Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the ecto-ATPases and CD39 ecto-apyrases

Smith, Thomas M.; Kirley, Terence L.

doi:10.1016/s0167-4838(98)00063-6

Cited by 176 publications

(179 citation statements)

References 44 publications

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“…COS-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% bovine serum and antibiotics and antimycotics (Invitrogen) [13]. Cells were transfected with a total of 4 μg, or 8 μg in co-transfection experiments, plasmid DNA per 100mm plate using Lipofectamine and PLUS reagents (Invitrogen) as previously described [13].…”

Section: Transient Transfection and Preparation Of Cos Cell Membranesmentioning

confidence: 99%

“…Cells were transfected with a total of 4 μg, or 8 μg in co-transfection experiments, plasmid DNA per 100mm plate using Lipofectamine and PLUS reagents (Invitrogen) as previously described [13]. Transfection with 4 μg, or 8 μg for co-transfection experiments, of empty pcDNA3 vector was performed as a control.…”

Section: Transient Transfection and Preparation Of Cos Cell Membranesmentioning

confidence: 99%

“…The COS-1 cells were harvested approximately 48 hours post-transfection. The crude cell membrane preparations were obtained as previously described [13].…”

Section: Transient Transfection and Preparation Of Cos Cell Membranesmentioning

confidence: 99%

“…Despite these differences in substrate specificity, all family members are characterized by the presence of seven highly conserved domains, called apyrase conserved regions (ACRs [11,12]) and by their dependence upon divalent cations (Mg 2+ or Ca 2+ ) for activity. Human NTPDase3 (aka HB6 [13] or CD39L3 [14]) is a membrane bound NTPDase that is characterized by two transmembrane domains, 7 potential N-glycosylation sites, and 10 extracellular cysteines that form 5 disulfide bonds [15]. NTPDase3, with an ATPase:ADPase ratio of 3-5 [13], is a functionally intermediate enzyme compared to the other two well-characterized cell surface NTPDases, NTPDase1 (ATPase:ADPase ratio ≈ 1) and NTPDase2 (NTPase:NDPase > 25:1).…”

Section: Introductionmentioning

confidence: 99%

“…Human NTPDase3 (aka HB6 [13] or CD39L3 [14]) is a membrane bound NTPDase that is characterized by two transmembrane domains, 7 potential N-glycosylation sites, and 10 extracellular cysteines that form 5 disulfide bonds [15]. NTPDase3, with an ATPase:ADPase ratio of 3-5 [13], is a functionally intermediate enzyme compared to the other two well-characterized cell surface NTPDases, NTPDase1 (ATPase:ADPase ratio ≈ 1) and NTPDase2 (NTPase:NDPase > 25:1).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of an alternative splice variant of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): A possible modulator of nucleotidase activity and purinergic signaling

Crawford¹,

Gaddie²,

Smith³

et al. 2007

Archives of Biochemistry and Biophysics

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Nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) is a cell surface, membrane-bound enzyme that hydrolyzes extracellular nucleotides, thereby modulating purinergic signaling. An alternatively spliced variant of NTPDase3 was obtained and analyzed. This alternatively spliced variant, termed "NTPDase3β", is produced through the use of an alternative terminal exon (exon 11) in place of the terminal exon (exon 12) in the full-length NTPDase3, now termed "NTPDase3α". This results in an expressed protein lacking the C-terminal cytoplasmic sequence, the C-terminal transmembrane helix, and apyrase conserved region 5. The cDNA encoding this truncated splice variant was detected in a human lung library by PCR. Like the full-length NTPDase3α, the alternatively spliced NTPDase3β was expressed in COS cells after transfection, but only the fulllength NTPDase3α is enzymatically active and properly trafficked to the plasma membrane. However, when the truncated NTPDase3β was co-transfected with full-length NTPDase3α, there was a significant reduction in the amount of NTPDase3α that was properly processed and trafficked to the plasma membrane as active enzyme, indicating that the truncated form interferes with normal biosynthetic processing of the full-length enzyme. This suggests a role for the NTPDase3β variant in the regulation of NTPDase3 nucleotidase activity, and therefore the control of purinergic signaling, in those cells and tissues expressing both NTPDase3α and NTPDase3β.

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Section: Transient Transfection and Preparation Of Cos Cell Membranesmentioning

confidence: 99%

Section: Transient Transfection and Preparation Of Cos Cell Membranesmentioning

confidence: 99%

“…The COS-1 cells were harvested approximately 48 hours post-transfection. The crude cell membrane preparations were obtained as previously described [13].…”

Section: Transient Transfection and Preparation Of Cos Cell Membranesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Characterization of an alternative splice variant of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): A possible modulator of nucleotidase activity and purinergic signaling

Crawford¹,

Gaddie²,

Smith³

et al. 2007

Archives of Biochemistry and Biophysics

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2‐Substituted thienotetrahydropyridine derivatives: Allosteric ectonucleotidase inhibitors

Schäkel

Mirza

Pietsch

et al. 2021

Archiv der Pharmazie

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The antithrombotic prodrugs ticlopidine and clopidogrel are thienotetrahydropyridine derivatives that are metabolized in the liver to produce thiols that irreversibly block adenosine diphosphate (ADP)-activated P2Y 12 receptors on thrombocytes. In their native, nonmetabolized form, both drugs were reported to act as inhibitors of ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39). CD39 catalyzes the extracellular hydrolysis of nucleoside tri-and diphosphates, mainly adenosine 5ʹ-triphosphate (ATP) and ADP, yielding adenosine monophosphate, which is further hydrolyzed by ecto-5ʹ-nucleotidase (CD73) to produce adenosine. While ATP has proinflammatory effects, adenosine is a potent anti-inflammatory, immunosuppressive agent. Inhibitors of CD39 and CD73 have potential as novel checkpoint inhibitors for the immunotherapy of cancer and infection. In the present study, we investigated 2-substituted thienotetrahydropyridine derivatives, structurally related to ticlopidine, as CD39 inhibitors. Due to their substituent on the 2-position, they will not be metabolically transformed into reactive thiols and can, therefore, be expected to be devoid of P2Y 12 receptorantagonistic activity in vivo. Several of the investigated 2-substituted thienotetrahydropyridine derivatives showed concentration-dependent inhibition of CD39.The most potent derivative, 32, showed similar CD39-inhibitory potency to ticlopidine, both acting as allosteric inhibitors. Compound 32 showed an improved selectivity profile: While ticlopidine blocked several NTPDase isoenzymes, 32 was characterized as a novel dual inhibitor of CD39 and CD73.

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Neuromodulation: Purinergic Signaling in Respiratory Control

Funk

2013

Comprehensive Physiology

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The main functions of the respiratory neural network are to produce a coordinated, efficient, rhythmic motor behavior and maintain homeostatic control over blood oxygen and CO2/pH levels. Purinergic (ATP) signaling features prominently in these homeostatic reflexes. The signaling actions of ATP are produced through its binding to a diversity of ionotropic P2X and metabotropic P2Y receptors. However, its net effect on neuronal and network excitability is determined by the interaction between the three limbs of a complex system comprising the signaling actions of ATP at P2Rs, the distribution of multiple ectonucleotidases that differentially metabolize ATP into ADP, AMP, and adenosine (ADO), and the signaling actions of ATP metabolites, especially ADP at P2YRs and ADO at P1Rs. Understanding the significance of purinergic signaling is further complicated by the fact that neurons, glia, and the vasculature differentially express P2 and P1Rs, and that both neurons and glia release ATP. This article reviews at cellular, synaptic, and network levels, current understanding and emerging concepts about the diverse roles played by this three-part signaling system in: mediating the chemosensitivity of respiratory networks to hypoxia and CO2/pH; modulating the activity of rhythm generating networks and inspiratory motoneurons, and; controlling blood flow through the cerebral vasculature.

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Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the ecto-ATPases and CD39 ecto-apyrases

Cited by 176 publications

References 44 publications

Characterization of an alternative splice variant of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): A possible modulator of nucleotidase activity and purinergic signaling

Characterization of an alternative splice variant of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): A possible modulator of nucleotidase activity and purinergic signaling

2‐Substituted thienotetrahydropyridine derivatives: Allosteric ectonucleotidase inhibitors

Neuromodulation: Purinergic Signaling in Respiratory Control

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