Position of Pro and Ser near Glu7.32 in the extracellular loop 3 of mammalian and nonmammalian gonadotropin-releasing hormone (GnRH) receptors is a critical determinant for differential ligand selectivity for mammalian GnRH and chicken GnRH-II

A Glu/Asp7.32 residue in the extracellular loop 3 of the mammalian GnRH receptor (GnRHR) is known to interact with Arg8 of mammalian GnRH (mGnRH), which may confer preferential ligand selectivity for mGnRH than for chicken GnRH-II (cGnRH-II). However, some nonmammalian GnRHRs also have the Glu/Asp residue at the same position, yet respond better to cGnRH-II than mGnRH. Amino acids flanking Glu/Asp7.32 are differentially arranged such that mammalian and nonmammalian GnRHRs have an S-E/D-P motif and P-X-S/Y motif, respectively. We presumed the position of Ser7.31 or Pro7.33 of rat GnRHR as a potential determinant for ligand selectivity. Either placing Pro before Glu7.32 or placing Ser after Glu7.32 significantly decreased the sensitivity and/or efficacy for mGnRH, but slightly increased that for cGnRH-II in several mutant receptors. Among them, those with a PEV, PES, or SES motif exhibited a marked decrease in sensitivity for mGnRH such that cGnRH-II had a higher potency than mGnRH, showing a reversed preferential ligand selectivity. Chimeric mGnRHs in which positions 5, 7, and/or 8 were replaced by those of cGnRH-II revealed a greater ability to activate these mutant receptors than mGnRH, whereas they were less potent to activate wild-type rat GnRHR than mGnRH. Interestingly, a mutant bullfrog type I receptor with the SEP motif exhibited an increased sensitivity for mGnRH but a decreased sensitivity for cGnRH-II. These results indicate that the position of Pro and Ser near Glu7.32 in the extracellular loop 3 is critical for the differential ligand selectivity between mammalian and nonmammalian GnRHRs.     

Comparative studies on the major features of insulin receptors in mammalian and non-mammalian liver membranes

1. The camel has insulin receptors that by multiple function criteria are very similar to those of the other mammals (rabbit and rat) and non-mammals (chicken and pigeon), with sharp pH dependence to insulin binding at pH 7.2-7.6. 2. Equilibrium binding was faster at higher temperatures (24-37 degrees C) than at lower (4 degrees C). 3. Binding data yielded curvilinear Scatchard plots with half maximal displacement of 125I-insulin at 9 x 10(-9) M, 2.5 x 10(-9) M, 6.3 x 10(-10) M for camel, rabbit, pigeon and chicken respectively, suggesting differences in mammalian and non-mammalian liver membranes. 4. Autoradiogram patterns showed the presence of an identical subunit structure with Mr 74,000 for all membranes studied. Pigeon membrane showed a band with Mr 110,000, the absence of which in other membranes could be due to the degradation factor or the concentration of disuccinimidyl suberate (DSS).     

Characterization of insulinase from mammalian and non-mammalian livers

The Michaelis constants (Km's) and maximum reaction velocities (Vmax's) for the degradation of beef insulin by livers from frogs, guinea pigs, rats, a rabbit, a dog and a pig were determined. The Km's for mammalian livers appear to be species-dependent and range from 0.25 microM to 0.65 microM. The Km for frog liver was somewhat lower, averaging 0.13 microM. The Km is independent of animal age, but the enzyme concentrations (Vmax) were greatly reduced in the fetal guinea pig and 3 day rat compared to the adult livers. There appears to be no relation between Km and the chemical dissimilarity between beef insulin and endogenous insulin of the species, since guinea pig liver insulinase had a Km (0.50 microM) intermediate between dog (0.47 microM) and pig (0.65 microM) liver insulinase although guinea pig insulin has a markedly different amino acid sequence and biologic activity.     

Bradykinin and its receptors in non-mammalian vertebrates

The generation of bradykinin (BK) in blood by the action of the kallikrein-kinin system has been studied intensively in mammals but the system has received relatively little attention in non-mammalian vertebrates. The plasma of crocodilians and Testudines (turtles and tortoises) contains all the components of the kallikrein-kinin system found in mammals (prekallikrein activator, prekallikrein, kininogen, and kininases) and activation results in generation of [Thr6]-BK. Plasma of birds and snakes probably lacks a prekallikrein activator analogous to mammalian Factor XII but treatment with exogenous proteases (pig pancreatic kallikrein and/or trypsin) generates [Thr6, Leu8]-BK (chicken), [Ala1, Thr6]-BK (python) and [Val1, Thr6]-BK (colubrid snakes). The skins of certain frogs, particularly of the genus Rana, contain very high concentrations of BK-related peptides but their pathway of biosynthesis involves the action of cellular endoproteinase(s) cleaving at the site of single arginyl residues rather than by the action of the kallikrein-kinin system. Evidence for a prekallikrein activator in fish plasma is lacking but treatment with exogenous proteases generates [Arg0, Trp5, Leu8]-BK (trout and cod), [Trp5]-BK (bowfin and gar), [Met1, Met5]-BK (sturgeon). The cardiovascular actions and effects upon gastrointestinal smooth muscle of these peptides in their species of origin differ markedly. For example, intra-arterial injections of the native BK peptides into unanesthetized fish produce transient hypertension in the cod, complex depressor and pressor responses in the trout and bowfin and hypotension in the sturgeon. Pharmacological studies in snakes and fish and with the recombinantally expressed chicken BK receptor have demonstrated that the BK receptors in the tissues of non-mammalian vertebrates have appreciably different ligand binding properties from the well-characterized mammalian B1 and B2 receptors.     

Homologue of mammalian apolipoprotein A-II in non-mammalian vertebrates

Although apolipoprotein with molecular weight 14 kDa （apo-14kDa） is associated with fish plasma highdensity lipoproteins （HDLs）, it remains to be determined whether apo-14 kDa is the homologue of mammalian apoA-II. We have obtained the full eDNA sequences that encode Japanese eel and rainbow trout apo-14 kDa. Homologues of Japanese eel apo-14 kDa sequence could be found in 14 fish species deposited in the DDBJ/EMBL/GenBank or TGI database. Fish apo- 14 kDa lacks propeptide and contains more internal repeats than mammalian apoA-II. Nevertheless, phylogenetic analysis allowed fish apo-14 kDa to be the homologue of mammalian apoA-II. In addition, in silico cloning of the TGI, Ensembl, or NCBI database revealed apoA-IIs in dog, chicken, green anole lizard, and African clawed frog whose sequences had not so far been available, suggesting both apoA-I and apoA-II as fundamental constituents of vertebrate HDLs.     

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His⁵,Trp⁷,Leu⁸]-GnRH, dogfish (df) GnRH; [His⁵,Asn⁸]-GnRH, catfish (cf) GnRH; and [His⁵,Trp⁷,Tyr⁸]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro ( )-isomers -arginine ( -Arg) or -naphthylalanine ( -Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of -Nal⁶ into [His⁵,Asn⁸]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of -Nal⁶ or -Arg⁶ into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [ -Nal⁶,Pro⁹NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [ -Nal⁶,Pro⁹NEt]-mGnRH (k_d = 0.40 ± 0.04 and 0.55 ± 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.     

GnRHs and GnRH receptors

GnRH is the pivotal hypothalamic hormone regulating reproduction. Over 20 forms of the decapeptide have been identified in which the NH2- and COOH-terminal sequences, which are essential for receptor binding and activation, are conserved. In mammals, there are two forms, GnRH I which regulates gonadotropin and GnRH II which appears to be a neuromodulator and stimulates sexual behaviour. GnRHs also occur in reproductive tissues and tumours in which a paracrine/autocrine role is postulated. GnRH agonists and antagonists are now extensively used to treat hormone-dependent diseases, in assisted conception and have promise as novel contraceptives. Non-peptide orally-active GnRH antagonists have been recently developed and may increase the flexibility and range of utility. As with GnRH, GnRH receptors have undergone co-ordinated gene duplications such that cognate receptor subtypes for respective ligands exist in most vertebrates. Interestingly, in man and some other mammals (e.g. chimp, sheep and bovine) the Type II GnRH receptor has been silenced. However, GnRH I and GnRH II still appear to have distinct roles in signalling differentially through the Type I receptor (ligand-selective-signalling) to have different downstream effects. The ligand-receptor interactions and receptor conformational changes involved in receptor activation have been partly delineated. Together, these findings are setting the scene for generating novel selective GnRH analogues with potential for wider and more specific application.     

Conformational constraint of mammalian,chicken, and salmon GnRHs,but not GnRH II,enhances binding at mammalian and nonmammalian receptors: evidence for preconfiguration of GnRH II

Mammalian GnRH (mGnRH) is believed to interact with mGnRH type I receptors in a beta-II' turn conformation involving residues 5-8. This conformation can be constrained by substitution of a D-amino acid at position 6 or by a lactam ring involving residues 6 and 7, thereby increasing receptor binding affinity. It has been proposed that this is not the case for non-mGnRH receptors. However, we show that this conformational constraint increases the binding affinity of mammalian, chicken, and salmon GnRH for the chicken and catfish receptors, as well as for the mouse receptor. Therefore, we conclude that the beta-II' turn conformation enhances ligand binding for non-mGnRH as well as mGnRH type I receptors. In contrast, most substitutions of a D-amino acid in position 6 have limited effect on binding affinity for GnRH II. We suggest that this ligand is preconfigured through intramolecular interactions, which accounts for its high binding affinity and total conservation of primary structure over 500 million years of evolution.     

Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling

Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.     

Identification of Darmstoff analogs as selective agonists and antagonists of lysophosphatidic acid receptors

Darmstoff describes a family of gut smooth muscle-stimulating acetal phosphatidic acids initially isolated and characterized from the bath fluid of stimulated gut over 50 years ago. Despite similar structural and biological profiles, Darmstoff analogs have not previously been examined as potential LPA mimetics. Here, we report a facile method for the synthesis of potassium salts of Darmstoff analogs. To understand the effect of stereochemistry on lysophosphatidic acid mimetic activity, synthesis of optically pure stereoisomers of selected Darmstoff analogs was achieved starting with chiral methyl glycerates. Each Darmstoff analog was evaluated for subtype-specific LPA receptor agonist/antagonist activity, PPARgamma activation, and autotaxin inhibition. From this study we identified compound 12 as a pan-antagonist and several pan-agonists for the LPA(1-3) receptors. Introduction of an aromatic ring in the lipid chain such as analog 22 produced a subtype-specific LPA(3) agonist with an EC(50) of 692 nM. Interestingly, regardless of their LPA(1/2/3) ligand properties all of the Darmstoff analogs tested activated PPARgamma. However, these compounds are weak inhibitors of autotaxin. The results indicate that Darmstoff analogs constitute a novel class of lysophosphatidic acid mimetics.     

Effects of salmon GnRH and chicken GnRH-II on testicular apoptosis in goldfish (Carassius auratus)

Apoptosis is a form of cell death, characterized by morphological and biochemical changes. Apoptosis occurs in the normal testis and in response to different agents. In this study, we investigated the effect of gonadotropin-releasing hormone (GnRH) in testicular apoptosis in the goldfish. GnRH is a decapeptide that is expressed in different tissues, including gonads in mammalian and non-mammalian species. While GnRH is considered to be a paracrine mediator of ovarian follicular atresia, the role of GnRH in the testis is less clear. In the present study, treatments with native salmon GnRH and chicken GnRH-II increased DNA fragmentation (a hallmark of apoptosis) in the mature goldfish testis. On the other hand, gonadotropin hormone was found to act as survival factor, by decreasing spontaneous and GnRH-induced DNA fragmentation in the goldfish testis. The results demonstrate that GnRH plays an important paracrine role in the control of apoptosis in the goldfish testis.     

GnRH and GnRH receptors: distribution,function and evolution

Gonadotropin‐releasing hormone (GnRH) was originally identified because of its essential role in regulating reproduction in all vertebrates. Since then, three phylogenetically related GnRH decapeptides have been characterized in vertebrates and invertebrates. Almost all tetrapods investigated have at least two GnRH forms (GnRH1 and GnRH2) in the central nervous system. From distributional and functional studies in vertebrates, GnRH1 in the hypothalamus projects predominantly to the pituitary and regulates reproduction via gonadotropin release. GnRH2, which is located in the midbrain, projects to the whole brain and is thought to be involved in sexual behaviour and food intake. GnRH3, located in the forebrain, has only been found in teleost fish and appears to be involved in sexual behaviour, as well as, in some fish species, gonadotropin release. Multiple GnRH receptors (GnRH‐Rs), G‐protein‐coupled receptors regulate endocrine functions and neural transmissions in vertebrates. Phylogenetic and structural analyses of coding sequences show that all vertebrate GnRH‐Rs cluster into two main receptor types comprised of four subfamilies. This suggests that at least two rounds of GnRH receptor gene duplications may have occurred in different groups within each lineage. Functional studies suggest that two particular subfamilies of GnRH receptors have independently evolved to act as species‐specific endocrine modulators in the pituitary, and these show the greatest variety in regulating neuron networks in the brain. Given the long evolutionary history of the GnRH system, it seems likely that much more remains to be understood about its roles in behaviour and function of vertebrates.     

Gonadotropin-releasing hormone (GnRH)-I and GnRH-II induce cell growth inhibition in human endometrial cancer cells: Involvement of integrin beta3 and focal adhesion kinase

Endometrial carcinoma is the most common neoplasm of the female genital tract, accounting for nearly one half of all gynecologic cancers in the Western world. Although intensive research on pathological phenomena of endometrial cancer is currently going on, but exact cause and biological aspects of this disease are not well described yet. In addition to well-documented roles of gonadotropin-releasing hormone (GnRH) in hypopituitary ovarian (HPO) axis, the agonistic or antagonistic analogs (or both) of GnRH have been shown to inhibit the proliferation of a variety of human gynecologic cancers. Thus, in the present study, we further examined the possibility that GnRH induces integrin beta3 and activation of focal adhesion kinase (FAK) through mitogen-activated protein kinases (MAPKs), ERK1/2 and p38, to inhibit the growth of HEC1A endometrial cancer cell line. As a result, both GnRH-I and GnRH-II resulted in a significant increase in integrin beta3 expression and evoked the activation of FAK in a time-dependent manner in these cells. In addition, these analogs induced an activation of ERK1/2 and p38 MAPK in a time-dependent manner as downstream pathways of FAK. It appears that GnRH-II has much greater effect on the activation of FAK, ERK1/2 and p38 compared to GnRH-I in these cells. Further, we demonstrated that the growth inhibition of HEC1A cells by GnRH-I or GnRH-II is involved in the activation of integrin-FAK and ERK1/2 and p38 MAPK pathways. Taken together, these results suggest that GnRH may be involved in the inhibition of endometrial cancer cell growth via activation of integrin beta3 and FAK as a direct effect. This knowledge could contribute to a better understanding of the mechanisms implicated in the therapeutic action of GnRH and its biomedical application for the treatment against endometrial cancer.     

Trypanocidal activity of peptidyl vinyl ester derivatives selective for inhibition of mammalian proteasome trypsin-like activity

Nine vinyl ester tripeptides selective for inhibition of mammalian proteasome trypsin-like activity were tested for in vitro activity against Trypanosoma brucei. Interestingly, two compounds showed trypanocidal activity in the low micromolar range without displaying cytotoxicity against human cells. However, the compounds did not inhibit the trypsin-like activity of the trypanosome proteasome although their effect correlates with inactivation of the chymotrypsin-like activity. This finding shows that the inhibitor sensitivities between mammalian and trypanosome proteasome are distinct. This difference may be exploited for rational anti-trypanosomal drug development.     

Effect of GnRH analogs in postnatal domestic cats

The aim of this study was to reproductively assess the clinical and hormonal effects of a GnRH agonist (AG) and an antagonist (AN) administered during the postnatal period in domestic cats. Forty-eight male and female postnatal kittens were randomly assigned to deslorelin acetate 1.6 mg subcutaneous (AG; n = 16), acyline 33 μg/100 g subcutaneous weekly for 3 months (AN; n = 16), or control (CO; n = 16) which remained untreated. The cats were followed up (behavioral observation, physical examination, fecal sexual steroid determinations, mating test, and pregnancy diagnosis) up to puberty. Puberty was delayed (weeks) in the AG animals (62.9 ± 3.5; P < 0.01) but not in the AN (15.5 ± 1.7; P > 0.05) when they were compared with CO kittens (13.4 ± 0.4). Fifteen (15/16) of the AN and CO animals, and only 11 of 16 cats of the AG group were fertile (P > 0.1). No differences were found in body weight (P > 0.1) and measurements (P > 0.1), libido (P > 0.1) and in the appearance of side effects (P > 0.1; except a pyometra in an AG female) among groups. In both AG- and AN-treated males (testosterone; P < 0.01) and females (estradiol-17β; P < 0.01) fecal hormone concentrations were lower than in CO group during the first five postnatal weeks but not later. It is concluded that the neonatal administration of these AG and AN decreased fecal sexual steroids during the first postnatal weeks causing, the agonists but not the antagonist, a significant, reversible delay in puberty appearance.     

Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigating odorant-OR relationships in mammals has been the inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput 'deorphanization' of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently expressed mammalian ORs in HEK293T cells. The stages of OR cell-surface expression include cell culture preparation, transfer of cells, transfection, immunocytochemistry or flow cytometry, odorant stimulation and luciferase assay. This protocol can be completed in a period of 3 d from the transfer of cells to cell-surface expression detection and/or measurement of functional activation.     

Pituitary somatostatin receptors: dissociation at the pituitary level of receptor affinity and biological activity for selective somatostatin analogs

High affinity and saturable binding of [125I-Tyr11]somatostatin (SS) is described in membrane homogenates from a pituitary transplantable tumor (GH4C1) rich in somatotrophs (KD for SS = 0.67 nM; Bmax = 30 fmol/mg protein). Binding characteristics and pharmacology are similar to those measured on normal pituitary membranes. The potency of various SS analogs highly correlates with that measured in in vitro bioassay for growth hormone. This suggests that those GH4C1 membranes are a good model for SS receptors on somatotrophs. Interestingly however, analogs in which the Asn5 is deleted (Des-Asn5) or D-Ser replaces Ser13 show dissociated potencies between the various assays: [D-Ser13] analogs are more potent in pituitary than in GH4C1 membranes binding assay. Des-Asn5-modified analogs are much more potent in both pituitary binding assays than in the bioassay. This could reflect a multiplicity of SS receptor subtypes in pituitary.     

Potent and selective inhibition of human cytochrome P450 3A4 by seco-pancratistatin structural analogs

seco-Derivatives of the anticancer agent pancratistatin bearing the 2S,3S,4S,5S configuration were accessed via a novel, highly diastereoselective anti-aldol reaction. Structure–activity relationships reveal important insights into the seco-pancratistatin pharmacophore as a potent and selective inhibitor of human cytochrome P450 3A4 (CYP3A4), and highlight features of concern in advancing a potent, selective anticancer agent in the pancratistatin series.     

Evolution of GnRH ligands and receptors in gnathostomata

Gonadotropin-releasing hormone (GnRH) is the final common signaling molecule used by the brain to regulate reproduction in all vertebrates. Until now, a total of 24 GnRH structural variants have been characterized from vertebrate, protochordate and invertebrate nervous tissue. Almost all vertebrates already investigated have at least two GnRH forms coexisting in the central nervous system. Furthermore, it is now well accepted that three GnRH forms are present both in early and late evolved teleostean fishes. The number and taxonomic distribution of the different GnRH variants also raise questions about the phylogenetic relationships between them. Most of the GnRH phylogenetic analyses are in agreement with the widely accepted idea that the GnRH family can be divided into three main groups. However, the examination of the gnathostome GnRH phylogenetic relationships clearly shows the existence of two main paralogous GnRH lineages: the 'midbrain GnRH" group and the "forebrain GnRH" group. The first one, represented by chicken GnRH-II forms, and the second one composed of two paralogous lineages, the salmon GnRH cluster (only represented in teleostean fish species) and the hypophysotropic GnRH cluster, also present in tetrapods. This analysis suggests that the two forebrain clades share a common precursor and reinforces the idea that the salmon GnRH branch has originated from a duplication of the hypophysotropic lineage. GnRH ligands exert their activity through G protein-coupled receptors of the rhodopsin-like family. As with the ligands, multiple GnRHRs are expressed in individual vertebrate species and phylogenetic analyses have revealed that all vertebrate GnRHRs cluster into three main receptor types. However, new data and a new phylogenetic analysis propose a two GnRHR type model, in which different rounds of gene duplications may have occurred in different groups within each lineage.