C-terminal Splice Research Articles

Phosphorylation of a Threonine Unique to the Short C-terminal Isoform of βII-Spectrin Links Regulation of α-β Spectrin Interaction to Neuritogenesis

Spectrin tetramers are cytoskeletal proteins required in the formation of complex animal tissues. Mammalian alphaII- and betaII-spectrin subunits form dimers that associate head to head with high affinity to form tetramers, but it is not known if this interaction is regulated. We show here that the short C-terminal splice variant of betaII-spectrin (betaIISigma2) is a substrate for phosphorylation. In vitro, protein kinase CK2 phosphorylates Ser-2110 and Thr-2159; protein kinase A phosphorylates Thr-2159. Antiphospho-Thr-2159 peptide antibody detected phosphorylated betaIISigma2 in Cos-1 cells. Immunoreactivity was increased in Cos-1 cells by treatment with forskolin, indicating that phosphorylation is promoted by elevated cAMP. The effect of forskolin was counteracted by the cAMP-dependent kinase inhibitor, H89. In vitro, protein kinase A phosphorylation of an active fragment of betaIISigma2 greatly reduced its interaction with alphaII-spectrin at the tetramerization site. Mutation of Thr-2159 to alanine eliminated inhibition by phosphorylation. Among the processes that require spectrin in mammals is the formation of neurites (incipient nerve axons). We tested the relationship of spectrin phosphorylation to neuritogenesis by transfecting the neuronal cell line, PC12, with enhanced green fluorescent protein-coupled fragments of betaIISigma2-spectrin predicted to act as inhibitors of spectrin tetramer formation. Both wild-type and T2159E mutant fragments allowed normal neuritogenesis in PC12 cells in response to nerve growth factor. The mutant T2159A inhibited neuritogenesis. Because the T2159A mutant represents a high affinity inhibitor of tetramer formation, we conclude that tetramers are requisite for neuritogenesis. Furthermore, because both the T2159E mutant and the wild-type allow neuritogenesis, we conclude that the short C-terminal betaII-spectrin is phosphorylated during this process.

Differential expression of a C-terminal splice variant of phosphatidylinositol transfer protein β lacking the constitutive-phosphorylated Ser262 that localizes to the Golgi compartment

Mammalian PITPbeta (phosphatidylinositol transfer protein beta) is a 272-amino-acid polypeptide capable of transferring PtdIns, PtdCho and SM (sphingomyelin) between membrane bilayers. It has been reported that Ser262 present in the C-terminus of PITPbeta is constitutively phosphorylated and determines Golgi localization. We provide evidence for the expression of an sp (splice) variant of PITPbeta (PITPbeta-sp2) where the C-terminal 15 amino acids of PITPbeta-sp1 are replaced by an alternative C-terminus of 16 amino acids. PITPbeta-sp1 is the product of the first 11 exons, whereas PITPbeta-sp2 is a product of the first 10 exons followed by the twelfth exon--exon 11 being 'skipped'. Both splice variants are capable of PtdIns and PtdCho transfer, with PITPbeta-sp2 being unable to transport SM. PITPbeta is ubiquitously expressed, with the highest amounts of PITPbeta found in HL60 cells and in rat liver; HL60 cells express only PITPbeta-sp1, whereas rat liver expresses both sp variants in similar amounts. In both cell types, PITPbeta-sp1 is constitutively phosphorylated and both the PtdIns and PtdCho forms of PITPbeta-sp1 are present. In contrast, PITPbeta-sp2 lacks the constitutively phosphorylated Ser262 (replaced with glutamine). Nonetheless, both PITPbeta variants localize to the Golgi and, moreover, dephosphorylation of Ser262 of PITPbeta-sp1 does not affect its Golgi localization. The presence of PITPbeta sp variants adds an extra level of proteome complexity and, in rat liver, the single gene for PITPbeta gives rise to seven distinct protein species that can be resolved on the basis of their charge differences.

Molecular Identification and Characterization of a Family of Kinases with Homology to Ca2+/Calmodulin-dependent Protein Kinases I/IV

Despite the critical importance of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1alpha and CL1beta were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2alpha and CL2beta contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca(2+)/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2.

P3-426: An analysis of spatial, functional and species differences in C-terminal splice variants of the 5-HT4 serotonin receptor

P3-426: An analysis of spatial, functional and species differences in C-terminal splice variants of the 5-HT4 serotonin receptor

Carboxy Terminus Splice Variation Alters ClC Channel Gating and Extracellular Cysteine Reactivity

CLH-3a and CLH-3b are Caenorhabditis elegans ClC channel splice variants that exhibit striking differences in voltage, Cl−, and H+ sensitivity. The major primary structure differences between the channels include a 71 amino acid CLH-3a N-terminal extension and a 270 amino acid extension of the CLH-3b C-terminus. Deletion of the CLH-3a N-terminus or generation of a CLH-3a/b chimera has no effect on channel gating. In contrast, deletion of a 169 amino acid C-terminal CLH-3b splice insert or deletion of the last 11 amino acids of cystathionine-β-synthase domain 1 gives rise to functional properties identical to those of CLH-3a. Voltage-, Cl−-, and H+-dependent gating of both channels are lost when their glutamate gates are mutated to alanine. Glutamate gate cysteine mutants exhibit similar degrees of inhibition by MTSET, but the inhibition time constant of CLH-3b is sevenfold greater than that of CLH-3a. Differences in MTSET inhibition are reversed by deletion of the same cytoplasmic C-terminal regions that alter CLH-3b gating. Our results indicate that splice variation of the CLH-3b cytoplasmic C-terminus alters extracellular structure and suggest that differences in the conformation of the outer pore vestibule and associated glutamate gate may account for differences in CLH-3a and CLH-3b gating.

Expression and Functional Characterization of the Human Ether-à-go-go-Related Gene (HERG) K+ Channel Cardiac Splice Variant in Xenopus laevis Oocytes

HERG C(Cardiac), a C-terminal splice variant of the human ether-à-go-go-related gene (HERG A), was identified and found to be 100% homologous to HERG(USO). Real-time polymerase chain reaction data indicated that in the human heart HERG C(Cardiac )mRNA was expressed eight times more than HERG A, whereas in human ventricular tissue it was expressed six times more than HERG A. A HERG C(Cardiac)-green fluorescence protein (GFP) construct was heterologously expressed in Xenopus oocytes. Confocal micrographs revealed that HERG C(Cardiac )was mainly expressed in the plasma membrane. HERG C(Cardiac) channel expressed in oocytes produced slower inactivating outward currents and faster deactivating tail currents than those of HERG A channel. Equal amounts of HERG A and HERG C(Cardiac) cRNA coinjected into oocytes formed intermediate HERG A + HERG C(Cardiac) heteromultimers, which was reconfirmed by immunoprecipitation experiments with a HERG A N-terminal antibody. These heteromultimers had different inactivation, deactivation and activation kinetics from those of HERG A and HERG C(Cardiac) channels. HERG A + HERG C(Cardiac) heteromultimers significantly reduced the model action potential mean amplitude and increased the fast and slow inactivation tau values of the action potential repolarization phase, suggesting involvement of HERG A and HERG C(Cardiac) heteromultimers in modulation of the refractory interval.

Role of phospholipase D2 in the agonist‐induced and constitutive endocytosis of G‐protein coupled receptors

We have recently shown that the mu-opioid receptor [MOR1, also termed mu-opioid peptide (MOP) receptor] is associated with the phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane. We further demonstrated that, in human embryonic kidney (HEK) 293 cells co-expressing MOR1 and PLD2, treatment with (D-Ala2, Me Phe4, Glyol5)enkephalin (DAMGO) led to an increase in PLD2 activity and an induction of receptor endocytosis, whereas morphine, which does not induce opioid receptor endocytosis, failed to activate PLD2. In contrast, a C-terminal splice variant of the mu-opioid receptor (MOR1D, also termed MOP(1D)) exhibited robust endocytosis in response to both DAMGO and morphine treatment. We report here that MOR1D also mediates an agonist-independent (constitutive) PLD2-activation facilitating agonist-induced and constitutive receptor endocytosis. Inhibition of PLD2 activity by over-expression of a dominant negative PLD2 (nPLD2) blocked the constitutive PLD2 activation and impaired the endocytosis of MOR1D receptors. Moreover, we provide evidence that the endocytotic trafficking of the delta-opioid receptor [DOR, also termed delta-opioid peptide (DOP) receptor] and cannabinoid receptor isoform 1 (CB1) is also mediated by a PLD2-dependent pathway. These data indicate the generally important role for PLD2 in the regulation of agonist-dependent and agonist-independent G protein-coupled receptor (GPCR) endocytosis.

Constitutive Expression of Pax5 in Cord Blood Progenitor Cells Rather Inhibits B Lymphopoiesis as Well as Myelopoiesis through the Exon 9-Dependent and Independent Mechanism.

Pax5 is a key regulator of B cell commitment and is indispensable throughout the early stages of B cell differentiation. Human B lymphopoiesis can be simulated by cultivating cord blood (CB) progenitor cells on the murine bone marrow stromal cell line such as HESS-5. When CB CD34+ cells are seeded onto HESS-5 cells and supplemented with SCF and G-CSF, the number of non-adherent myeloid cells initially increases, reaching a maximum during the 3rd week, and decreases rapidly thereafter. On the other hand, foci of small B-lymphoid cells beneath the stromal layer appear during the 2nd week and increase in size until the 6th week. In this culture system, we have identified two C-terminal splice variants of Pax5; one skips exon 9 without subsequent frameshift (del9), and the other has a frameshift insert between exon 8 and 9, resulting in novel C-terminal sequences (ins8′). By quantitative RT-PCR analysis, del9 mRNA was barely detected in CB CD34+ cells and thereafter upregulated in progenitor B cells during the culture. In contrast, wild-type Pax5 (wt) and ins8′ mRNA could not be detected until the 3rd week. Then, wt mRNA was rapidly upregulated 100-fold or more abundantly than del9 and ins8′ mRNA (Fig. 1). Transactivation potential of Pax5 isoforms was examined with a reporter plasmid containing high-affinity Pax5 binding sites from the CD19 gene linked to the minimal TATA-like promoter. As a result, del9 showed constantly higher (up to 3.5-fold) activity, but ins8′ had slightly lower activity than wt Pax5. Furthermore, TLE4, a Groucho co-repressor family member and a known Pax5 partner, inhibited the transcriptional activity of wt Pax5 but not del9 or ins8′ isoform, suggesting its interaction with the exon 9 sequences of Pax5. We next examined the impact of Pax5 on growth and differentiation of CB CD34+ cells by retroviral transduction of each isoform as well as EGFP. After infection, sorted EGFP + cells were subjected to the colony-forming assay and the co-culture assay on HESS-5 cells. Erythroid or mixed colony formation was almost completely inhibited by either isoform. GM-colony formation was moderately inhibited by del9 and ins8′ isoforms, but profoundly suppressed by wt Pax5. In coculture with HESS-5 cells, the number of non-adherent myeloid cells was reduced by Pax5 isoforms in a similar manner to the results of GM-colony formation. Unexpectedly, after 6 weeks of culture, the number of CD19+ adherent lymphoid cells decreased more profoundly in the culture transduced with wt Pax5 than those with del9 and ins8′ isoforms (Fig 2). These results suggest that constitutive expression of Pax5 in cord blood progenitor cells substantially inhibits not only myelopoiesis but also B lymphopoiesis through the exon 9-dependent and independent mechanism. We also raise the possibility that developmentally regulated concentration of Pax5 isoforms may be crucial for human B lymphopoiesis. [Display omitted]

Novel splice variants of human ADAR2 mRNA: Skipping of the exon encoding the dsRNA-binding domains, and multiple C-terminal splice sites

We report here two previously unknown alternative splice sites in the mRNA of human adenosine deaminase acting on RNA type 2 (ADAR2), an RNA editing enzyme. One splices out the whole of exon 2, which encodes two double-stranded RNA-binding domains (dsRBDs), resulting in a frameshift that introduces a stop codon in the downstream exon. This variant accounts for between 13% and 20% of the total ADAR2 mRNA in each developmental stage and brain region examined, even though its translated product is not expressed at levels that are detectable by Western blot analysis. The other new splice site is located in exon 9, 83 nucleotides downstream of the stop codon for the long C-terminus, resulting in a new 3′ untranslated region (UTR) that is about 80 bp longer than the previously reported short C-terminus. The variant produced by this splice site has a stop codon at the same site as that in ADAR2 mRNA containing canonical exons 9 and 10, and is predicted to be translated as an enzymatically active ADAR2 protein. With these two additional splice sites, a total of 48 mRNA variants are theoretically possible, because each splicing event occurs independently. Among them, variants containing the long C-terminus are translated in human brains in situ, implying that alternative splicing in the 3′ UTR of ADAR2 might regulate translational efficiency and mRNA stability in vivo.

Calcium-activated potassium channel of the tobacco hornworm,Manduca sexta: molecular characterization and expression analysis

Large-conductance calcium- and voltage-gated potassium channels (BK or Slowpoke) serve as dynamic integrators linking electrical signaling and intracellular activity. These channels can mediate many different Ca2+-dependent physiological processes including the regulation of neuronal and neuroendocrine cell excitability and muscle contraction. To gain insights into the function of BK channels in vivo, we isolated a full-length cDNA encoding the alpha subunit of a Slowpoke channel from the tobacco hornworm, Manduca sexta (msslo). Amino acid sequence comparison of the deduced Manduca protein revealed at least 80% identity to the insect Slo channels. The five C-terminal alternative splice regions are conserved, but the cloned cDNA fragments contained some unique combinations of exons E, G and I. Our spatial profile revealed that transcript levels were highest in skeletal muscle when compared with the central nervous system (CNS) and visceral muscle. The temporal profile suggested that msslo expression is regulated developmentally in a tissue- and regional-specific pattern. The levels of msslo transcripts remain relatively constant throughout metamorphosis in the CNS, transiently decline in the heart and are barely detectable in the gut except in adults. A dramatic upregulation of msslo transcript levels occurs in thoracic but not abdominal dorsal longitudinal body wall muscles (DLM), suggesting that the msSlo current plays an important role in the excitation or contractile properties of the phasic flight muscle. Our developmental profile of msslo expression suggests that msSlo currents may contribute to the changes in neural circuits and muscle properties that produce stage-specific functions and behaviors.

Crystal Structures of an Intein from the Split dnaE Gene of Synechocystis sp. PCC6803 Reveal the Catalytic Model Without the Penultimate Histidine and the Mechanism of Zinc Ion Inhibition of Protein Splicing

The first naturally occurring split intein was found in the dnaE gene of Synechocystis sp. PCC6803 and belongs to a subclass of inteins without a penultimate histidine residue. We describe two high-resolution crystal structures, one derived from an excised Ssp DnaE intein and the second from a splicing-deficient precursor protein. The X-ray structures indicate that His147 in the conserved block F activates the side-chain N(delta) atom of the intein C-terminal Asn159, leading to a nucleophilic attack on the peptide bond carbonyl carbon atom at the C-terminal splice site. In this process, Arg73 appears to stabilize the transition state by interacting with the carbonyl oxygen atom of the scissile bond. Arg73 also seems to substitute for the conserved penultimate histidine residue in the formation of an oxyanion hole, as previously identified in other inteins. The finding that the precursor structure contains a zinc ion chelating the highly conserved Cys160 and Asp140 reveals the structural basis of Zn2+-mediated inhibition of protein splicing. Furthermore, it is of interest to observe that the carbonyl carbon atom of Asn159 and N(eta) of Arg73 are 2.6 angstroms apart in the free intein structure and 10.6 angstroms apart in the precursor structure. The orientation change of the aromatic ring of Tyr-1 following the initial acyl shift may be a key switching event contributing to the alignment of Arg73 and the C-terminal scissile bond, and may explain the sequential reaction property of the Ssp DnaE intein.

Pharmacological Characterization of Human and Murine Neuropeptide S Receptor Variants

We have recently shown that Neuropeptide S (NPS) can promote arousal and induce anxiolytic-like effects after central administration in rodents. Another study reported a number of natural polymorphisms in the human NPS receptor gene. Some of these polymorphisms were associated with increased risk of asthma and possibly other forms of atopic diseases, but the physiological consequences of the mutations remain unclear. One of the polymorphisms produces an Asn-Ile exchange in the first extracellular loop of the receptor protein, and a C-terminal splice variant of the NPS receptor was found overexpressed in human asthmatic airway tissue. We sought to study the pharmacology of the human receptor variants in comparison with the murine receptor protein. Here, we report that the N107I polymorphism in the human NPS receptor results in a gain-of-function characterized by an increase in agonist potency without changing binding affinity in NPSR Ile107. In contrast, the C-terminal splice variant of the human NPS receptor shows a pharmacological profile similar to NPSR Asn107. The mouse NPS receptor, which also carries an Ile residue at position 107, displays an intermediate pharmacological profile. Structure-activity relationship studies show that the amino terminus of NPS is critical for receptor activation. The altered pharmacology of the Ile107 isoform of the human NPS receptor implies a mechanism of enhanced NPS signaling that might have physiological significance for brain function as well as peripheral tissues that express NPS receptors.

Identification of Four Novel Exon 5 Splice Variants of the Mouse μ-Opioid Receptor Gene: Functional Consequences of C-Terminal Splicing

The rat mu-opioid receptor clone in which novel exon 5 was found in the place of exon 4 (MOR-1B) was one of the first MOR-1 variants described. We now have identified the mouse homolog of the rat MOR-1B as well as four additional variants derived from splicing from exon 3 into different sites within exon 5. The sequences of all of the variants were identical except for the intracellular tip of the C terminus encoded by exon 5, where each variant predicted a unique amino acid sequence ranging from 2 to 39 amino acids. All of the mMOR-1B variants were selective for mu-opioids in receptor-binding assays, as anticipated, because they all have identical binding pockets defined by the transmembrane domains. However, the relative potency and efficacy of mu-agonists to each other varied from variant to variant in guanosine 5'-O-(3-[35S]thio)triphosphate-binding studies, as shown by morphine-6beta-glucuronide, which was the most efficacious agent against mouse MOR-1B1 (mMOR-1B1) and the least efficacious agent against mMOR-1B2. mMOR-1B4 was quite unusual. Although mMOR-1B4 was mu-selective in receptor-binding studies and antagonists labeled mMOR-1B4 well, the binding affinities of most of the mu-agonists were far lower than those seen with mMOR-1, suggesting that the 39 amino acids at the C terminus of mMOR-1B4 influences the conformation of the receptor and its ligand recognition site itself either directly or through its interactions with other proteins. In conclusion, alterations in the amino acid sequence of the C terminus do not alter the mu-specificity of the receptor but they can influence the binding characteristics, efficacy, and potency of mu-opioids.

Novel mechanism of a C-terminal intronic splicing mutation in type 2 long QT syndrome

Novel mechanism of a C-terminal intronic splicing mutation in type 2 long QT syndrome

Serotonin 5‐HT4 receptors and their mRNAs in rat and guinea pig brain: Distribution and effects of neurotoxic lesions

Serotonin 5-HT4 receptors are widely distributed in the periphery and in brain, where they modulate the release of various neurotransmitters and have been implicated in learning and memory. Nine C-terminal splice variants of this receptor have been cloned in mammalian species. In the rat, three such variants have been described: 5-HT4(a), 5-HT4(b), and 5-HT4(e). In the present study, we have examined several aspects of the distribution of these receptors in brain. First, we provide, in rat and guinea pig, a detailed comparison of the distribution of 5-HT4 receptors labeled by the antagonist [125I]-SB 207710 with the distribution of their encoding mRNA visualized by in situ hybridization histochemistry (ISHH). The results suggest that, in several projection systems (striato-nigral and striato-pallidal pathways, projection from dentate granule cells to field CA3, habenulo-interpeduncular pathway), 5-HT4 receptors are located both somatodendritically and axonally. Second, we have analyzed the distribution of mRNA for the three known rat splice variants by reverse transcription-polymerase chain reaction (RT-PCR) and by ISHH. RT-PCR indicates that all three variants are widely distributed, with 5-HT4(b) mRNA being present in all regions examined (olfactory tubercle, striatum, hippocampus, inferior colliculus, substantia nigra, parietal cortex) and 5-HT4(a) and 5-HT4(e) showing a somewhat more restricted distribution. In other regions (periaqueductal gray, reticular formation, medial septum, diagonal band), faint ISHH signals are observed for 5-HT4(a)+4(e) mRNAs, whereas 5-HT4(b) mRNA signals are almost undetectable. Finally, neurotoxic lesions of basal ganglia components in guinea pig also indicate a location of these receptors on terminals of striatal projection neurons.

Association of CaV1.3 L-Type Calcium Channels with Shank

Neurons express multiple types of voltage-gated calcium (Ca2+) channels. Two subtypes of neuronal L-type Ca2+ channels are encoded by CaV1.2 and CaV1.3 pore-forming subunits. Both CaV1.2 and CaV1.3 subunits contain class I PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-binding consensus at their C termini. In yeast two-hybrid screen of rat brain cDNA library with the C-terminal bait of CaV1.3a (long C-terminal splice variant) L-type Ca2+ channel subunit, we isolated multiple clones of postsynaptic adaptor protein Shank. We demonstrated a specific association of CaV1.3a C termini, but not of CaV1.2 C termini, with Shank PDZ domain in vitro. We further demonstrated that the proline-rich region present in C termini of CaV1.3a subunit binds to Shank Src homology 3 domain. We established that CaV1.3a and Shank localized to postsynaptic locations in cultured rat hippocampal neurons. By expressing epitope-tagged recombinant CaV1.3 subunits in rat hippocampal neuronal cultures, we demonstrated that the presence of Shank-binding motifs in CaV1.3a sequence is both necessary and sufficient for synaptic clustering of CaV1.3 L-type Ca2+ channels. In experiments with dominant-negative peptides and dihydropyridine-resistant CaV1.3a mutants, we demonstrated an importance of Shank-binding motif in CaV1.3a sequence for phosphorylated cAMP response element-binding protein (pCREB) signaling in cultured hippocampal neurons. Our results directly link CaV1.3 neuronal L-type Ca2+ channels to macromolecular signaling complex formed by Shank and other modular adaptor proteins at postsynaptic density and provide novel information about the role played by CaV1.3 L-type Ca2+ channels in pCREB signaling.

The effects of aging on different C-terminal splice forms of the ζ1(NR1) subunit of the N-methyl- d-aspartate receptor in mice

Changes in NMDA receptors in the prefrontal/frontal cortex and hippocampus of C57BL/6 mice during aging show a relationship to declines in spatial learning. The present study was designed to determine whether aging influences the mRNA expression of different splice forms of the ζ1 subunit of the NMDA receptor. We examined the mRNA of 4 C-terminal splice forms with the use of in situ hybridization. The ζ1-1 splice form (+C1 and +C2 cassettes) overall showed a maintenance of mRNA density from 3 to 10 months of age, followed by a significant decline by 26 months of age. In contrast, the mRNA for the ζ1-3 splice form [+C1 and +C2′(−C2)] showed significant declines between 3- and 10-month-old mice. These declines were maintained in the old mice. The ζ1-2 splice form (−C1 and +C2) showed a near-significant decrease in expression during aging across all brain regions. The ζ1-4 subunit mRNA [−C1 and +C2′ (−C2)] showed no significant changes with increased age. These results indicate that there is a differential effect of aging on different splice variants of the ζ1 subunit of the NMDA receptor and those that are affected show a different temporal pattern of aging. This heterogeneity has implications for producing imbalances in the modulation of the remaining receptors.

E1A Activates Transcription of p73 and Noxa to Induce Apoptosis

p73, a member of the p53 family of proteins, transcriptionally activates a number of genes involved in the control of cell cycle and apoptosis. Overexpression of p73 was detected in a large number of primary head and neck cancers, and in the established cell lines examined, these all contained inactivating p53 mutations. The significance of p73 overexpression in the pathogenesis of head and neck cancer is currently unclear. We have shown that the expression of adenovirus 5 E1A in a panel of head and neck cancer cell lines induces apoptosis independently of their p53 status. In this study we examined the role of p73 and its transcriptional targets in E1A-mediated induction of apoptosis. E1A expression resulted in significant activation of the TAp73 promoter but had no effect on the alternative, DeltaNp73 promoter. E1A also increased expression of endogenous TAp73 mRNA and protein. E1A mutants lacking the p300- and/or pRB-binding sites showed reduced ability to activate the TAp73 promoter. Additionally, mutations in the E2F1-binding sites in the TAp73 promoter impaired activation by E1A. Importantly, expression of the 13S isoform of E1A substantially induced the p53 apoptotic target Noxa in several p53-deficient cancer cell lines. Our results indicate that E1A activation of p73 and the p53 apoptotic target Noxa can occur in the absence of a functional p53. This activation is likely to play a key role in the mechanism of p53-independent apoptosis induced by E1A in some cancers and may provide an avenue for future cancer therapies.

C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation.

The present study provides functional characterization of alternative splicing of the NTPDase2 (ecto-nucleoside triphosphate diphosphohydrolase-2) involved in the regulation of extracellular nucleotide concentrations in a range of organ systems. A novel NTPDase2beta isoform produced by alternative splicing of the rat NTPDase2 gene provides an extended intracellular C-terminus and distinguishes itself from NTPDase2alpha isoform in gaining several intracellular protein kinase CK2 (casein kinase 2) phosphorylation sites and losing the intracellular protein kinase C motif. The plasmids containing NTPDase2alpha or NTPDase2beta cDNA were used to stably transfect Chinese-hamster ovary-S cells. Imaging studies showed that NTPDase2alpha was predominantly membrane-bound, whereas NTPDase2beta had combined cell surface and intracellular localization. alpha and beta isoforms showed variations in divalent cation dependence and substrate specificity for nucleoside-5'-triphosphates and nucleoside-5'-diphosphates. NTPDase2beta exhibited reduced ATPase activity and no apparent ADPase activity. NTPDase2 isoforms demonstrated similar sensitivity to inhibitors such as suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid, and differential regulation by protein kinases. NTPDase2beta was up-regulated by intracellular protein kinase CK2 phosphorylation, whereas NTPDase2alpha activity was down-regulated by protein kinase C phosphorylation. The results demonstrate that alternative coding of the intracellular C-terminal domain contributes distinctive phenotypic variation with respect to extracellular nucleotide specificity, hydrolysis kinetics, protein kinase-dependent intracellular regulation and protein trafficking. These findings advance the molecular physiology of this enzyme system by characterizing the contribution of the C-terminal domain to many of the enzyme's signature properties.

Microarray and real-time RT-PCR analyses of differential human gene expression patterns induced by severe acute respiratory syndrome (SARS) coronavirus infection of Vero cells

Vero E6 African green monkey kidney cells are highly susceptible to infection with the newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV), and they are permissive for rapid viral replication, with resultant cytopathic effects. We employed cDNA microarray analysis to characterize the cellular transcriptional responses of homologous human genes at 12 h post-infection. Seventy mRNA transcripts belonging to various functional classes exhibited significant alterations in gene expression. There was considerable induction of heat shock proteins that are crucial to the immune response mechanism. Modified levels of several transcripts involved in pro-inflammatory and anti-inflammatory processes exemplified the balance between opposing forces during SARS pathogenesis. Other genes displaying altered transcription included those associated with host translation, cellular metabolism, cell cycle, signal transduction, transcriptional regulation, protein trafficking, protein modulators, and cytoskeletal proteins. Alterations in the levels of several novel transcripts encoding hypothetical proteins and expressed sequence tags were also identified. In addition, transcription of apoptosis-related genes DENN and hIAP1 was upregulated in contrast to FAIM. Elevated Mx1 expression signified a strong host response to mediate antiviral resistance. Also expressed in infected cells was the C-terminal alternative splice variant of the p53 tumor suppressor gene encoding a modified truncated protein that can influence the activity of wild-type p53. We observed the interplay between various mechanisms to favor virus multiplication before full-blown apoptosis and the triggering of several pathways in host cells in an attempt to eliminate the pathogen. Microarray analysis identifies the critical host–pathogen interactions during SARS-CoV infection and provides new insights into the pathophysiology of SARS.