A small sequence in the third intracellular loop of the VPAC1 receptor is responsible for its efficient coupling to the calcium effector

2002 ◽  
Vol 30 (4) ◽  
pp. 447-450 ◽  
Author(s):  
I. Langer ◽  
P. Vertongen ◽  
J. Perret ◽  
M. Waelbroeck ◽  
P. Robberecht
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Intracellular Loop ◽  
Ovary Cells ◽  
The Third ◽  
Vpac1 Receptor ◽  
Third Intracellular Loop ◽  
Efficient Coupling ◽  
Similar Density ◽  
In Cells

The stimulatory effect of vasoactive intestinal peptide (VIP) on the intracellular calcium concentration ([Ca2+]i) has been investigated in Chinese hamster ovary cells stably transfected with the reporter gene aequorin, and expressing human VPAC1, VPAC2, chimaeric VPAC1/VPAC2 or mutated receptors. The VIP-induced increase in [Ca2+]i was linearly correlated with receptor density, and was higher in cells expressing VPAC1 receptors than in cells expressing a similar density of VPAC2 receptors. The study was performed to establish the receptor sequence responsible for this difference. VPAC1/VPAC2 chimaeric receptors were first used for broad positioning: those receptors having the third intracellular loop (IC3) of the VPAC1 or the VPAC2 receptor behaved, in this respect, phenotypically like VPAC1 and VPAC2 receptors respectively. Replacement in the VPAC2 receptor of the sequence comprising residues 315–318 (VGGN) within IC3 by its VPAC1 receptor counterpart (residues 328–331; IRKS) and the introduction of VGGN instead of IRKS into VPAC1 was sufficient to mimic VPAC1 and VPAC2 receptor characteristics respectively. Thus a small sequence in the IC3 domain of the VPAC1 receptor is responsible for the efficient agonist-stimulated increase in [Ca2+]i.

scholarly journals A Small Sequence in the Third Intracellular Loop of the VPAC1 Receptor Is Responsible for Its Efficient Coupling to the Calcium Effector

2002 ◽  
Vol 16 (5) ◽  
pp. 1089-1096 ◽  
Author(s):  
Ingrid Langer ◽  
Pascale Vertongen ◽  
Jason Perret ◽  
Magali Waelbroeck ◽  
Patrick Robberecht
Keyword(s):  
Intracellular Loop ◽  
The Third ◽  
Vpac1 Receptor ◽  
Third Intracellular Loop ◽  
Efficient Coupling

Influence of metallothionein-1 localization on its function

2001 ◽  
Vol 355 (2) ◽  
pp. 473-479 ◽  
Author(s):  
Marilyne LEVADOUX-MARTIN ◽  
John E. HESKETH ◽  
John H. BEATTIE ◽  
Heather M. WALLACE
Keyword(s):  
Dna Damage ◽  
Uv Light ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Protective Role ◽  
S Phase ◽  
Coding Region ◽  
Ovary Cells ◽  
In Cells

Metallothioneins (MTs) have a major role to play in metal metabolism, and may also protect DNA against oxidative damage. MT protein has been found localized in the nucleus during S-phase. The mRNA encoding the MT-1 isoform has a perinuclear localization, and is associated with the cytoskeleton; this targeting, due to signals within the 3′-untranslated region (3′-UTR), facilitates nuclear localization of MT-1 during S-phase [Levadoux, Mahon, Beattie, Wallace and Hesketh (1999) J. Biol. Chem. 274, 34961-34966]. Using cells transfected with MT gene constructs differing in their 3′-UTRs, the role of MT protein in the nucleus has been studied. Chinese hamster ovary cells were transfected with either the full MT gene (MTMT cells) or with the MT 5′-UTR and coding region linked to the 3′-UTR of glutathione peroxidase (MTGSH cells). Cell survival following exposure to oxidative stress and chemical agents was higher in cells expressing the native MT gene than in cells where MT localization was disrupted, or in untransfected cells. Also, MTMT cells showed less DNA damage than MTGSH cells in response to either hydrogen peroxide or mutagen. After exposure to UV light or mutagen, MTMT cells showed less apoptosis than MTGSH cells, as assessed by DNA fragmentation and flow cytometry. The data indicate that the perinuclear localization of MT mRNA is important for the function of MT in a protective role against DNA damage and apoptosis induced by external stress.

scholarly journals DistinctC-mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3

2017 ◽  
Vol 114 (10) ◽  
pp. 2574-2579 ◽  
Author(s):  
Aleksandra Shcherbakova ◽  
Birgit Tiemann ◽  
Falk F. R. Buettner ◽  
Hans Bakker
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
The Third ◽  
The Stability ◽  
Thrombospondin Type 1 Repeats

Thrombospondin type 1 repeats (TSRs) occur in diverse proteins involved in adhesion and signaling. The two extracellular TSRs of the netrin receptor UNC5A contain WxxWxxWxxC motifs that can beC-mannosylated on all tryptophans. A singleC-mannosyltransferase (dumpy-19, DPY-19), modifying the first two tryptophans, occurs inCaenorhabditis elegans, but four putative enzymes (DPY-19–like 1–4, DPY19L1–4) exist in mammals. Single and triple CRISPR-Cas9 knockouts of the three homologs that are expressed in Chinese hamster ovary cells (DPY19L1, DPY19L3, and DPY19L4) and complementation experiments with mouse homologs showed that DPY19L1 preferentially mannosylates the first two tryptophans and DPY19L3 prefers the third, whereas DPY19L4 has no function in TSR glycosylation. Mannosylation by DPY19L1 but not DPY19L3 is required for transport of UNC5A from the endoplasmic reticulum to the cell surface. In vertebrates, a newC-mannosyltransferase has apparently evolved to increase glycosylation of TSRs, potentially to increase the stability of the structurally essential tryptophan ladder or to provide additional adhesion functions.

scholarly journals Control of the Cardiac Muscarinic K+Channel by β-Arrestin 2

2001 ◽  
Vol 276 (15) ◽  
pp. 11691-11697 ◽  
Author(s):  
Z. Shui ◽  
I. A. Khan ◽  
T. Haga ◽  
J. L. Benovic ◽  
M. R. Boyett
Keyword(s):  
Muscarinic Receptor ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
K Channel ◽  
Receptor Kinase ◽  
Ovary Cells ◽  
M2 Muscarinic Receptor ◽  
Receptor Channel ◽  
In Cells

Control of the cardiac muscarinic K+current (iK,ACh) by β-arrestin 2 has been studied. In Chinese hamster ovary cells transfected with m2 muscarinic receptor, muscarinic K+channel, receptor kinase (GRK2), and β-arrestin 2, desensitization of iK,AChduring a 3-min application of 10 μmACh was significantly increased as compared with that in cells transfected with receptor, channel, and GRK2 only (fade in current increased from 45 to 78%). The effect of β-arrestin 2 was lost if cells were not co-transfected with GRK2. Resensitization (recovery from desensitization) of iK,AChin cells transfected with β-arrestin 2 was significantly slowed (time constant increased from 34 to 232 s). Activation and deactivation of iK,AChon application and wash-off of ACh in cells transfected with β-arrestin 2 were significantly slowed from 0.9 to 3.1 s (time to half peak iK,ACh) and from 6.2 to 13.8 s (time to half-deactivation), respectively. In cells transfected with a constitutively active β-arrestin 2 mutant, desensitization occurred in the absence of agonist (peak current significantly decreased from 0.4 ± 0.05 to 0.1 ± 0.01 nA). We conclude that β-arrestin 2 has the potential to play a major role in desensitization and other aspects of the functioning of the muscarinic K+channel.

scholarly journals Evidence for an ethanolamine cycle: differential recycling of the ethanolamine moiety of phosphatidylethanolamine derived from phosphatidylserine and ethanolamine

1995 ◽  
Vol 310 (2) ◽  
pp. 673-679 ◽  
Author(s):  
Y J Shiao ◽  
J E Vance
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Specific Radioactivity ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Time Period ◽  
In Cells

Evidence is presented for the operation of an ethanolamine-phosphatidylethanolamine (PtdEtn) cycle in Chinese hamster ovary cells. PtdEtn was labelled with [3H]ethanolamine and radioactivity was chased by incubation with 1 mM unlabelled ethanolamine. Radioactivity in [3H]PtdEtn gradually declined over a 23 h time period. In contrast, when the cells were incubated in medium lacking unlabelled ethanolamine, radioactivity in PtdEtn remained constant for at least 23 h. These observations suggest that the ethanolamine moiety is continuously released from PtdEtn and recycled back into PtdEtn. In cells incubated without unlabelled ethanolamine, labelled ethanolamine released from PtdEtn is re-incorporated into PtdEtn without significant dilution. In contrast, in cells incubated with unlabelled ethanolamine the specific radioactivity of the intracellular ethanolamine pool decreases as a result of dilution by the exogenous ethanolamine, hence radioactivity in PtdEtn gradually declines. Similar results were obtained for confluent and non-confluent cells. Our data also demonstrate that when PtdEtn is derived from phosphatidylserine decarboxylation, the ethanolamine cycle operates only in actively dividing, and not in confluent, cells, implying that PtdEtn derived from different biosynthetic origins [i.e. from decarboxylation of phosphatidylserine or from ethanolamine (most likely via the CDP-ethanolamine pathway)] is metabolized differently.

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