Therapeutic benefits of central LH receptor agonism in the APP/PS1 AD model involve trophic and immune regulation and are reproductive status dependent.

The mechanisms that underly reproductive hormone effects on cognition, neuronal plasticity, and AD risk, particularly in relation to gonadotropin LH receptor (LHCGR) signaling, remain poorly understood. To address this gap in knowledge and clarify the impact of circulating steroid hormones on the therapeutic effects of CNS LHCGR activation, we delivered the LHCGR agonist human chorionic gonadotropin (hCG) intracerebroventricularly (ICV) and evaluated functional, structural, plasticity-related signaling cascades, Aß pathology, and transcriptome differences in reproductively intact and ovariectomized (OVX) APP/PS1 AD female mice. Here we demonstrate that CNS hCG delivery restored function to wild-type levels only in OVX APP/PS1 mice. Spine density was increased in all hCG treated groups independently of reproductive status. Notably, increases in BDNF signaling and cognition, were selectively upregulated only in the OVX hCG-treated group. RNA sequencing analyses identified a significant increase in peripheral myeloid and pro-inflammatory genes within the hippocampi of the OVX group that were completely reversed by hCG treatment, identifying a potential mechanism underlying the selective therapeutic effect of LHCGR activation. Interestingly, in intact mice, hCG administration mimicked the effects of gonadectomy. Together, our findings indicate that CNS LHCGR agonism in the post-menopausal context is beneficial through trophic and immune mechanisms. Our findings also underscore the presence of a steroid-LHCGR mechanistic interaction that is unexplored yet potentially meaningful to fully understand "post-menopausal" brain function and CNS hormone treatment response.

Comparative Study of the Restoring Effect of Metformin, Gonadotropin, and Allosteric Agonist of Luteinizing Hormone Receptor on Spermatogenesis in Male Rats with Streptozotocin-Induced Type 2 Diabetes Mellitus.

We compared the effectiveness of human chorionic gonadotropin (hCG; 5 days, 20 IU/rat/day), allosteric luteinizing hormone receptor agonist TP04 (5 days, 20 mg/kg/day), and metformin (28 days, 120 mg/kg/day) in restoring spermatogenesis in male rats with type 2 diabetes mellitus. hCG and TP04 increased the levels of testosterone and expression of the steroidogenic protein StAR, the number of spermatogenic cells, thickness of the seminal epithelium, and the number and motility of mature sperm that were reduced in diabetic rats, though they did not reduce the number of defective spermatozoa. Metformin had a weak effect on steroidogenesis, but was not inferior to luteinizing hormone receptor agonist by its restorative effect on spermatogenesis and also reduced the number of defective forms of spermatozoa. Thus, the spermatogenesis-restoring effect of metformin and luteinizing hormone receptor agonist in type 2 diabetes mellitus are comparable, despite different mechanisms of action.

Stimulation of Ovulation in Immature Female Rats Using Orthosteric and Allosteric Luteinizing Hormone Receptor Agonists.

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) are widely used for the treatment of reproductive disorders and for controlled ovulation induction, but their use is limited by side effects. Allosteric agonists of the LH/hCG receptor, including thieno[2,3-d]thienopyrimidine TP03 developed by us, can become an alternative. TP03 (50 mg/rat, i.p.) when administered to immature female rats treated 48 h before with Follimag has been shown to increase progesterone levels (maximum 8 h post-treatment) and induce ovulation, as indicated by the appearance at 24 h corpus luteum (8.6 ± 0.5 per ovary). In terms of its activity, TP03 is comparable to hCG, although it acts more moderately. In the ovaries, unlike hCG, TP03 does not lead to an increase in the expression of vascular endothelial growth factor, which can cause ovarian hyperstimulation syndrome. Thus, TP03 is a promising drug as an ovulation inducer and ovarian steroidogenesis stimulator.

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro.

The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

The Effect of Low-Molecular-Weight Allosteric Agonist of Luteinizing Hormone Receptor on Functional State of the Testes in Aging and Diabetic Rats.

Human chorionic gonadotropin that is widely used for improving spermatogenesis. The effect of chorionic gonadotropin is mediated through luteinizing hormone receptor. Treatment with gonadotropin is associated with undesirable effects due to hyperactivation of testosterone production and luteinizing hormone receptor desensitization. A promising alternative could be low-molecular-weight agonists of luteinizing hormone receptors, but their effects on spermatogenesis have not been investigated. Here we analyzed the effect of a thieno[2,3-d]pyrimidines (TP), 4-((3-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno [2,3-d]pyrimidine-4-yl) phenyl)carbamoyl)pyridine 1-oxide (TP22), an allosteric agonist of luteinizing hormone receptors, on the seminiferous tubules and spermatogenic cells in 4- and 18-month-old male rats and in animals with diabetes mellitus. TP22 and gonadotropin were administered in daily doses of 15 mg/kg and 20 U/rat for 5 days. Blood testosterone level, morphology of the seminiferous tubules, and the number of germ cells in them were estimated. Being comparable by the efficiency to gonadotropin, TP22 increased the testosterone level in all the studied groups of rats and restored epithelium thickness in the seminiferous tubules and the number of spermatogonia and pachytenic spermatocytes that are reduced in aging and diabetes, but, unlike gonadotropin, did not suppress the expression of luteinizing hormone receptor. The efficacy of TP22 as a stimulator of testicular spermatogenesis has been demonstrated both under normal conditions and in age-related and diabetes-associated reproductive dysfunctions.

Rescue of Function of Mutant Luteinising Hormone Receptors with Deficiencies in Cell Surface Expression, Hormone Binding, and Hormone Signalling.

INTRODUCTION: G protein-coupled receptor (GPCR) mutations are implicated in many diseases. Most inactivating mutations cause receptor misfolding and prevent trafficking to the plasma membrane. Pharmacological chaperones can "rescue" cell surface expression of such mutants, presumably by stabilising correct folding of the nascent protein. OBJECTIVE: Here we examine the scope of intracellularly retained luteinising hormone receptor (LHR) mutants that can be "rescued" by the pharmacological chaperone LHR-Chap, and whether this allosteric agonist can also restore the function of mutant LHRs with deficiencies in hormone binding or hormone-induced signalling. METHODS: Mutant LHRs were expressed in HEK 293-T cells. Cell surface expression/localisation, hormone binding, and hCG/LHR-Chap signalling were determined by ELISA, radioligand binding, and inositol phosphate accumulation assays, respectively. Molecular modelling predicted LHR-Chap interactions. RESULTS: LHR-Chap increased cell surface expression of a subset of retained mutants located in transmembrane helices predicted to be stabilised by LHR-Chap binding. For 3 (T4613.47I, L5024.61P, and S6167.46Y) hCG-responsiveness was increased following treatment. LHRs with mutations in the hormone-binding site (C131ECDR and I152ECDT) or in the hinge region (E354HingeK) had good cell surface expression but poor response to hormone stimulation, yet were responsive to allosteric activation by LHR-Chap. CONCLUSIONS: LHR-Chap, in addition to rescuing cell surface expression of intracellularly retained LHR mutants, can rescue function in mutant receptors with binding and signalling deficiencies that have normal cell surface expression. This demonstration of rescue of multiple elements of LHR dysfunction arising from inactivating mutations offers exceptional potential for treating patients with diseases arising from GPCR mutations in general.

The Effects of Separate and Combined Treatment of Male Rats with Type 2 Diabetes with Metformin and Orthosteric and Allosteric Agonists of Luteinizing Hormone Receptor on Steroidogenesis and Spermatogenesis.

In men with type 2 diabetes mellitus (T2DM), steroidogenesis and spermatogenesis are impaired. Metformin and the agonists of luteinizing hormone/human chorionic gonadotropin(hCG)-receptor (LH/hCG-R) (hCG, low-molecular-weight allosteric LH/hCG-R-agonists) can be used to restore them. The aim was to study effectiveness of separate and combined administration of metformin, hCG and 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP3) on steroidogenesis and spermatogenesis in male rats with T2DM. hCG (15 IU/rat/day) and TP3 (15 mg/kg/day) were injected in the last five days of five-week metformin treatment (120 mg/kg/day). Metformin improved testicular steroidogenesis and spermatogenesis and restored LH/hCG-R-expression. Compared to control, in T2DM, hCG stimulated steroidogenesis and StAR-gene expression less effectively and, after five-day administration, reduced LH/hCG-R-expression, while TP3 effects changed weaker. In co-administration of metformin and LH/hCG-R-agonists, on the first day, stimulating effects of LH/hCG-R-agonists on testosterone levels and hCG-stimulated expression of StAR- and CYP17A1-genes were increased, but on the 3-5th day, they disappeared. This was due to reduced LH/hCG-R-gene expression and increased aromatase-catalyzed estradiol production. With co-administration, LH/hCG-R-agonists did not contribute to improving spermatogenesis, induced by metformin. Thus, in T2DM, metformin and LH/hCG-R-agonists restore steroidogenesis and spermatogenesis, with metformin being more effective in restoring spermatogenesis, and their co-administration improves LH/hCG-R-agonist-stimulating testicular steroidogenesis in acute but not chronic administration.

Comparative Study of the Steroidogenic Effects of Human Chorionic Gonadotropin and Thieno[2,3-D]pyrimidine-Based Allosteric Agonist of Luteinizing Hormone Receptor in Young Adult, Aging and Diabetic Male Rats.

Low-molecular-weight agonists of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor (LHCGR), which interact with LHCGR transmembrane allosteric site and, in comparison with gonadotropins, more selectively activate intracellular effectors, are currently being developed. Meanwhile, their effects on testicular steroidogenesis have not been studied. The purpose of this work is to perform a comparative study of the effects of 5-amino-N-tert-butyl-4-(3-(1-methylpyrazole-4-carboxamido)phenyl)-2-(methylthio)thieno[2,3-d] pyrimidine-6-carboxamide (TP4/2), a LHCGR allosteric agonist developed by us, and hCG on adenylyl cyclase activity in rat testicular membranes, testosterone levels, testicular steroidogenesis and spermatogenesis in young (four-month-old), aging (18-month-old) and diabetic male Wistar rats. Type 1 diabetes was caused by a single streptozotocin (50 mg/kg) injection. TP4/2 (20 mg/kg/day) and hCG (20 IU/rat/day) were administered for 5 days. TP4/2 was less effective in adenylyl cyclase stimulation and ability to activate steroidogenesis when administered once into rats. On the 3rd-5th day, TP4/2 and hCG steroidogenic effects in young adult, aging and diabetic rats were comparable. Unlike hCG, TP4/2 did not inhibit LHCGR gene expression and did not hyperstimulate the testicular steroidogenesis system, moderately increasing steroidogenic proteins gene expression and testosterone production. In aging and diabetic testes, TP4/2 improved spermatogenesis. Thus, during five-day administration, TP4/2 steadily stimulates testicular steroidogenesis, and can be used to prevent androgen deficiency in aging and diabetes.

Conservation of Steroidogenic Effect of the Low-Molecular-Weight Agonist of Luteinizing Hormone Receptor in the Course of Its Long-Term Administration to Male Rats.

Abstract-It was shown that the thienopyrimidine derivative TP03, a low-molecular-weight agonist of the luteinizing hormone receptor (LHR), during the treatment of male rats for 7 days steadily increased the production of testosterone (T), whose elevated level was retained for 7 days, and increased the expression of the gene for LHR, which indicates the maintenance of the sensitivity of Leydig cells to gonadotropins. At the same time, the steroidogenic effect of human chorionic gonadotropin (hCG), which significantly increased the T level on the first day of administration, was further weakened, which was accompanied by a decrease in the expression of the gene for LHR in the testes, indicating the development of resistance of Leydig cells to hCG. Along with this, in the case of hCG administration, a compensatory increase in the expression of genes of the steroidogenic enzymes, such as cytochrome P450scc and dehydrogenase 3ß-HSD, was shown in the testes, while in the case of TP03 administration this effect was absent.

CNS luteinizing hormone receptor activation rescues ovariectomy-related loss of spatial memory and neuronal plasticity.

Ovariectomy (OVX), a menopause model, leads to cognition and neuronal plasticity deficits that are rescued by estrogen administration or downregulation of pituitary luteinizing hormone (LH). LH is present in the brain. However, whether LH levels differ across brain regions, change across reproductive stages, or whether brain-specific LHR signaling play a role in OVX-related cognitive and neuroplasticity losses is completely unknown. To address this, we measured brain LH in cycling and OVX C57Bl/6 across brain regions and determined whether OVX-related functional and plasticity deficits could be rescued by intracerebroventricular administration of the LHR agonist (hCG). Here, we show that while pituitary LH is increased in OVX, brain LH is decreased, primarily in spatial memory and navigation areas. Furthermore, intracerebroventricular hCG delivery after OVX rescued dendritic spine density and spatial memory. In vitro, we show that hCG increased neurite outgrowth in primary hippocampal neurons in a receptor-specific manner. Taken together, our data suggest that loss of brain LH signaling is involved in cognitive and plasticity losses associated with OVX and loss of ovarian hormones.

Regulation of Luteinizing Hormone Receptor mRNA Expression in the Ovary: The Role of miR-122.

The expression of luteinizing hormone receptor (LHR) in the mammalian ovary is regulated in response to changes in the secretion of follicle-stimulating hormone and luteinizing hormone by the anterior pituitary, at least in part, through posttranscriptional mechanisms. The steady-state levels of LHR mRNA are maintained by controlling its rate of degradation by an RNA-binding protein designated as LHR mRNA-binding protein (LRBP). LRBP forms a complex with LHR mRNA and targets it for degradation in the p bodies. miR-122, an 18 nucleotide noncoding RNA, regulates the expression of LRBP. Thus, the levels of miR-122 determine the cellular levels of LHR mRNA expression. This phenomenon has been examined during the induction of LHR mRNA expression that occurs during follicle maturation in response to rising levels of FSH. In this situation, miR-122 and LRBP levels decrease as LHR mRNA expression undergoes downregulation in response to preovulatory LH surge. miR-122 expression as well as LRBP levels show robust increases. The mechanism of induction of LRBP by miR-122 has also been discussed.

Effects of gonadotropin-releasing hormone agonist on human chorionic gonadotropin activity in granulosa cells of immature female rats.

Although the expression of gonadotropin-releasing hormone (GnRH) in the ovaries is well established, its physiological role remains unknown. The aim of this study was to determine whether ovarian GnRH mediates the actions of human chorionic gonadotropin (hCG) in the granulosa cells of immature female rats. Follicular growth was induced by administration of pregnant mare serum gonadotropin (PMSG, 15 IU/0.15 ml) on day 25 after birth, and hCG (20 IU/0.2 ml) was administered on day 27 revealing the increase of plasma progesterone level. Primary cultures of granulosa cells were established from large follicles 2 days after PMSG treatment. Progesterone synthesis was augmented by hCG in a dose-dependent manner. Annexin A5 (ANXA5), a biomarker of GnRH, was expressed in the granulosa-luteal cells after hCG treatment, as shown by immunohistochemistry, suggesting that hCG treatment induced GnRH action. The GnRH mRNA level was increased by hCG, and treatment with GnRH agonist (GnRHa) increased ANXA5 mRNA levels in the primary cultures of granulosa cells. Concomitant incubation of GnRH (10-7 M) or GnRHa (fertirelin acetate, 10-8 M) with hCG suppressed progesterone synthesis during a 3 h incubation period. The mRNA expression of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) was synergistically stimulated and suppressed by hCG and GnRHa, respectively. GnRHa stimulated p21 expression, and GnRHa and hCG synergistically reduced the mRNA expression levels of p27 and FOXO1. These data suggest that GnRH induced by LH may have a role for the LH-mediated luteinization of granulosa cells. In addition, ANXA5 may be involved in GnRH action. GnRH-ANXA5 would be an important mechanism in cell differentiation.

[The decrease in the basal and luteinizing hormone receptor agonist-stimulated production of testosterone in aging male rats.]

The aging leads to a weakening of the steroid function of the testes and a decrease in their sensitivity to gonadotropins. However, the mechanisms of this are poorly understood. The aim of this work was to study the stimulating effects of human chorionic gonadotropin (hCG) and TP03, a low-molecular-weight agonist of luteinizing hormone (LH)/hCG receptor, on testosterone (T) production and the expression of steroidogenic proteins in young (3 months) and aging (15 months) male rats, and to investigate the activity of the adenylyl cyclase system in the membranes isolated from the testes of rats. The treatment with hCG (100 IU/rat/day) and TP03 (15 mg/rat/day) was carried out for 3 days. In the testes of aging rats the stimulation of adenylyl cyclase (AC) by gonadotropin and guanine nucleotide was decreased, indicating a weakening of the coupling of LH/hCG receptor and Gs protein, the main components of the adenylyl cyclase system regulating the steroidogenesis. In elderly rats, the T level in the blood and the expression of the Star, Cyp11a1 and Cyp17a1 genes encoding the StAR protein and the steroidogenic enzymes cytochromes P450scc and P450-17α in the testes were decreased. With increasing age, the stimulating effect of hCG and TP03 on the T production was weakened, despite the different mechanisms of their action on LH/hCG receptor. The treatment of both young and aging rats with hCG led to an increase in the expression of the genes encoding StAR, P450scc and dehydrogenase 3ß-HSD, while in aging rats, in addition, the expression of the Hsd17B gene was increased and in young rats the expression of the genes encoding Р450-17α and 17ß-HSD was reduced. The treatment of in young rats with TP03 led to an increase in the Star and Cyp17a1 expression, and the TP03 treatment of aging rats increased the Star and Hsd17B expression. Thus, in the testes of aging rats, the coupling between LH/hCG receptor and Gs-protein and the sensitivity of LH/hCG receptor to agonists were weakened, which leads to a decrease in the hCG- and TP03-induced production of T, and the basal and LH/hCG receptor agonists-stimulated levels of gene expression for some steroidogenic proteins were changed.

Therapeutic Potential of Human Chorionic Gonadotropin Against Overactive Bladder.

Overactive bladder (OAB) is a common form of urinary incontinence, resulting from spontaneous and random contractions of the urinary bladder. The affected individuals have an uncontrollable urge to urinate and experience incontinence and nocturia, which can greatly reduce the quality of daily life. There are several drugs for the treatment, and all of them have serious side effects. The following findings suggested that human chorionic gonadotropin (hCG) has a therapeutic potential that is worth investigating for the treatment of OAB. The finding are (1) human detrusor muscle contains hCG receptors, (2) detrusor muscle becomes quiescent during pregnancy, (3) hCG can inhibit detrusor muscle contractions induced by cholinergic stimulation in rats, and (4) hCG can mimic the anticholinergic drug on detrusor muscle contractions.

Embryonic Poly(A)-Binding Protein (EPAB) Is Required for Granulosa Cell EGF Signaling and Cumulus Expansion in Female Mice.

Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos before zygotic genome activation. EPAB is required for translational activation of maternally stored mRNAs in the oocyte and Epab(-/-) female mice are infertile due to impaired oocyte maturation, cumulus expansion, and ovulation. The aim of this study was to characterize the mechanism of follicular somatic cell dysfunction in Epab(-/-) mice. Using a coculture system of oocytectomized cumulus oophorus complexes (OOXs) with denuded oocytes, we found that when wild-type OOXs were cocultured with Epab(-/-) oocytes, or when Epab(-/-) OOXs were cocultured with WT oocytes, cumulus expansion failed to occur in response to epidermal growth factor (EGF). This finding suggests that oocytes and cumulus cells (CCs) from Epab(-/-) mice fail to send and receive the necessary signals required for cumulus expansion. The abnormalities in Epab(-/-) CCs are not due to lower expression of the oocyte-derived factors growth differentiation factor 9 or bone morphogenetic protein 15, because Epab(-/-) oocytes express these proteins at comparable levels with WT. Epab(-/-) granulosa cells (GCs) exhibit decreased levels of phosphorylated MEK1/2, ERK1/2, and p90 ribosomal S6 kinase in response to lutenizing hormone and EGF treatment, as well as decreased phosphorylation of the EGF receptor. In conclusion, EPAB, which is oocyte specific, is required for the ability of CCs and GCs to become responsive to LH and EGF signaling. These results emphasize the importance of oocyte-somatic communication for GC and CC function.

[New achievements in the development and study of the mechanisms of action of the low molecular weight agonists of receptors of the thyroid-stimulating and the luteinizing hormones].

Pituitary glycoprotein hormones, luteinizing (LH) and thyroid-stimulating (TSH), exert their regulatory effects on cells through the G protein-coupled receptors, specifically binding to their extracellular domain. There is an alternative way of activation of LH and TSH receptors, when low molecular weight organic molecules bind to an allosteric site of the receptors which is localized within their transmembrane channel. Low molecular weight agonists have many advantages over glycoprotein hormones, among them a high efficiency not only in the case of the parenteral but also in the oral administration, low immunogenicity, chemical stability, and a low cost. Unlike pituitary glycoprotein hormones with the agonistic activity, low molecular weight compounds may be either agonists or inverse agonists and neutral antagonists. Recently it was shown that low molecular weight agonists of LH receptor are able to stimulate its mutant forms by restoring the processing of receptor in a cell, and by increasing its sensitivity to LH, which is important for the treatment of reproductive dysfunctions caused by mutations in the LH receptor. This review summarizes the recent achievements that are linked with the development of low molecular weight regulators of TSH and LH receptors and the study of their mechanisms of action. It also presents the author' data concerning the creation of new low molecular weight agonists of LH receptor based on the thienopyrimidine structure, which are effective both in vitro, and in vivo in different ways of administration.

The Presence of Human Chorionic Gonadotropin/Luteinizing Hormone Receptors in Pancreatic ß-Cells.

We investigated the possible presence of functional human chorionic gonadotropin (hCG)/luteinizing hormone (LH) receptors in ß-cells of pancreas, using a combination of techniques on hCG/LH receptor knockout mice, immortalized rat insulinoma cells, and human pancreatic islets. The results showed the presence of receptors and their activation resulted in a dose-dependent increase in glucose-induced release of insulin. These findings place hCG and LH among the regulators of insulin release with potential implications for insulin-level changes during the periods of altered hCG and LH secretion.

LH and hCG action on the same receptor results in quantitatively and qualitatively different intracellular signalling.

Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED(50): 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.

[The secondary structure of peptides derived from the third intracellular loop of the serpentine type receptors and its interrelation with their biological activity].

We and other authors have shown that synthetic peptides corresponding to regions of the third intracellular loop (ICL-3) of receptors of the serpentine type are capable of activating G-protein signaling cascades and trigger them in the absence of hormone. To create on the basis of these peptides the selective regulators of hormonal signaling systems the relationship between their biological activity and secondary structure are studied. It is assumed that most suitable is a helical conformation, which allows the peptide effectively interact with signaling proteins. The aim of this study was to test the biological activity and secondary structure of synthesized by us linear peptides and their dimeric and palmitoylated analogs, corresponding to C-terminal region of the ICL-3 of luteinizing hormone receptor (LHR) and 5-hydroxytryptamine receptor of the type 6 (5-HT6R). It is shown that LHR-peptides at the micromolar concentrations stimulate the basal activity of adenylyl cyclase (AC) and the GTP-binding of G-proteins in the plasma membranes of rat testes, while 5-HT6R-peptides activate AC and G-proteins in the synaptosomal membranes of rat brain. The action of peptides is tissue-specific and observed in the tissues where there are homologous receptors. The most effective were palmitoylated peptides. LHR-peptide reduced the AC stimulatory effect of human chorionic gonadotropin, while 5-HT6R-peptides the effect of 5-HT6R-agonist, EMD-386088, and the action of the peptides was not found in the case of non-homologous receptors. Using circular dichroism spectroscopy it is shown that in neutral (pH 7) and acidic (pH 2) medium all the peptides are exist predominantly in the antiparallel beta-sheet (37-42%) and disordered conformations (33-35%). In alkaline medium (pH 10) in the case palmitoylated peptides the increase of the contribution of the helical conformation to 12-27% was observed. In the presence of trifluoroethanol (10-80%), a helix-forming solvent, the contribution of helical conformation for the majority of peptides was slightly increased (for palmitoylated analogs to 14%), however, in this case the antiparallel beta-sheet and disordered conformation prevailed. The conclusion was made that the lack of clearly expressed ability to form helices in peptides derived the ICLs of receptors did not significantly affect their activity. This is consistent with proposed mechanism of peptides action, whereby peptide interacts with the complementary regions of homologous receptor that does not require the helix formation.