Oxytocin-stimulated phosphoinositide hydrolysis in human myometrial cells: involvement of pertussis toxin-sensitive and -insensitive G-proteins

Phaneuf, S; Europe‐Finner, G. Nicholas; Varney, Mark A.; Mackenzie, I. Z.; Watson, Steve P.; Bernal, A. López

doi:10.1677/joe.0.1360497

Cited by 147 publications

(93 citation statements)

References 24 publications

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“…Myometrial samples were taken from the upper corpus region (termed upper segment) and from close to the cervix (termed lower uterine segment). Primary myocyte isolation and culture were essentially as described by Phaneuf et al (39). Primary myocytes were cultured with complete D-valine medium (Invitrogen), whereas the immortalized pregnant human myometrial cells (PHM1-41) and HeLa cells were cultured in Dulbecco's Glutamax II modified Eagle's medium (Sigma), supplemented with 10% fetal calf serum, penicillin (1 unit/ml), and streptomycin (1 ng/ml), and cultured in T75 flasks under standard conditions at 37°C with 5% CO2.…”

Section: Methodsmentioning

confidence: 99%

Alternative Splicing of the Adenylyl Cyclase Stimulatory G-protein Gαs Is Regulated by SF2/ASF and Heterogeneous Nuclear Ribonucleoprotein A1 (hnRNPA1) and Involves the Use of an Unusual TG 3′-Splice Site

Pollard

Krainer²,

Robson

et al. 2002

Journal of Biological Chemistry

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The factors involved in regulating alternative splicing of the human adenylyl cyclase stimulatory G-protein G␣ s in different cell types remain undefined. We have designed a G␣ s minigene that retains the signals required for G␣ s alternative splicing in vivo. Employing transient transfection of human myometrial smooth muscle cells and HeLa cells, as well as in vitro splicing assays, we have provided evidence that the antagonistic splicing factors SF2/ASF and hnRNPA1 act as potent regulators of G␣ s isoform expression in these cells. Both SF2/ASF and hnRNPA1 control the selection of competing 5-splice sites and respectively promote inclusion or skipping of the small cassette-type exon 3 of G␣ s transcripts, resulting in the generation of G␣ s -long and G␣ sshort mRNA isoforms. We have also provided evidence that SF2/ASF and hnRNPA1 play a role in 3-splice site selection involving the use of a non-canonical TG 3-splice site preceding exon 4. Using a score-matrix analysis to identify putative exonic enhancer sequences (ESEs), we found multiple high score ESE motifs for SF2/ASF, SC35, and SRp40 in exon 3 of G␣ s . These results suggest that tissue-specific expression of SF2/ASF and hnRNPA1 governs the expression of alternative isoforms of G␣ s in these different cells types.The GTP-binding protein G␣ s is the primary stimulatory component of adenylyl cyclase in most cell types and has also been associated with activation of dihydropyridine-sensitive voltage-gated calcium channels (1) in skeletal muscle as well as inactivation of cardiac sodium channels (2). Two ubiquitously expressed forms of G␣ s have been identified via ADP-ribosylation with cholera toxin or by Western blotting (3, 4). These proteins migrate in SDS-PAGE with apparent molecular masses of 52 and 45 kDa, depending on the experimental conditions, and have been designated the long and short isoforms of G␣ s , respectively (3-5). Both isoforms of G␣ s are generated by alternative splicing of a single precursor mRNA transcript.In humans, a single copy of the G␣ s gene is found on chromosome 20 and is composed of 13 exons separated by 12 introns, altogether spanning a 20-kb region of genomic DNA (6).Cloning of the human G␣ s gene and G␣ s complementary DNAs showed that the short form of G␣ s is distinguished from the long form by the exclusion of the 45-bp exon 3, which encodes 15 amino acids. Similar studies have pointed to the existence of two additional G␣ s mRNA species. These isoforms may be produced by the use of an unusual alternative TG 3Ј-splice site, instead of the consensus AG 3Ј-splice site upstream of exon 4, resulting in the inclusion of an extra triplet coding for a serine residue after amino acids 87 and 72 of the long and short forms of G␣ s , respectively (6) (Fig. 1). It has been suggested that inclusion of this extra serine residue into G␣ s proteins confers additional consensus sequence sites for phosphoregulation by protein kinases C and A (7, 8). Tissue-dependent alternative splicing of the G␣ s precursor transcript may therefore r...

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Section: Methodsmentioning

confidence: 99%

Alternative Splicing of the Adenylyl Cyclase Stimulatory G-protein Gαs Is Regulated by SF2/ASF and Heterogeneous Nuclear Ribonucleoprotein A1 (hnRNPA1) and Involves the Use of an Unusual TG 3′-Splice Site

Pollard

Krainer²,

Robson

et al. 2002

Journal of Biological Chemistry

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“…The OTR-G protein coupling in human myometrium is well described and OTR has been shown to couple with both G αq/11 and G αi to collectively regulate diverse cellular functions through complex signalling pathways [52]. In human amnion, OTR was found to initiate its pro-inflammatory signal transduction via G αi -dependent signalling [53], whilst mainly G αq/11 coupling has been found to be involved in OT-induced myometrial contractions [52].…”

Section: Inflammationmentioning

confidence: 99%

Advances in the role of oxytocin receptors in human parturition

Kim

Bennett

Terzidou

2017

Molecular and Cellular Endocrinology

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“…Accordingly, selective oxytocin analogues capable of blocking this pathway are employed in the treatment of preterm labor (9). The physiological role played by OTRs coupled to G i in myometrial cells is far less clear, although it was shown some years ago to also lead to an increase of cell contractility (10). Recently, a specific role for OTR coupled to G i has emerged in MDCK and HEK293 cells stably transfected with the human OTR, where OT inhibits cell growth in a pertussis toxin (PTx)-sensitive manner, thus suggesting a key role of OTR-G i coupling in mediating an anti-proliferative effect (11).…”

Section: S-labeled Guanosine 5-3-o-(thio) Trisphosphate ([ 35 S]gtp␥smentioning

confidence: 99%

The Oxytocin Receptor Antagonist Atosiban Inhibits Cell Growth via a “Biased Agonist” Mechanism

Reversi

Rimoldi

Marrocco

et al. 2005

Journal of Biological Chemistry

114

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In human myometrial cells, the promiscuous coupling of the oxytocin receptors (OTRs) to G q and G i leads to contraction. However, the activation of OTRs coupled to different G protein pathways can also trigger opposite cellular responses, e.g. OTR coupling to G i inhibits, whereas its coupling to G q stimulates, cell proliferation. WAF1/CIP1 induction, the same signaling events leading to oxytocin-mediated cell growth inhibition via a G i pathway. Finally, atosiban exposure did not cause OTR internalization and led to only a modest decrease (20%) in the number of high affinity cell membrane OTRs, two observations consistent with the finding that atosiban did not lead to any desensitization of the oxytocin-induced activation of the G q -phospholipase C pathway. Taken together, these observations indicate that atosiban acts as a "biased agonist" of the human OTRs and thus belongs to the class of compounds capable of selectively discriminating only one among the multiple possible active conformations of a single G protein-coupled receptor, thereby leading to the selective activation of a unique intracellular signal cascade.

show abstract

Oxytocin-stimulated phosphoinositide hydrolysis in human myometrial cells: involvement of pertussis toxin-sensitive and -insensitive G-proteins

Cited by 147 publications

References 24 publications

Alternative Splicing of the Adenylyl Cyclase Stimulatory G-protein Gαs Is Regulated by SF2/ASF and Heterogeneous Nuclear Ribonucleoprotein A1 (hnRNPA1) and Involves the Use of an Unusual TG 3′-Splice Site

Alternative Splicing of the Adenylyl Cyclase Stimulatory G-protein Gαs Is Regulated by SF2/ASF and Heterogeneous Nuclear Ribonucleoprotein A1 (hnRNPA1) and Involves the Use of an Unusual TG 3′-Splice Site

Advances in the role of oxytocin receptors in human parturition

The Oxytocin Receptor Antagonist Atosiban Inhibits Cell Growth via a “Biased Agonist” Mechanism

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