Functional characterisation of an ovine endometrial oxytocin receptor cDNA transiently expressed in Cos-7 cells

1996 ◽  
Vol 149 (3) ◽  
pp. 389-396 ◽  
Author(s):  
P R Riley ◽  
D R E Abayasekara ◽  
H J Stewart ◽  
A P F Flint
Keyword(s):  
G Protein ◽  
Arginine Vasopressin ◽  
Signal Transduction Pathway ◽  
Oxytocin Receptor ◽  
Response Threshold ◽  
Induced Response ◽  
Dependent Manner ◽  
Coding Region ◽  
Pi Turnover ◽  
Functional Characterisation

Abstract The entire coding region of an ovine endometrial oxytocin receptor (OTR) cDNA was generated by PCR, subcloned into the SV40 major late promoter expression vector pSVLJ and transiently expressed in Cos-7 cells. A specific OTR antagonist, 125I-labelled d(CH2)5 [Tyr(Me)2,Thr4,Tyr-NH29]-vasotocin (OTA), was used to describe the binding kinetics of the expressed receptor which had a Kd of 4·5 nm and Bmax of 2·4 nm/mg protein (6·8 × 105 receptor molecules/transfected cell). The functional properties of the expressed OTR were determined by measuring oxytocin-induced phosphoinositide (PI) hydrolysis. Oxytocin increased PI turnover in OTR transfected cells fourfold in excess of residual endogenous activity, and stimulated phospholipase C (PLC) activity in a dose- and time-dependent manner, confirming that the expressed OTR cDNA was functional. Arginine vasopressin also stimulated PI turnover in a dose-dependent manner; thresholds of responses to oxytocin and arginine vasopressin were 10−9 m and 10−7 m respectively. OTA did not increase PI turnover and competitively inhibited the oxytocin-induced response. Direct activation of the pathway by aluminium fluoride and guanosine (3′-Othio)-triphosphate (GTPγS) confirmed that the OTR was G-protein linked. Co-incubation of GTPγS with oxytocin shifted the PI-response threshold from 10−7 m to 10−9 m and significantly increased the level of response, suggesting that maximum PI turnover was agonist-dependent. The G-protein involved in mediating the signal transduction pathway was pertussis toxin-insensitive and, therefore, probably a member of the Gq subfamily. The PLC inhibitor, U73122, had no effect on oxytocin-induced PI turnover, consistent with the response in endometrial tissue. These data suggest that the signalling pathway mediated by expressed OTR is similar to that attributed to OTR occupancy in ovine endometrium. Journal of Endocrinology (1996) 149, 389–396

scholarly journals Structure and regulation of the mDot1 gene, a mouse histone H3 methyltransferase

2004 ◽  
Vol 377 (3) ◽  
pp. 641-651 ◽  
Author(s):  
Wenzheng ZHANG ◽  
Yoshihide HAYASHIZAKI ◽  
Bruce C. KONE
Keyword(s):  
Alternative Splicing ◽  
Fluorescent Protein ◽  
Expressed Sequence Tag ◽  
Collecting Duct ◽  
Signal Transduction Pathway ◽  
Dependent Manner ◽  
Coding Region ◽  
Duct Cells ◽  
Collecting Duct Cells

Recently, a new class of histone methyltransferases that plays an indirect role in chromatin silencing by targeting a conserved lysine residue in the nucleosome core was described, namely the Dot1 (disruptor of telomeric silencing) family [Feng, Wang, Ng, Erdjument-Bromage, Tempst, Struhl and Zhang (2002) Curr. Biol. 12, 1052–1058; van Leeuwen, Gafken and Gottschling (2002) Cell (Cambridge, Mass.) 109, 745–756; Ng, Feng, Wang, Erdjument-Bromage, Tempst, Zhang and Struhl (2002) Genes Dev. 16, 1518–1527]. In the present study, we report the isolation, genomic organization and in vivo expression of a mouse Dot1 homologue (mDot1). Expressed sequence tag analysis identified five mDot1 mRNAs (mDot1a–mDot1e) derived from alternative splicing. mDot1a and mDot1b encode 1540 and 1114 amino acids respectively, whereas mDot1c–mDot1e are incomplete at the 5´-end. mDot1a is closest to its human counterpart (hDot1L), sharing 84% amino acid identity. mDot1b is truncated at its N- and C-termini and contains an internal deletion. The five mDot1 isoforms are encoded by 28 exons on chromosome 10qC1, with exons 24 and 28 further divided into two and four sections respectively. Alternative splicing occurs in exons 3, 4, 12, 24, 27 and 28. Northern-blot analysis with probes corresponding to the methyltransferase domain or the mDot1a-coding region detected 7.6 and 9.5 kb transcripts in multiple tissues, but only the 7.6 kb transcript was evident in mIMCD3-collecting duct cells. Transfection of mDot1a–EGFP constructs (where EGFP stands for enhanced green fluorescent protein) into human embryonic kidney (HEK)-293T or mIMCD3 cells increased the methylation of H3-K79 but not H3-K4, -K9 or -K36. Furthermore, DMSO induced mDot1 gene expression and methylation specifically at H3-K79 in mIMCD3 cells in a time- and dose-dependent manner. Collectively, these results add new members to the Dot1 family and show that mDot1 is involved in a DMSO-mediated signal-transduction pathway in collecting duct cells.

scholarly journals cAMP activates calcium signalling via phospholipase C to regulate cellulase production in the filamentous fungus Trichoderma reesei

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yumeng Chen ◽  
Xingjia Fan ◽  
Xinqing Zhao ◽  
Yaling Shen ◽  
Xiangyang Xu ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Phospholipase C ◽  
Trichoderma Reesei ◽  
Filamentous Fungus ◽  
Calcium Signalling ◽  
Signal Transduction Pathway ◽  
Cellulase Production ◽  
Signalling Pathway ◽  
Dependent Manner ◽  
Transduction Pathway

Abstract Background The filamentous fungus Trichoderma reesei is one of the best producers of cellulase and has been widely studied for the production of cellulosic ethanol and bio-based products. We previously reported that Mn2+ and N,N-dimethylformamide (DMF) can stimulate cellulase overexpression via Ca2+ bursts and calcium signalling in T. reesei under cellulase-inducing conditions. To further understand the regulatory networks involved in cellulase overexpression in T. reesei, we characterised the Mn2+/DMF-induced calcium signalling pathway involved in the stimulation of cellulase overexpression. Results We found that Mn2+/DMF stimulation significantly increased the intracellular levels of cAMP in an adenylate cyclase (ACY1)-dependent manner. Deletion of acy1 confirmed that cAMP is crucial for the Mn2+/DMF-stimulated cellulase overexpression in T. reesei. We further revealed that cAMP elevation induces a cytosolic Ca2+ burst, thereby initiating the Ca2+ signal transduction pathway in T. reesei, and that cAMP signalling causes the Ca2+ signalling pathway to regulate cellulase production in T. reesei. Furthermore, using a phospholipase C encoding gene plc-e deletion strain, we showed that the plc-e gene is vital for cellulase overexpression in response to stimulation by both Mn2+ and DMF, and that cAMP induces a Ca2+ burst through PLC-E. Conclusions The findings of this study reveal the presence of a signal transduction pathway in which Mn2+/DMF stimulation produces cAMP. Increase in the levels of cAMP activates the calcium signalling pathway via phospholipase C to regulate cellulase overexpression under cellulase-inducing conditions. These findings provide insights into the molecular mechanism of the cAMP–PLC–calcium signalling pathway underlying cellulase expression in T. reesei and highlight the potential applications of signal transduction in the regulation of gene expression in fungi.

scholarly journals Mot3, a Zn Finger Transcription Factor That Modulates Gene Expression and Attenuates Mating Pheromone Signaling in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 879-892 ◽  
Author(s):  
Anatoly V Grishin ◽  
Michael Rothenberg ◽  
Maureen A Downs ◽  
Kendall J Blumer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Signaling Pathway ◽  
G Protein ◽  
Binding Sites ◽  
Dependent Manner ◽  
Pheromone Response ◽  
Mating Pheromone ◽  
Pheromone Signaling ◽  
Yeast Genes

Abstract In the yeast Saccharomyces cerevisiae, mating pheromone response is initiated by activation of a G protein- and mitogen-activated protein (MAP) kinase-dependent signaling pathway and attenuated by several mechanisms that promote adaptation or desensitization. To identify genes whose products negatively regulate pheromone signaling, we screened for mutations that suppress the hyperadaptive phenotype of wild-type cells overexpressing signaling-defective G protein β subunits. This identified recessive mutations in MOT3, which encodes a nuclear protein with two Cys2-His2 Zn fingers. MOT3 was found to be a dosage-dependent inhibitor of pheromone response and pheromone-induced gene expression and to require an intact signaling pathway to exert its effects. Several results suggested that Mot3 attenuates expression of pheromone-responsive genes by mechanisms distinct from those used by the negative transcriptional regulators Cdc36, Cdc39, and Mot2. First, a Mot3-lexA fusion functions as a transcriptional activator. Second, Mot3 is a dose-dependent activator of several genes unrelated to pheromone response, including CYC1, SUC2, and LEU2. Third, insertion of consensus Mot3 binding sites (C/A/T)AGG(T/C)A activates a promoter in a MOT3-dependent manner. These findings, and the fact that consensus binding sites are found in the 5′ flanking regions of many yeast genes, suggest that Mot3 is a globally acting transcriptional regulator. We hypothesize that Mot3 regulates expression of factors that attenuate signaling by the pheromone response pathway.

scholarly journals Density-dependent nerve growth factor regulation of Gs-alpha RNA in pheochromocytoma 12 cells.

1990 ◽  
Vol 10 (6) ◽  
pp. 3277-3279 ◽  
Author(s):  
G Tjaden ◽  
A Aguanno ◽  
R Kumar ◽  
D Benincasa ◽  
R M Gubits ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
G Protein ◽  
Alpha Subunit ◽  
Low Density ◽  
Dependent Manner ◽  
Nerve Growth ◽  
Density Dependent ◽  
Stimulatory G Protein ◽  
Gs Alpha

Nerve growth factor (NGF) affects levels of the alpha subunit of the stimulatory G protein (Gs-alpha) in pheochromocytoma 12 cells in a bidirectional, density-dependent manner. Cells grown at high density responded to NGF treatment with increased levels of Gs-alpha mRNA and protein. Conversely, in cells grown in low-density cultures, levels of this mRNA were lowered by NGF treatment.

scholarly journals OsPRR37 Alternatively Promotes Heading Date Through Suppressing the Expression of Ghd7 in the Japonica Variety Zhonghua 11 under Natural Long-Day Conditions

Rice ◽  
2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yong Hu ◽  
Xin Zhou ◽  
Bo Zhang ◽  
Shuangle Li ◽  
Xiaowei Fan ◽  
...  
Keyword(s):  
Functional Divergence ◽  
Heading Date ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Coding Region ◽  
Japonica Variety ◽  
Long Day ◽  
Nucleotide Mutation ◽  
Alternative Function ◽  
Pr Domain

AbstractHeading date is an important agronomic trait of rice (Oryza sativa L.) and is regulated by numerous genes, some of which exhibit functional divergence in a genetic background-dependent manner. Here, we identified a late heading date 7 (lhd7) mutant that flowered later than wild-type Zhonghua 11 (ZH11) under natural long-day (NLD) conditions. Map-based cloning facilitated by the MutMap strategy revealed that LHD7 was on the same locus as OsPRR37 but exhibited a novel function as a promoter of heading date. A single-nucleotide mutation of G-to-A in the coding region caused a substitution of aspartic acid for glycine at site 159 within the pseudo-receiver (PR) domain of OsPRR37. Transcriptional analysis revealed that OsPRR37 suppressed Ghd7 expression in both ZH11 background under NLD conditions and the Zhenshan 97 background under natural short-day conditions. Consistently, the expression of Ehd1, Hd3a and RFT1 was enhanced by OsPRR37 in the ZH11 background. Genetic analysis indicated that the promotion of heading date and reduction in grain yield by OsPRR37 were partially dependent on Ghd7. Further investigation showed that the alternative function of OsPRR37 required an intact Ghd7-related regulatory pathway involving not only its upstream regulators OsGI and PhyB but also its interacting partner Hd1. Our study revealed the distinct role of OsPRR37 in the ZH11 background, which provides a more comprehensive understanding of OsPRR37 function and enriches the theoretical bases for improvement of rice heading date in the future.

scholarly journals G-Protein β Subunit of Cochliobolus heterostrophus Involved in Virulence, Asexual and Sexual Reproductive Ability, and Morphogenesis

2004 ◽  
Vol 3 (6) ◽  
pp. 1653-1663 ◽  
Author(s):  
Sherif Ganem ◽  
Shun-Wen Lu ◽  
Bee-Na Lee ◽  
David Yu-Te Chou ◽  
Ruthi Hadar ◽  
...  
Keyword(s):  
Map Kinase ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Cochliobolus Heterostrophus ◽  
Wild Type ◽  
Heterotrimeric G Protein ◽  
Reproductive Ability ◽  
Pathway Gene ◽  
Mitogen Activated Protein ◽  
Nuclear Degradation

ABSTRACT Previous work established that mutations in mitogen-activated protein (MAP) kinase (CHK1) and heterotrimeric G-protein α (Gα) subunit (CGA1) genes affect the development of several stages of the life cycle of the maize pathogen Cochliobolus heterostrophus. The effects of mutating a third signal transduction pathway gene, CGB1, encoding the Gβ subunit, are reported here. CGB1 is the sole Gβ subunit-encoding gene in the genome of this organism. cgb1 mutants are nearly wild type in vegetative growth rate; however, Cgb1 is required for appressorium formation, female fertility, conidiation, regulation of hyphal pigmentation, and wild-type virulence on maize. Young hyphae of cgb1 mutants grow in a straight path, in contrast to those of the wild type, which grow in a wavy pattern. Some of the phenotypes conferred by mutations in CGA1 are found in cgb1 mutants, suggesting that Cgb1 functions in a heterotrimeric G protein; however, there are also differences. In contrast to the deletion of CGA1, the loss of CGB1 is not lethal for ascospores, evidence that there is a Gβ subunit-independent signaling role for Cga1 in mating. Furthermore, not all of the phenotypes conferred by mutations in the MAP kinase CHK1 gene are found in cgb1 mutants, implying that the Gβ heterodimer is not the only conduit for signals to the MAP kinase CHK1 module. The additional phenotypes of cgb1 mutants, including severe loss of virulence on maize and of the ability to produce conidia, are consistent with CGB1 being unique in the genome. Fluorescent DNA staining showed that there is often nuclear degradation in mature hyphae of cgb1 mutants, while comparable wild-type cells have intact nuclei. These data may be genetic evidence for a novel cell death-related function of the Gβ subunit in filamentous fungi.

scholarly journals Inhibition by somatostatin of amylase secretion induced by calcium and cyclic AMP in rat pancreatic acini

1994 ◽  
Vol 304 (2) ◽  
pp. 531-536 ◽  
Author(s):  
H Ohnishi ◽  
T Mine ◽  
I Kojima
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Pancreatic Acini

It has recently been shown that somatostatin inhibits amylase secretion from isolated pancreatic acini by reducing cyclic AMP (cAMP) production [Matsushita, Okabayashi, Hasegawa, Koide, Kido, Okutani, Sugimoto and Kasuga (1993) Gastroenterology 104, 1146-1152]. To date, however, little is known as to the other mechanism(s) by which somatostatin inhibits amylase secretion in exocrine pancreas. To investigate the action of somatostatin independent of cAMP generation, we examined the effect of somatostatin in isolated rat pancreatic acini stimulated by 1 microM calcium ionophore A23187 and 1 mM 8-bromo-cyclic AMP (8Br-cAMP). Somatostatin inhibited amylase secretion evoked by a combination of A23187 and 8Br-cAMP in a dose-dependent manner. The maximum inhibition was obtained by 10(-7) M somatostatin, and at this concentration somatostatin inhibited the effect of A23187 and 8Br-cAMP by approximately 30%. In electrically permeabilized acini, an elevation of free calcium concentration resulted in an increase in amylase secretion and cAMP enhanced the secretion evoked by calcium. cAMP shifted the dose-response curve for calcium-induced secretion leftwards and elevated the peak value of secretion. Somatostatin inhibited the effect of cAMP on calcium-induced amylase secretion by shifting the dose-response curve to the right. To determine the involvement of a G-protein(s), we examined the effect of somatostatin in acini pretreated with pertussis toxin. Pretreatment of acini with pertussis toxin completely blocked somatostatin-inhibition of amylase-secretion evoked by A23187 and 8Br-cAMP. These results indicate that somatostatin decreases amylase secretion induced by cAMP and calcium by reducing the calcium sensitivity of exocytosis. A pertussis toxin-sensitive G-protein is also involved in this step.

scholarly journals Valine-279 Deletion–Mutation on Arginine Vasopressin Receptor 2 Causes Obstruction in G-Protein Binding Site: A Clinical Nephrogenic Diabetes Insipidus Case and Its Sub-Molecular Pathogenic Analysis

Biomedicines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 301
Author(s):  
Ming-Chun Chen ◽  
Yu-Chao Hsiao ◽  
Chun-Chun Chang ◽  
Sheng-Feng Pan ◽  
Chih-Wen Peng ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Protein Binding ◽  
Diabetes Insipidus ◽  
Binding Site ◽  
G Protein ◽  
Arginine Vasopressin ◽  
Nephrogenic Diabetes Insipidus ◽  
Md Simulations ◽  
Protein Binding Site ◽  
Vasopressin Receptor

Congenital nephrogenic diabetes insipidus (CNDI) is a genetic disorder caused by mutations in arginine vasopressin receptor 2 (AVPR2) or aquaporin 2 genes, rendering collecting duct cells insensitive to the peptide hormone arginine vasopressin stimulation for water reabsorption. This study reports a first identified AVPR2 mutation in Taiwan and demonstrates our effort to understand the pathogenesis caused by applying computational structural analysis tools. The CNDI condition of an 8-month-old male patient was confirmed according to symptoms, family history, and DNA sequence analysis. The patient was identified to have a valine 279 deletion–mutation in the AVPR2 gene. Cellular experiments using mutant protein transfected cells revealed that mutated AVPR2 is expressed successfully in cells and localized on cell surfaces. We further analyzed the pathogenesis of the mutation at sub-molecular levels via long-term molecular dynamics (MD) simulations and structural analysis. The MD simulations showed while the structure of the extracellular ligand-binding domain remains unchanged, the mutation alters the direction of dynamic motion of AVPR2 transmembrane helix 6 toward the center of the G-protein binding site, obstructing the binding of G-protein, thus likely disabling downstream signaling. This study demonstrated that the computational approaches can be powerful tools for obtaining valuable information on the pathogenesis induced by mutations in G-protein-coupled receptors. These methods can also be helpful in providing clues on potential therapeutic strategies for CNDI.

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