scholarly journals Revisiting and Questioning Functional Rescue between Dimerized LH Receptor Mutants

2012 ◽  
Vol 26 (4) ◽  
pp. 655-668 ◽  
Author(s):  
Meilin Zhang ◽  
Rongbin Guan ◽  
Deborah L. Segaloff
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Hormone Receptors ◽  
Resonance Energy ◽  
Extracellular Domain ◽  
Glycoprotein Hormone ◽  
Lh Receptor ◽  
Defective Mutant

Abstract The glycoprotein hormone receptors are G protein-coupled receptors containing a large extracellular domain fused to a prototypical serpentine domain. cis-activation occurs when binding of hormone to the extracellular domain stabilizes the serpentine domain in an active conformation. Studies by others suggested that these receptors can also signal by trans-activation, where hormone binding to one receptor protomer activates the serpentine domain of an associated protomer, as documented by the partial rescue of hormone-dependent signaling when a binding defective mutant is coexpressed with a signaling defective mutant. However, our characterizations of several LH receptor (LHR) mutants used in previous studies differ markedly from those originally reported. Also, when examining a pair of LHR mutants previously shown to functionally rescue in vitro as well as in vivo, in addition to finding that the properties of the individual mutants differ significantly from those originally described, we determined that when this pair of mutants was coexpressed in vitro, quantitative analyses did not indicate functional rescue. Additional data are presented that provide a plausible alternate explanation for the apparent in vivo trans-activation that was reported. Finally, using LHR mutants that we have documented to be expressed at the cell surface but to lack human chorionic gonadotropin binding activity or to be severely impaired in their ability to activate Gs, we did not observe functional rescue of human chorionic gonadotropin-stimulated cAMP when the mutants were coexpressed, even though bioluminescence resonance energy transfer analyses confirmed that the coexpressed mutants formed dimers. Taken altogether, our data substantively question the concept of functional rescue between LHR mutants.

scholarly journals Biological Properties of a Novel Follicle-Stimulating Hormone/Human Chorionic Gonadotropin Chimeric Gonadotropin

Endocrinology ◽  
2006 ◽  
Vol 147 (9) ◽  
pp. 4205-4212 ◽  
Author(s):  
Louise M. Garone ◽  
Elena Ammannati ◽  
Theresa S. Brush ◽  
David J. Fischer ◽  
Enrico Gillio Tos ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Biological Properties ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Recombinant Human Fsh

A chimeric recombinant human gonadotropin, termed C3, demonstrates both follitropic and lutropic bioactivities. The α-subunit construct for C3 is comprised of the recombinant wild-type human glycoprotein hormone α-subunit. The β-subunit DNA construct for C3 encodes residues 1–145 from human chorionic gonadotropin (hCG)-β with the exceptions that FSHβ amino acid 88 (D) is substituted for hCGβ amino acid 94 (R) and FSHβ amino acids 95–108 (TVRGLGPSYCSFGE) are substituted for hCGβ amino acids 101–114 (GGPKDHPLTCDDPR). C3 is a potent FSH and LH agonist able to bind and to signal through FSH and LH receptors in vitro. In in vivo bioassays optimized to quantify each type of activity, C3 was found to have lutropin and follitropin potencies at levels similar to those of recombinant human LH and recombinant human FSH, respectively. In immature rats, C3 was sufficient to support the maturation of normal ovarian follicles. Moreover, a significant portion of follicles matured by C3 ruptured in response to an ovulatory hCG stimulus and gave rise to morphologically normal oocytes. Furthermore, a low dose of C3 promoted weight gain in the rodent uterus, suggesting it also supported preparation for implantation without histological evidence of excessive luteinization of the ovary. In summary, the biological properties of C3 indicate that its chimeric nature has resulted in a fully functional, dual-acting human gonadotropin.

scholarly journals Modulation of Ligand Selectivity Associated with Activation of the Transmembrane Region of the Human Follitropin Receptor

2004 ◽  
Vol 18 (8) ◽  
pp. 2061-2073 ◽  
Author(s):  
Lucia Montanelli ◽  
Joost J. J. Van Durme ◽  
Guillaume Smits ◽  
Marco Bonomi ◽  
Patrice Rodien ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Hormone Receptors ◽  
Transmembrane Helix ◽  
High Sensitivity ◽  
Site Directed Mutagenesis ◽  
Constitutive Activity ◽  
Glycoprotein Hormone ◽  
Functional Specificity ◽  
Ligand Selectivity

Abstract Recently, three naturally occurring mutations in the serpentine region of the FSH receptor (FSHr) (D567N and T449I/A) have been identified in three families with spontaneous ovarian hyperstimulation syndrome (OHSS). All mutant receptors displayed abnormally high sensitivity to human chorionic gonadotropin and, in addition, D567N and T449A displayed concomitant increase in sensitivity to TSH and detectable constitutive activity. In the present study, we have used a combination of site-directed mutagenesis experiments and molecular modeling to explore the mechanisms responsible for the phenotype of the three OHSS FSHr mutants. Our results suggest that all mutations lead to weakening of interhelical locks between transmembrane helix (TM)-VI and TM-III, or TM-VI and TM-VII, which contributes to maintaining the receptor in the inactive state. They also indicate that broadening of the functional specificity of the mutant FSHr constructs is correlated to their increase in constitutive activity. This relation between basal activity and functional specificity is a characteristic of the FSHr, which is not shared by the other glycoprotein hormone receptors. It leads to the interesting suggestion that different pathways have been followed during primate evolution to avoid promiscuous stimulation of the TSHr and FSHr by human chorionic gonadotropin. In the hFSHr, specificity would be exerted both by the ectodomain and the serpentine portion.

Human chorionic gonadotropin improves endometrial receptivity by increasing the expression of homeobox A10

2020 ◽  
Vol 26 (6) ◽  
pp. 413-424
Author(s):  
Mengchen Zhu ◽  
Shanling Yi ◽  
Xiaomin Huang ◽  
Junan Meng ◽  
Haixiang Sun ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Embryo Implantation ◽  
Endometrial Receptivity ◽  
Endometrial Stromal Cells ◽  
Healthy Control ◽  
Hcg Receptor ◽  
Methyltransferase 1

Abstract Homeobox A10 (HOXA10) is a characterized marker of endometrial receptivity. The mechanism by which hCG intrauterine infusion promotes embryo implantation is still unclear. This study seeks to investigate whether hCG improves endometrial receptivity by increasing expression of HOXA10. HOXA10 expression with human chorionic gonadotropin stimulation was analyzed in vitro and in vivo. Our results demonstrate that HOXA10 was decreased in the endometria of recurrent implantation failure patients compared to that in the healthy control fertile group, also we observed that hCG intrauterine infusion increased endometrial HOXA10 expression. HOXA10, blastocyst-like spheroid expansion area was increased, whereas DNA (cytosine-5-)-methyltransferase 1 was decreased when human endometrial stromal cells (hESCs) were treated with 0.2 IU/ml of hCG for 48 h. HOXA10 promoter methylation was also reduced after hCG treatment. Collagen XV (ColXV) can repress the expression of DNA (cytosine-5-)-methyltransferase 1, and hCG treatment increased the expression of ColXV. However, when the hESCs were treated with LH/hCG receptor small interfering RNA to knock down LH/hCG receptor, hCG treatment failed to repress DNA (cytosine-5-)-methyltransferase 1 expression or to increase ColXV expression. Our findings suggest that hCG may promote embryo implantation by increasing the expression of HOXA10.

Inhibition of tumor growth in vitro and in vivo by a monoclonal antibody against human chorionic gonadotropin β

2007 ◽  
Vol 114 (2) ◽  
pp. 94-102 ◽  
Author(s):  
Ning Yu ◽  
Wei Xu ◽  
Zhenggang Jiang ◽  
Qinghua Cao ◽  
Yiwei Chu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Tumor Growth ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Inhibition Of Tumor Growth

Effects of the antiandrogen hydroxyflutamide on progesterone secretion by preovulatory rat follicles in vivo and in vitro

1991 ◽  
Vol 69 (9) ◽  
pp. 1288-1293 ◽  
Author(s):  
Yallampalli Chandrasekhar ◽  
David T. Armstrong
Keyword(s):  
Luteinizing Hormone ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Serum Progesterone ◽  
Antagonistic Action ◽  
Lh Surge ◽  
Progesterone Secretion ◽  
Preovulatory Follicles

Serum and ovarian progesterone levels and in vitro production of progesterone by preovulatory follicles were measured on proestrus in pregnant mare's serum gonadotropin (PMSG) primed immature rats in which the luteinizing hormone (LH) surge and ovulation were blocked by administration of the antiandrogen hydroxyflutamide. Serum progesterone levels observed at 12:00 on proestrus were significantly elevated, twofold above those observed in vehicle-treated controls, by in vivo administration of 5 mg hydroxyflutamide 4 h earlier. In control rats, proestrous progesterone did not increase until 16:00, in parallel with rising LH levels of the LH surge. No LH surge occurred in the hydroxyflutamide-treated rats, ovulation was blocked, and serum progesterone declined throughout the afternoon of proestrus, from the elevated levels present at 12:00. Administration of human chorionic gonadotropin (hCG) at 11:00 advanced the elevation of serum progesterone by 2 h in vehicle-treated controls and prevented the decline in progesterone levels in hydroxyfiutamide-treated rats. The patterns of change in ovarian tissue concentrations with time and treatment were essentially similar to those observed for serum progesterone. In in vitro experiments, progesterone secretion during 24 h culture of preovulatory follicles obtained on PMSG-induced proestrus was significantly increased, sixfold, by addition to the culture media of 370 μM but not of 37 μM hydroxyflutamide. Testosterone (50 nM) and hCG (20 mIU/mL) caused 26- and 14-fold increases, respectively, in progesterone secretion by cultured follicles. Hydroxyflutamide significantly reduced the stimulatory effect of testosterone but not of hCG on progesterone secretion in vitro. These results suggest that the antiandrogen hydroxyflutamide stimulates progesterone secretion, both in vivo and in vitro, through an initial androgen-agonistic action, before its antagonistic action is expressed. Its androgen-antagonistic action is responsible for its ability to inhibit testosterone-induced progesterone secretion in vitro. Its failure to reduce hCG-stimulated progesterone secretion in vivo and in vitro indicates that the latter stimulation is exerted independently of, and not as a consequence of, androgen action. The decrease in serum progesterone levels on the afternoon of proestrus therefore appears to be a consequence rather than a cause of the absence of an LH surge in the hydroxyflutamide-treated rats. It is concluded that the inhibitory effect of hydroxyflutamide on the preovulatory LH surge and ovulation is due not to inhibition of progesterone secretion at the ovarian level but most likely to neuroendocrine site(s) of action of the inhibitor.Key words: antiandrogen, hydroxyflutamide, progesterone, luteinizing hormone, ovulation, human chorionic gonadotropin.

Reduction of Testicular Human Chorionic Gonadotropin Receptors by Human Chorionic Gonadotropin in vivo and in vitro

1988 ◽  
Vol 29 (4) ◽  
pp. 156-161 ◽  
Author(s):  
Mikio Namiki ◽  
Masaya Kitamura ◽  
Norio Nonomura ◽  
Masahiro Nakamura ◽  
Akihiko Okuyama ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Gonadotropin Receptors

Free α-Subunit, Free β-Subunit of Human Chorionic Gonadotropin (hCG), and Intact hCG in Sera of Healthy Individuals and Testicular Cancer Patients

1992 ◽  
Vol 38 (3) ◽  
pp. 370-376 ◽  
Author(s):  
S Madersbacher ◽  
R Klieber ◽  
K Mann ◽  
C Marth ◽  
M Tabarelli ◽  
...  
Keyword(s):  
Testicular Cancer ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Serum Concentrations ◽  
Healthy Individuals ◽  
Pathological Conditions

Abstract To determine the serum concentrations of human chorionic gonadotropin (hCG), its free beta-subunit (hCG beta), and the free alpha-subunit (free alpha) common to all human glycoprotein hormones under physiological and pathological conditions, we developed monoclonal antibody-based immunoenzymometric assays. Free alpha-subunit was detected in the sera of all healthy individuals of both sexes; hCG was measurable in sera of 54% of the men, and 46% were positive for free hCG beta; in nonpregnant women, 69.5% were positive for hCG, 68.4% for the free beta-subunit. Pathological conditions, i.e., hCG-producing tumors, were studied in vitro and in vivo. In vitro, the concentrations of hCG, free hCG beta, and free alpha in tissue-culture supernates of a choriocarcinoma cell-line ("JAR") showed a parallel pattern during time-course analysis. In vivo, in long-term follow-up studies of 13 patients with testicular cancer, serum concentrations of the three analytes paralleled each other, whether the disease was in remission or not. Because of a selective increase of free hCG beta and free alpha in 27% of seminomatous tumor patients and in 13% of the nonseminomatous patients, the percentage of tumor-marker-positive sera was increased from 15% to 42% and 57% to 70%, respectively, by the additional measurement of free hCG beta and free alpha. Thus hCG, free hCG beta, and free alpha are physiologically present in a high percentage of the sera from healthy men, and the determination of free hCG beta and free alpha, although not of prognostic value, improves the diagnostic possibilities in patients with testicular cancer.

scholarly journals Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

1999 ◽  
Vol 22 (3) ◽  
pp. 273-283 ◽  
Author(s):  
C Sen Gupta ◽  
RR Dighe
Keyword(s):  
Pichia Pastoris ◽  
Structure Function ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Recombinant Expression ◽  
Expression System ◽  
Biologically Active ◽  
Maximal Response ◽  
Glycoprotein Hormone

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalently associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hCGalpha and hCGbeta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Co-expression of both subunits in the same cell resulted in secretion of heterodimeric hCG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hCG. The level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermentor. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hCG and is suitable for structure-function studies of the hormone.

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