scholarly journals A Crucial Role for Gαq/11, But Not Gαi/o or Gαs, in Gonadotropin-Releasing Hormone Receptor-Mediated Cell Growth Inhibition

2008 ◽  
Vol 22 (11) ◽  
pp. 2520-2530 ◽  
Author(s):  
Colin D. White ◽  
Marla Coetsee ◽  
Kevin Morgan ◽  
Colleen A. Flanagan ◽  
Robert P. Millar ◽  
...  
Keyword(s):  
Cell Growth ◽  
Crucial Role ◽  
Direct Evidence ◽  
Gnrh Receptor ◽  
Binding Assays ◽  
Erk Activation ◽  
Cellular Effects ◽  
Mapk Cascades ◽  
Gtpγs Binding ◽  
Gonadotropin Releasing Hormone Receptor

Abstract GnRH acts on its cognate receptor in pituitary gonadotropes to regulate the biosynthesis and secretion of gonadotropins. It may also have direct extrapituitary actions, including inhibition of cell growth in reproductive malignancies, in which GnRH activation of the MAPK cascades is thought to play a pivotal role. In extrapituitary tissues, GnRH receptor signaling has been postulated to involve coupling of the receptor to different G proteins. We examined the ability of the GnRH receptor to couple directly to Gαq/11, Gαi/o, and Gαs, their roles in the activation of the MAPK cascades, and the subsequent cellular effects. We show that in Gαq/11-negative cells stably expressing the GnRH receptor, GnRH did not induce activation of ERK, jun-N-terminal kinase, or P38 MAPK. In contrast to Gαi or chimeric Gαqi5, transfection of Gαq cDNA enabled GnRH to induce phosphorylation of ERK, jun-N-terminal kinase, and P38. Furthermore, no GnRH-mediated cAMP response or inhibition of isoproterenol-induced cAMP accumulation was observed. In another cellular background, [35S]GTPγS binding assays confirmed that the GnRH receptor was unable to directly couple to Gαi but could directly interact with Gαq/11. Interestingly, GnRH stimulated a marked reduction in cell growth only in cells expressing Gαq, and this inhibition could be significantly rescued by blocking ERK activation. We therefore provide direct evidence, in multiple cellular backgrounds, that coupling of the GnRH receptor to Gαq/11, but not to Gαi/o or Gαs, and consequent activation of ERK plays a crucial role in GnRH-mediated cell death.

The ERK pathway involves positive and negative regulations of HT-29 colorectal cancer cell growth by extracellular zinc

2003 ◽  
Vol 285 (6) ◽  
pp. G1181-G1188 ◽  
Author(s):  
Ki-Sook Park ◽  
Nam-Gu Lee ◽  
Ki-Hoo Lee ◽  
Jeong Taeg Seo ◽  
Kang-Yell Choi
Keyword(s):  
Colorectal Cancer ◽  
Signal Transduction ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Cyclin D1 ◽  
Growth Regulation ◽  
Erk Pathway ◽  
Differential Growth ◽  
Erk Activation ◽  
Ht 29

Dietary zinc is an important trace element in the body and is related to both cell proliferation and growth arrest. A recent study found that extracellular zinc-sensing receptors trigger intracellular signal transduction in HT-29 human colorectal cancer cells. However, the signaling mechanism causing this growth regulation by extracellular zinc is not clearly understood. At 10- and 100-μM levels of ZnCl2 treatment, HT-29 cell growth and proliferation increased and decreased, respectively, in a minimally serum-starved medium (MSSM). A lack of significant increase in intracellular zinc levels after zinc treatment suggested that this differential growth regulation of HT-29 cells by extracellular zinc is acquired by receptor-mediated signal transduction. Moreover, this zinc-induced growth regulation was differentially affected by PD-98059, suggesting the involvement of the ERK pathway. Transient ERK activation and subsequent cyclin D1 induction were observed on adding 10 μM ZnCl2 in MSSM in the presence of cell proliferation. On the other hand, prolonged ERK activity was observed with a subsequent increase of cyclin D1 and p21Cip/WAF1 on adding 100 μM ZnCl2 in MSSM, and this was associated with nonproliferation. Moreover, this ERK activation and cyclin D1 and p21Cip/WAF1 induction were abolished by PD-98059 pretreatment. The differential regulations of cell growth, ERK activities, and cyclin D1 and p21Cip/WAF1 inductions were also observed in serum-enriched medium containing higher zinc concentrations. Therefore, differential cell cycle regulator induction occurs by a common ERK pathway in the differential growth regulation of HT-29 cells by extracellular zinc.

scholarly journals EBF recommendation on practical management of critical reagents for antidrug antibody ligand-binding assays

Bioanalysis ◽  
2019 ◽  
Vol 11 (19) ◽  
pp. 1787-1798 ◽  
Author(s):  
Susanne Pihl ◽  
Barry WA van der Strate ◽  
Michaela Golob ◽  
Janka Ryding ◽  
Laurent Vermet ◽  
...  
Keyword(s):  
Life Cycle ◽  
Ligand Binding ◽  
Crucial Role ◽  
Binding Assays ◽  
Antidrug Antibody ◽  
Regulatory Guidelines ◽  
Minimum Requirements ◽  
Antidrug Antibodies ◽  
Immunogenicity Assays

Immunogenicity (ISI) assays are required to measure antidrug antibodies that are generated against biotherapeutic modalities. As for any ligand-binding assays, critical reagents (CR) play a crucial role in immunogenicity assays, as the robustness and reliability of an assay are defined by the quality and long-term availability of these reagents. The current regulatory guidelines do not provide clear directions on how to implement and verify lot-to-lot changes of CR during an assay life cycle, or the acceptance criteria that should be used when implementing new lots of CR. These aspects were extensively discussed within the European Bioanalysis Forum community. In this paper, CR for immunogenicity assays are identified and the minimum requirements for introducing new lots of CR in immunogenicity assays are described.

Interaction Of Folk Medicinal Plant Extracts With Human ɑ2-Adrenoceptor Subtypes

2002 ◽  
Vol 57 (3-4) ◽  
pp. 332-338 ◽  
Author(s):  
Ammar Saleem ◽  
Mia Engström ◽  
Siegfried Wurster ◽  
Juha-Matti Savola ◽  
Kalevi Pihlaja
Keyword(s):  
Medicinal Plant ◽  
Binding Activity ◽  
Binding Assays ◽  
Peganum Harmala ◽  
Plant Materials ◽  
Drug Candidates ◽  
Medicinal Plant Extracts ◽  
Gtpγs Binding ◽  
Competition Binding ◽  
Specific Ligand

Forty-two extracts of folk medicinal plant organs from Pakistan were tested in competition binding assays for their interaction with the specific ligand recognition sites on the human α2-adrenoceptor subtypes α2A, α2B and α2C. Strong binding of the extracts (40 mg/ml) from Acacia nilotica (L.) Delile leaves (88-98% displacement of radiolabel) and Peganum harmala seeds (89-96% displacement) on three subtypes prompted us to extract these plant materials with 40% and 80% methanol, ethanol, and acetone. The extraction results indicated an absence of α2-adrenoceptor binding activity in the stalk of A. nilotica and A. tortils, whereas the leaves of both plants contained activity. The extracts of A. nilotica leaves showed a slight, but consistent, preference for the α2C-adrenoceptor, whereas the leaves of A. tortils were slightly more active on the α2B subtype. The extract of P. harmala stalks was less active than that of its seeds. The binding activities of A. nilotica leaves and P. harmala seeds were mainly concentrated in the water and 30% methanol fractions and further sub-fractions. In a functional activity assay, the active fractions inhibited epinephrine-stimulated 35S-GTPγS binding, thus indicating a predominantly antagonistic nature of the compounds with α2-adrenoceptor affinity in these fractions. Among the known major alkaloids of P. harmala (demissidine, harmaline, harmine, 6-methoxyharmalan, and norharmane), only 6-methoxyharmalan showed moderate affinity (dissociation constant (Ki) of 530 ± 40 nᴍ for α2A subtype). This study is a first systematic attempt towards the discovery of potential drug candidates from these plant materials for treating α2-adrenoceptor related diseases

scholarly journals Cellular characterization of a new irreversible inhibitor of S-adenosylmethionine decarboxylase and its use in determining the relative abilities of individual polyamines to sustain growth and viability of L1210 cells

1989 ◽  
Vol 259 (2) ◽  
pp. 325-331 ◽  
Author(s):  
D L Kramer ◽  
R M Khomutov ◽  
Y V Bukin ◽  
A R Khomutov ◽  
C W Porter
Keyword(s):  
Cell Growth ◽  
Supporting Cell ◽  
Enzyme Specificity ◽  
Irreversible Inhibitor ◽  
Enzyme Active Site ◽  
Adenosylmethionine Decarboxylase ◽  
Cellular Effects ◽  
L1210 Cells ◽  
Growth Inhibitory ◽  
Cell Growth And Viability

S-(5'-Deoxy-5'-adenosyl)methylthioethylhydroxylamine (AMA) is an irreversible inhibitor of S-adenosylmethionine (AdoMet) decarboxylase, which is designed to bind covalently the pyruvate residue at the enzyme active site. In the present study the cellular effects of AMA were characterized for the first time in cultured L1210 leukaemia cells. At the approximate IC50 (concn. giving 50% inhibition; 100 microM), AMA decreased spermidine and spermine by more than 80% at 48 h while increasing putrescine more than 10-fold. As an indication of enzyme specificity, growth inhibition was fully prevented with exogenous spermidine. When compared with the irreversible inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO), at similar growth-inhibitory concentrations, AMA was less cytotoxic, as determined by colony-formation efficiency. In combination with AMA, DFMO eliminated the rise in putrescine and decreased growth in an additive manner. The near-total depletion of intracellular polyamine pools achieved with the drug combination provided an opportunity to examine the relative abilities of individual polyamines to support growth and viability. Of the three exogenously supplied polyamines, only spermidine fully sustained cell growth and viability at control values during incubations totalling 120 h. By contrast, spermine supported growth at 23% of control and viability at 8%. Putrescine was similarly ineffective, supporting growth at 13% of control and viability at 7%. The data indicate that, in L1210 cells, spermidine is apparently the preferred polyamine in growth-related functions and is capable of fully supporting cell growth by itself. However, because spermine and putrescine can also support growth to some extent, maximum interference with growth and viability is best achieved by strategies which deplete all three polyamine pools.

Direct evidence for a single-step cold radiation-induced synthesis of acetonitrile and isoacetonitrile from the 1:1 CH4∙∙∙HCN complex: is it a missing link between inorganic and prebiotic astrochemistry?

2021 ◽  
Author(s):  
Anastasia D. Volosatova ◽  
Mariia A Lukianova ◽  
Pavel V Zasimov ◽  
Vladimir I. Feldman
Keyword(s):  
Interstellar Medium ◽  
Crucial Role ◽  
Direct Evidence ◽  
Prebiotic Chemistry ◽  
Single Step ◽  
Missing Link ◽  
Radiation Induced

Nitriles are important constituents of the extraterrestrial media. Nitriles are supposed to play a crucial role in the prebiotic chemistry occurring in the interstellar medium. In this work, we have...

scholarly journals Small molecule piperazinyl-benzimidazole antagonists of the gonadotropin-releasing hormone (GnRH) receptor

MedChemComm ◽  
2017 ◽  
Vol 8 (10) ◽  
pp. 1965-1969 ◽  
Author(s):  
Richard Fjellaksel ◽  
Marc Boomgaren ◽  
Rune Sundset ◽  
Ira H. Haraldsen ◽  
Jørn H. Hansen ◽  
...  
Keyword(s):  
Small Molecule ◽  
Hormone Receptor ◽  
Gnrh Receptor ◽  
Gonadotropin Releasing Hormone ◽  
Synthesis And Characterization ◽  
Releasing Hormone ◽  
Gonadotropin Releasing Hormone Receptor

In this communication, we report the synthesis and characterization of a library of small molecule antagonists of the human gonadotropin releasing hormone receptor based upon the 2-(4-tert-butylphenyl)-4-piperazinyl-benzimidazole scaffold via Cu-catalysed azide alkyne cycloaddition.

scholarly journals Conserved Amino Acid Residues that Are Important for Ligand Binding in the Type I Gonadotropin-Releasing Hormone (GnRH) Receptor Are Required for High Potency of GnRH II at the Type II GnRH Receptor

2007 ◽  
Vol 21 (1) ◽  
pp. 281-292 ◽  
Author(s):  
Sipho Mamputha ◽  
Zhi-liang Lu ◽  
Roger W. Roeske ◽  
Robert P. Millar ◽  
Arieh A. Katz ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Gnrh Receptor ◽  
Type I ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Binding Assays ◽  
Antagonist Activity ◽  
Conserved Residues ◽  
Gnrh Receptors ◽  
Mutant Receptors

Abstract GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp2.61(98), Asn2.65(102), and Lys3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp2.61(97) to Glu or Ala, Asn2.65(101) to Ala, or Lys3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp2]-GnRH I showed lesser decreases in potency than GnRH I at the Asp2.61(97)Glu mutant. In contrast, [Trp2]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn2.65(101)Ala mutant compared with analogs lacking the C⋕O group of Gly10NH2. This suggests that Asn2.65(101) recognizes Gly10NH2 of GnRH II. GnRH agonists showed large decreases in potency at the Lys3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.

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