N-Methyl-D-aspartate Receptors Expressed in a Nonneuronal Cell Line Mediate Subunit-specific Increases in Free Intracellular Calcium

Grant, Elfrida R.; Bacskai, Brian J.; Pleasure, David E; Pritchett, Dolan B.; Gallagher, Michael J.; Kendrick, Shelley J.; Kricka, Larry J.; Lynch, David R.

doi:10.1074/jbc.272.1.647

Cited by 42 publications

(39 citation statements)

References 69 publications

(41 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6), which is known to mediate calcium-dependent gene expression (39), as mentioned earlier. Most compelling, Act induced changes in the phosphorylation status of the NMDA receptor, NR1, which has been shown to regulate intracellular levels of calcium in neuronal cells (3,16,17,65) As expected, Act did induce calcium mobilization in human intestinal epithelial cells (Fig. 7), which could be important in the production of inflammatory mediators, such as IL-8, and crucial in the pathogenesis of Aeromonas gastrointestinal infections.…”

Section: Discussionmentioning

confidence: 99%

Microarray and Proteomics Analyses of Human Intestinal Epithelial Cells Treated with theAeromonas hydrophilaCytotoxic Enterotoxin

et al. 2005

View full text Add to dashboard Cite

We performed microarray analyses on RNA from human intestinal epithelial (HT-29) cells treated with the cytotoxic enterotoxin (Act) of Aeromonas hydrophila to examine global cellular transcriptional responses. Based on three independent experiments, Act upregulated the expression of 34 genes involved in cell growth, adhesion, signaling, immune responses (including interleukin-8 [IL-8] production), and apoptosis. We verified the upregulation of 14 genes by real-time reverse transcriptase-PCR and confirmed Act-induced production of IL-8 by enzyme-linked immunosorbent assay on supernatants from nonpolarized and polarized HT-29 cells. Maximal production of IL-8 in response to Act required the presence of intracellular calcium, since chelation of calcium with BAPTA-AM significantly reduced Act-induced IL-8 production in HT-29 cells. We also examined activation of mitogen-activated protein kinases and, as demonstrated by Western blot analysis of apical side-treated polarized HT-29 cells, Act induced phosphorylation of p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase 1/2. In addition, KinetWorks proteomics screening of whole-cell lysates revealed Act-induced phosphorylation of cyclic AMP-response element binding protein (CREB), c-Jun, adducin, protein kinase C, and signal transducer and activator of transcription 3 (STAT3) and decreased phosphorylation of protein kinase Bα, v-raf-1 murine leukemia viral oncogene homolog 1 (i.e., Raf1), and STAT1. We verified activation of CREB and activator protein 1 in polarized cells by gel shift assay. This is the first description of human intestinal epithelial cell transcriptional alterations, phosphorylation or activation of signaling molecules, cytokine production, and calcium mobilization in response to this toxin

show abstract

Section: Discussionmentioning

confidence: 99%

Microarray and Proteomics Analyses of Human Intestinal Epithelial Cells Treated with theAeromonas hydrophilaCytotoxic Enterotoxin

et al. 2005

View full text Add to dashboard Cite

show abstract

“…To determine whether calpain-mediated proteolysis alters NMDA receptor function, the effects of calpain inhibition on NMDA receptor-mediated calcium influx and agonist-induced intracellular calcium transients were studied. Like 125 I-MK-801 binding, these assays serve as markers of physiologically active receptors, because channel activity in this heterologous expression system is only present when a receptor contains both NR1 and NR2 subunits (Grant et al, 1997). Calcium uptake from the media in NR1a/2A cells cotransfected with calpastatin was increased over NR1a/2A/vectortransfected cells by 50% (Fig.…”

Section: Identification Of Calpain In Hek293tmentioning

confidence: 97%

“…Calcium uptake, calpain activation, and calcium imaging were routinely performed at this point. Ketamine (500 M) was added to the media during transfection to prevent NMDA receptor activation as previously described (Grant et al, 1997). Using HEK293t cells, the transfection efficiency is ϳ70%.…”

Section: Methodsmentioning

confidence: 99%

Proteolysis of theN-Methyl-d-Aspartate Receptor by Calpain in Situ

Guttmann

Sokol²,

Baker³

et al. 2002

J Pharmacol Exp Ther

Self Cite

View full text Add to dashboard Cite

N-Methyl-D-aspartate (NMDA) receptors are calcium-permeable glutamate receptors that play putative roles in learning, memory, and excitotoxicity. NMDA receptor-mediated calcium entry can activate the calcium-dependent protease calpain, leading to substrate degradation. The major NMDA receptor 2 (NR2) subunits of the receptor are in vitro substrates for calpain at selected sites in the C-terminal region. In the present study, we assessed the ability of calpain-mediated proteolysis to modulate the NR1a/2A subtype in a heterologous expression system. Human embryonic kidney (HEK293t) cells, which endogenously express calpain, were cotransfected with NR1a/2A in addition to the calpain inhibitor calpastatin or empty vector as control. Receptor activation by glutamate and glycine as co-agonists led to calpain activation as measured by succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-aminomethyl coumarin (Suc-LLVY-AMC). Calpain activation also resulted in the degradation of NR2A and decreased binding of 125 I-MK-801 ( 125 I-dizocilpine) to NR1a/2A receptors. No stable N-terminal fragment of the NMDA receptor was formed after calpain activation, suggesting calpain regulation of NMDA receptor levels in ways distinct from that previously observed with in vitro cleavage. NR2 subunit constructs lacking the final 420 amino acids were not degraded by calpain. Agonist-stimulated NR1a/2A-transfected cells also had decreased calcium uptake and produced lower changes in agonist-stimulated intracellular calcium compared with cells cotransfected with calpastatin. Calpastatin had no effect on either calcium uptake or intracellular calcium levels when the NR2A subunit lacked the final 420 amino acids. These studies demonstrate that NR2A is a substrate for calpain in situ and that this proteolytic event can modulate NMDA receptor levels.

show abstract

“…Treatments were performed 18 -24 h following transfection. Ketamine (500 M) was included in the media during transfection to prevent NMDA receptor activation as previously described (25).…”

mentioning

confidence: 99%

“…Transfection was performed using calcium phosphate as previously described (25). Treatments were performed 18 -24 h following transfection.…”

mentioning

confidence: 99%

Fyn-mediated Phosphorylation of NR2B Tyr-1336 Controls Calpain-mediated NR2B Cleavage in Neurons and Heterologous Systems

Hsu

Gleichman

et al. 2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Cleavage of the intracellular carboxyl terminus of the N-methyl-D-aspartate (NMDA) receptor 2 subunit (NR2) by calpain regulates NMDA receptor function and localization. Here, we show that Fyn-mediated phosphorylation of NR2B controls calpain-mediated NR2B cleavage. In cultured neurons, calpainmediated NR2B cleavage is significantly attenuated by blocking NR2B phosphorylation of Tyr-1336, but not Tyr-1472, via inhibition of Src family kinase activity or decreasing Fyn levels by small interfering RNA. In HEK cells, mutation of Tyr-1336 eliminates the potentiating effect of Fyn on calpainmediated NR2B cleavage. The potentiation of NR2B cleavage by Fyn is limited to cell surface receptors and is associated with calpain translocation to plasma membranes during NMDA receptor activation. Finally, reducing full-length NR2B by calpain does not decrease extrasynaptic NMDA receptor function, and truncated NR1/2B receptors similar to those generated by calpain have electrophysiological properties matching those of wild-type receptors. Thus, the Fyncontrolled regulation of NMDA receptor cleavage by calpain may play critical roles in controlling NMDA receptor properties during synaptic plasticity and excitotoxicity. The N-methyl-D-aspartate (NMDA) 2 receptor is a calciumpermeable ionotropic glutamate receptor requiring glutamate, glycine, and membrane depolarization for activation. These receptors play crucial roles in brain development, excitatory neurotransmission, and synaptic plasticity but also may contribute to the pathogenesis of neurological disorders, including ischemia, epilepsy, and Huntington's disease (1-4). NMDA receptors are made from heteromeric assemblies of different subunits called NR1 and NR2 (NR2A to -D) (5-8). The NR2B subunit is essential for both neonatal and mature NMDA receptors and is highly expressed in the entire embryonic brain and the adult forebrain (9). In the hippocampus, the major neonatal receptor is believed to contain two NR1 and two NR2B subunits, whereas the major receptor in more mature synapses probably contains two NR1 subunits, an NR2A subunit, and an NR2B subunit. Analysis of NR2B-null mice has suggested the physiological importance of NR2B, since the absence of NR2B-containing NMDA receptors in NR2B Ϫ/Ϫ mice leads to perinatal death, whereas mice lacking NR2A, NR2C, and NR2D subtypes are viable (10 -12).NR2B subunits have long C-terminal tails that link NR2B to intracellular pathways. Like NR2B-deficient mice, mice expressing NR2B subunits with truncated C-terminal domains die shortly after birth (13), showing the physiological importance of the intracellular region of NR2B. Therefore, processes that modify the C-terminal region of NR2B may alter the physiological events mediated by NR2B-containing receptors. Calpain and tyrosine kinases regulate NMDA receptors through the C-terminal domain. Calpain, a Ca 2ϩ -dependent neutral cysteine protease, cleaves the C-terminal domain of NR2B in HEK cells, neuronal cultures, and animal models of ischemia and seizures (14 -17). In acutely...

show abstract

N-Methyl-D-aspartate Receptors Expressed in a Nonneuronal Cell Line Mediate Subunit-specific Increases in Free Intracellular Calcium

Cited by 42 publications

References 69 publications

Microarray and Proteomics Analyses of Human Intestinal Epithelial Cells Treated with theAeromonas hydrophilaCytotoxic Enterotoxin

Microarray and Proteomics Analyses of Human Intestinal Epithelial Cells Treated with theAeromonas hydrophilaCytotoxic Enterotoxin

Proteolysis of theN-Methyl-d-Aspartate Receptor by Calpain in Situ

Fyn-mediated Phosphorylation of NR2B Tyr-1336 Controls Calpain-mediated NR2B Cleavage in Neurons and Heterologous Systems

Contact Info

Product

Resources

About