125I-human Chorionic Gonadotropin Binding Research Articles

Structure-function studies of the human thyrotropin receptor. Inhibition of binding of labeled thyrotropin (TSH) by synthetic human TSH receptor peptides

We have probed the hormone binding regions of the entire putative extracellular domain of the human thyrotropin (TSH) receptor (hTSHr) using synthetic peptides. A series of 26 overlapping peptides comprising the complete sequence of the extracellular domain of hTSHr was synthesized. Each peptide (20 amino acid residues each) was tested for its ability to interact with TSH, as evidenced by inhibition of binding of labeled hormone to native, membrane bound TSH receptors. Four of the 26 peptides interacted with labeled TSH and inhibited its binding to thyroid membranes. The most potent of these peptides was 256-275, which inhibited 125I-bovine TSH binding with an IC50 of 31.7 +/- 1.3 microM. The remaining peptides were 16-35 (351 +/- 9.4 microM), 106-125 (282 +/- 20.5 microM), and 226-245 (951 +/- 245 microM). An additional peptide, 286-305, showed minimal activity, and the remaining 21 peptides showed no activity. Peptides 256-275, 106-125, and 16-35 also inhibited binding of 125I-human chorionic gonadotropin to ovarian membrane receptors, suggesting that those regions of the receptor are involved in binding of a common glycoprotein hormone structure such as the alpha-subunit. In contrast, peptides 226-245 and 286-305 did not inhibit human chorionic gonadotropin binding, suggesting that these two regions are involved in hormone-specific activity. Of interest is the finding that the latter two peptides are from regions of TSHr that are largely dis-homologous to the lutropin receptor, whereas the former three, with the exception of 16-35, are from regions that are largely homologous between the two receptors. The data suggest that multiple, discontinuous regions of the extracellular domain of hTSHr are involved in the binding of the hormone. Furthermore, the binding regions are localized to TSHr-specific sequences as well as to regions that are highly homologous to LHr. This suggests that homologous regions of the two receptors are likely to perform similar functions in the interaction with their specific hormone, suggesting that those regions may be involved in binding of the glycoprotein hormone common alpha-subunit.

Isolation of luteinising hormone receptor binding inhibitor from bovine corpus luteum

A luteinising hormone receptor binding inhibitor (LHRBI) has been purified from bovine corpus luteum (CL). Steroid-free extract of the CL was subjected to successive chromatographies on Sephadex G-50, Q-Sepharose, Orange A dye and metal chelate affinity columns followed by high performance-reverse phase and gel filtration columns. Purification was monitored by the ability of the fractions to inhibit the binding of 125I-human chorionic gonadotropin (hCG) to porcine granulosa cells in vitro. The final isolate showed an 8000-fold enrichment of activity. It was also capable of inhibiting porcine granulosa cell secretion of estradiol and progesterone (P) in vitro. Administration of LHRBI into follicle-stimulating hormone (FSH)-stimulated, immature rats strongly inhibited the ovarian ovulatory response to hCG as revealed by decreased P levels and the number of ova released. The M(r) of LHRBI as assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was ca. 15 kDa and the pI was between 5.0 and 5.5.

Evidence for the rapid internalization and recycling of lutropin receptors in rat testis Leydig cells.

A study into the binding of 125I-human chorionic gonadotropin (hCG) to the lutropin (LH) receptor in rat testis Leydig cells, and subsequent internalization of the hormone-receptor complex, has been carried out. The results show that there is rapid internalization of the hormone-receptor complex; 240 receptors/cell (from a total of approx. 4000 receptors/cell) were internalized each minute in the first hour after exposure to hCG. Radioactivity was released from the cell 1 h after internalization and was found to be associated with highly degraded hCG. The endocytic process was found to have two temperature-sensitive steps. At 4 degrees C, movement of the hormone-receptor complex inside the cell did not occur, and at 21 degrees C hormone accumulated within the cytoplasm but was not degraded or released from the cell. At 34 degrees C, internalization, degradation and loss of the degraded hormone from the cell occurred. These processes appeared to reach a steady state after 2 h. Even though there is rapid internalization of the hormone-receptor complex following exposure to hCG, the binding sites on the cell surface were maintained for at least 4 h. The number of binding sites on the cell surface was not decreased by a protein synthesis inhibitor but was reduced to undetectable levels by monensin. This compound inhibits acidification of endocytic vesicles, which is known to be an important prerequisite to receptor cycling. It is concluded that, in the rat testis Leydig cells, following binding of hCG to the LH receptor there is rapid internalization of the complex and that recycling of the receptor occurs to the cell surface. This process may be essential in maintaining the capacity of the Leydig cell to bind fresh hormone.

Studies with purified immature porcine Leydig cells in primary culture.

The steroidogenic capacity of purified immature porcine Leydig cells in culture was studied over several days. The cells were obtained by fractionating crude testicular interstitial cell suspensions on a discontinuous Percoll gradient (d = 1.037, 1.042, 1.052, 1.098 g/ml), and characterized by specific binding of 125I-human chorionic gonadotropin (hCG), testosterone (T) and cyclic adenosine 3':5'-monophosphate (cAMP) production in response to hCG, and the enzymatic determination of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. The Leydig cells were recovered in a density band between 1.052-1.068 g/ml and grown in a chemically defined medium (Mather et al., 1981). In the absence of hCG, T production was low throughout the 6 days of culture. However, in response to hCG (10 mIU/ml), the cultured Leydig cells showed a progressive increase in T synthesis, which reached a maximum at Days 3-4. 8-Br-cAMP (1 mM) induced a comparable rise in T production to that obtained with hCG throughout the culture period. In contrast, 8-Br-cAMP induced a near maximal increase in dehydroepiandrosterone (DHEA) production from Day 1. This paper demonstrates that purified immature porcine Leydig cells in primary culture are a valuable model to study the ontogeny of Leydig cell function.

In vitro examination of LH-HCG receptors in human corpora lutea of the menstrual cycle and pregnancy.

Binding of 125I-human chorionic gonadotropin (HCG) to homogenates of the human corpora lutea of the menstrual cycle and pregnancy was examined in vitro. While corpora lutea of the menstrual cycle bound 125I-HCG specifically, the corpora lutea of pregnancy from 8 weeks' gestation bound little or none of the added 125I-HCG. In the corpus luteum of the menstrual cycle the dissociation constant for HCG and the number of binding sites were analyzed by a Scatchard plot. However, from these observations along with the findings of earlier reports, it may be suggested that LH-HCG receptors remain reduced in number in the corpus luteum of pregnancy.

Gonadotropin binding and stimulation of cyclic adenosine 3':5'-monophosphate and testosterone production in isolated Leydig cells.

Gonadotropin binding and stimulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) formation and testosterone synthesis were studied in collagenase-dispersed interstitial cells from the adult rat testis. Binding of 125I-human chorionic gonadotropin (hCG) by isolated Leydig cells was of high affinity (Ka = 10(10) M-1) and low capacity, equivalent to approximately 6000 sites/cell. The binding data were consistent with the presence of a single order of receptors, with no interaction between binding sites. Stimulation of testosterone synthesis by increasing concentrations of hCG was completely dissociated from changes in cyclic AMP formation, and maximum activation of steroidogenesis was induced by hCG concentrations which had no effect upon cyclic AMP production. Kinetic analysis of gonadotropin-induced responses in dispersed Leydig cells also showed a marked dissociation between steroidogenesis and cyclic nucleotide formation. Low concentrations of hCG caused maximum stimulation of testosterone production which was not accompanied by a rise in cyclic AMP formation at any time after addition of gonadotropin. Higher concentrations of hCG caused marked elevations of cyclic AMP at progressively earlier time intervals, but did not alter the 20 to 30 min lag period required for induction of testosterone synthesis. These observations indicated that occupancy of gonadotropin receptors occurs over a much wider range of hCG concentration than that required for maximum steroidogenesis.

Characterization and Solubilization of Gonadotropin Receptor of Bovine Corpus Luteum

Plasma membranes isolated from bovine corpora lutea showed specific binding with 125I-human chorionic gonadotropin and 125I-human luteinizing hormone; unlabeled hormones competitively inhibited the binding. The binding of the 125I-human chorionic gonadotropin to the receptor was a saturable phenomenon and the half-saturation was attained at a concentration of 4 x 10-10 m . Maximum hormone receptor binding was obtained within 15 min at pH 7.2 and 37°. The rate constants of association and dissociation of human chorionic gonadotropin to the receptor determined at 37° were 2.8 x 106 m -1 s -1 and 2.1 x 10-5 s -1, respectively. The dissociation constant calculated from these rates was 8.0 x 10-10 m . At 4° the rate of association was extremely slow and the hormone receptor complex was stable up to 48 hours. The dissociation constants obtained from equilibrium data were calculated to be 1.5 x 10-10 m for human chorionic gonadotropin and the number of binding sites were approximately 6.3 x 10-15 per mg of tissue. The binding of 125I-human chorionic gonadotropin to the plasma membranes was optimum at pH 7.2 and was reduced at acidic and basic pH. The binding was also inhibited at high concentrations of CaCl2, MgCl2, and NaCl, and in the presence of guanidine HCl and urea. The inhibition of binding at pH 5 and 9 and up to 1 m concentration of various ions was reversible. Treatment of plasma membrane with phospholipase C, neuraminidase, glucosidase, trypsin, and α-chymotrypsin did not decrease the binding of 125I-human chorionic gonadotropin. Pepsin and phospholipase A inhibited the binding of 125I-human chorionic gonadotropin to plasma membranes. The plasma membranes were solubilized in detergents. The proteins reprecipitated from the solution by ethanol-ammonium acetate retained the ability to bind 125I-human chorionic gonadotropin. Solubilization of the plasma membranes in 6 m guanidine HCl resulted in the separation of protein and lipid components. Fractionation of the protein component was achieved by gel filtration on a column of Sepharose 4B in 6 m guanidine HCl and ion exchange chromatography on DEAE-cellulose in 3 m urea. The receptor activity of the purified fractions could be reconstituted by emulsification with the lipid fraction, indicating the requirement of lipids to retain the native conformation of the receptor. The molecular weight of the active material estimated from Sepharose 4B column in 6 m guanidine HCl and polyacrylamide gel electrophoresis in sodium dodecyl sulfate was between 30,000 to 70,000. This partially purified protein may represent the regulatory subunit of the gonadotropin receptor.