnancy; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011974:Receptors, LH / Q000032:analysis*; D013737:Testis / Q000032:analysis
D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D008345:Manganese; D004229:Dithiothreitol