Isoforms of the Na-K-2Cl cotransporter in murine TAL II. Functional characterization and activation by cAMP

1999 ◽  
Vol 276 (3) ◽  
pp. F359-F366 ◽  
Author(s):  
Consuelo Plata ◽  
David B. Mount ◽  
Verena Rubio ◽  
Steven C. Hebert ◽  
Gerardo Gamba
Keyword(s):  
Amino Acids ◽  
Xenopus Oocytes ◽  
Functional Characterization ◽  
Inhibitory Effect ◽  
Tracer Uptake ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Thick Ascending Limb ◽  
Alternatively Spliced ◽  
Uptake Medium

The functional properties of alternatively spliced isoforms of the mouse apical Na+-K+-2Cl−cotransporter (mBSC1) were examined, using expression in Xenopus oocytes and measurement of22Na+or86Rb+uptake. A total of six isoforms, generated by the combinatorial association of three 5′ exon cassettes (A, B, and F) with two alternative 3′ ends, are expressed in mouse thick ascending limb (TAL) [see companion article, D. B. Mount, A. Baekgaard, A. E. Hall, C. Plata, J. Xu, D. R. Beier, G. Gamba, and S. C. Hebert. Am. J. Physiol. 276 ( Renal Physiol. 45): F347–F358, 1999]. The two 3′ ends predict COOH-terminal cytoplasmic domains of 129 amino acids (the C4 COOH terminus) and 457 amino acids (the C9 terminus). The three C9 isoforms (mBSC1-A9/F9/B9) all express Na+-K+-2Cl−cotransport activity, whereas C4 isoforms are nonfunctional in Xenopus oocytes. Activation or inhibition of protein kinase A (PKA) does not affect the activity of the C9 isoforms. The coinjection of mBSC1-A4 with mBSC1-F9 reduces tracer uptake, compared with mBSC1-F9 alone, an effect of C4 isoforms that is partially reversed by the addition of cAMP-IBMX to the uptake medium. The inhibitory effect of C4 isoforms is a dose-dependent function of the alternatively spliced COOH terminus. Isoforms with a C4 COOH terminus thus exert a dominant negative effect on Na+-K+-2Cl−cotransport, a property that is reversed by the activation of PKA. This interaction between coexpressed COOH-terminal isoforms of mBSC1 may account for the regulation of Na+-K+-2Cl−cotransport in the mouse TAL by hormones that generate cAMP.

Isoforms of the Na-K-2Cl cotransporter in murine TAL I. Molecular characterization and intrarenal localization

1999 ◽  
Vol 276 (3) ◽  
pp. F347-F358 ◽  
Author(s):  
David B. Mount ◽  
Allan Baekgaard ◽  
Amy E. Hall ◽  
Consuelo Plata ◽  
Jason Xu ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Transmembrane Domain ◽  
Alternative Polyadenylation ◽  
Functional Expression ◽  
Mouse Kidney ◽  
Dominant Negative ◽  
Thick Ascending Limb ◽  
Chromosome 2 ◽  
Negative Function ◽  
Alternatively Spliced

We have identified several alternatively spliced cDNAs encoding mBSC1, an apical bumetanide-sensitive Na+-K+-2Cl−cotransporter from mouse kidney. Two full-length clones were isolated, designated C4 and C9, predicting proteins of 770 and 1,095 amino acids, respectively. The C4 isoforms are generated by utilization of an alternative polyadenylation site located within the intron between exons 16 and 17 of the mBSC1 gene on chromosome 2; the resultant transcripts predict a truncated COOH terminus ending in a unique 55 amino acid sequence. The predicted C4 and C9 COOH termini differ in the distribution of putative phosphorylation sites for both protein kinase A and C. Independent splicing events involve three previously described cassette exons, which are predicted to encode most of the second transmembrane domain. A total of six different isoforms are expressed, generated by the combinatorial association of three cassette exons and two alternative 3′ ends. C9-specific and C4-specific antibodies detect proteins of ∼150 and 120 kDa, respectively, in mouse kidney. Immunofluorescence and immunohistochemistry indicate expression of both COOH-terminal isoforms within the thick ascending limb of the loop of Henle (TAL). However, staining with the C4 antibody is more heterogeneous, with a decreased proportion of positive cells in the cortical TAL. Functional expression in Xenopus oocytes indicates a dominant negative function for C4 isoforms [companion study, C. Plata, D. B. Mount, V. Rubio, S. C. Hebert, and G. Gamba. Am. J. Physiol. 276 ( Renal Physiol. 45): F347–F358, 1999], and the differential expression of these isoforms may contribute to functional heterogeneity of Na+-K+-2Cl−cotransport in mouse TAL.

scholarly journals A Mutation Linked with Bartter's Syndrome Locks Kir 1.1a (Romk1) Channels in a Closed State

1999 ◽  
Vol 114 (5) ◽  
pp. 685-700 ◽  
Author(s):  
Thomas P. Flagg ◽  
Margaret Tate ◽  
Jean Merot ◽  
Paul A. Welling
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
K Channel ◽  
Bartter’S Syndrome ◽  
Wild Type ◽  
Bartter's Syndrome ◽  
Closed State ◽  
Negative Effect

Mutations in the inward rectifying renal K+ channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. One disease-causing mutation removes the last 60 amino acids (332–391), implicating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated channels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles of this domain were systematically evaluated. When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion construct was indistinguishable from the GFP–wild-type channel, demonstrating that mutant channels are expressed on the oocyte plasma membrane in a nonconductive or locked-closed conformation. Incremental reconstruction of the COOH terminus identified amino acids 332–351 as the critical residues for restoring channel activity and uncovered the nature of the functional defect. Mutant channels that are truncated at the extreme boundary of the required domain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies the extreme COOH terminus as an important subunit interaction domain, controlling the efficiency of oligomerization. Collectively, these observations provide a mechanistic basis for the loss of function in one particular Bartter's-causing mutation and identify a structural element that controls open-state occupancy and determines subunit oligomerization. Based on the overlapping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.

scholarly journals Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

2011 ◽  
Vol 300 (5) ◽  
pp. C1055-C1064 ◽  
Author(s):  
Jun-Jie Tong ◽  
Peter J. Minogue ◽  
Wenji Guo ◽  
Tung-Ling Chen ◽  
Eric C. Beyer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Xenopus Oocytes ◽  
Dominant Negative ◽  
Wild Type ◽  
Gap Junction Channels ◽  
Dominant Negative Inhibitor ◽  
Intercellular Channels ◽  
Gap Junctional

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.

A Mutant (Arg327→His) GPIIb Associated to Thrombasthenia Exerts a Dominant Negative Effect in Stably Transfected CHO Cells

1996 ◽  
Vol 76 (03) ◽  
pp. 292-301 ◽  
Author(s):  
Milagros Ferrer ◽  
Marta Fernandez-Pinel ◽  
Consuelo Gonzalez-Manchon ◽  
Jose Gonzalez ◽  
Matilde S Ayuso ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cho Cells ◽  
Functional Characterization ◽  
Surface Expression ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Glycoprotein Complex ◽  
Negative Effect

SummaryThis work reports the structural and functional characterization of the platelet glycoprotein complex GPIIb-IIIa (integrin αIIbβ3) in a patient of type II Glanzmann thrombasthenia, bearing a homozygous G→A base transition at position 1074 of GPIIb that results in an Arg327→His substitution.CHO cells stably transfected with cDNA encoding His327GPIIb showed a drastic reduction in the surface expression of αIIbβ3 complex relative to control cells transfected with wild type GPIIb. Immunopre-cipitation analysis demonstrated that GPIIb synthesis, heterodimeriza-tion, and short term maturation were not impeded, suggesting that conformational changes dependent on Arg327 of GPIIb may play an essential role in either the rate of maturation and/or transport of heterodimers to the cell surface.Cotransfection of CHO cells with equimolar amounts of cDNAs encoding wild type and mutant His327-GPIIb led to a marked reduction in the surface expression of αIIbβ3. This novel observation of a dominant-negative effect of the mutant His327αIIb subunit provides a molecular basis for the reduced platelet αIIbβ3 content observed in the heterozygous offspring.

A Novel Mutation in EFNB1, Probably with a Dominant Negative Effect, Underlying Craniofrontonasal Syndrome

2006 ◽  
Vol 43 (2) ◽  
pp. 152-154 ◽  
Author(s):  
Vorasuk Shotelersuk ◽  
Pichit Siriwan ◽  
Surasawadee Ausavarat
Keyword(s):  
Amino Acids ◽  
Novel Mutation ◽  
Clinical Manifestations ◽  
Dominant Negative ◽  
Soluble Form ◽  
Dominant Negative Effect ◽  
Truncated Protein ◽  
Frontonasal Dysplasia ◽  
Thai Woman ◽  
Negative Effect

Craniofrontonasal syndrome (CFNS) is an X-linked disorder whose main clinical manifestations include coronal craniosynostosis and frontonasal dysplasia. Very recently, CFNS was shown to be caused by mutations in EFNB1 encoding ephrin-B1, and 20 mutations have been described. We report a Thai woman with CFNS, in whom a novel mutation was discovered: c.685_686insG, in exon 5 of EFNB1. It is the first insertion and the most 3′ point mutation in EFNB1 reported to date. The mutation is expected to result in a truncated ephrin-B1 of 230 amino acids, composed of a nearly complete extracellular part of ephrin-B1 with no transmembrane and cytoplasmic domains. This truncated protein might become a soluble form of the ligand, which previously was shown to be able to bind to receptors, but fail to cluster and to activate them—in other words, acting as a dominant negative protein. Nonetheless, further studies to detect the protein are needed to substantiate the hypothesis.

scholarly journals Functional Characterization of Nuclear Trafficking Signals in Pseudorabies Virus pUL31

2014 ◽  
Vol 89 (4) ◽  
pp. 2002-2012 ◽  
Author(s):  
Lars Paßvogel ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Walter Fuchs ◽  
Thomas C. Mettenleiter
Keyword(s):  
Nuclear Export ◽  
Pseudorabies Virus ◽  
Functional Characterization ◽  
Nuclear Export Signal ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Nuclear Targeting ◽  
Nuclear Egress ◽  
C Terminus ◽  
Negative Effect

ABSTRACTThe herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-ΔNLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes.IMPORTANCEThe molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.

scholarly journals Deletion Analysis of the Flagellar Switch Protein FliG of Salmonella

2000 ◽  
Vol 182 (11) ◽  
pp. 3022-3028 ◽  
Author(s):  
May Kihara ◽  
Gabriele U. Miller ◽  
Robert M. Macnab
Keyword(s):  
Amino Acids ◽  
Dominant Negative ◽  
Bacterial Motility ◽  
Dominant Negative Effect ◽  
Flagellar Motor ◽  
Wild Type ◽  
Terminal Fragment ◽  
N Terminus ◽  
Negative Effect ◽  
Dominance Properties

ABSTRACT The flagellar motor/switch complex, consisting of the three proteins FliG, FliM, and FliN, plays a central role in bacterial motility and chemotaxis. We have analyzed FliG, using 10-amino-acid deletions throughout the protein and testing the deletion clones for their motility and dominance properties and for interaction of the deletion proteins with the MS ring protein FliF. Only the N-terminal 46 amino acids of FliG (segments 1 to 4) were important for binding to FliF; consistent with this, an N-terminal fragment consisting of residues 1 to 108 bound FliF strongly, whereas a C-terminal fragment consisting of residues 109 to 331 did not bind FliF at all. Deletions in the region from residues 37 to 96 (segments 4 to 9), 297 to 306 (segment 30), and 317 to 326 (segment 32) permitted swarming, though not at wild-type levels; all other deletions caused paralyzed or, more commonly, nonflagellate phenotype. Except for those near the N terminus, deletions had a dominant negative effect on wild-type cells.

scholarly journals Tumor-derived Mutations in the TRAIL Receptor DR5 Inhibit TRAIL Signaling through the DR4 Receptor by Competing for Ligand Binding

2007 ◽  
Vol 282 (38) ◽  
pp. 28189-28194 ◽  
Author(s):  
Lianghua Bin ◽  
Jacqueline Thorburn ◽  
Lance R. Thomas ◽  
Peter E. Clark ◽  
Robin Humphreys ◽  
...  
Keyword(s):  
Point Mutations ◽  
Inhibitory Effect ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Agonistic Antibody ◽  
Signaling Complex ◽  
Trail Receptors ◽  
Negative Effect ◽  
Mutant Receptors ◽  
Trail Signaling

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a cytokine that preferentially induces apoptosis in tumor cells compared with normal cells through two receptors (DR4 and DR5). Somatic mutations in these receptors have been found in different kinds of cancer; however, it is poorly understood how the mutations affect signaling. We found that point mutations (L334F, E326K, E338K, and K386N) that were identified in human tumors result in the DR5 receptor losing its ability to form a functional death-inducing signaling complex and induce apoptosis. The mutant receptors also have a “dominant negative” effect whereby they inhibit the ability of TRAIL to induce apoptosis through functional DR4 receptors. This dominant negative mechanism is achieved through competition for TRAIL binding as shown by experiments where the ability of the mutant DR5 receptor to bind with the ligand was abolished, thus restoring TRAIL signaling through DR4. The inhibitory effect on signaling through the wild-type DR4 protein can be overcome if the inhibitory mechanism is bypassed by using a DR4-agonistic antibody that is not subject to this competition. This study provides a molecular basis for the use of specific therapeutic agonists of TRAIL receptors in people whose tumors harbor somatic DR5 mutations.

Dominant-Negative Effect of NPM-RAR on NPM Mediated Regulation of p53.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1456-1456
Author(s):  
Robert L. Redner ◽  
Erin M. Swaney ◽  
Elizabeth A. Rush
Keyword(s):  
Amino Acids ◽  
Dominant Negative ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Dominant Negative Effect ◽  
Response Element ◽  
Genetic Event ◽  
Cos Cells ◽  
Control Plasmid ◽  
P53 Response

Abstract We have been studying the variant Acute Promyelocytic Leukemia (APL) translocations with a view towards identifying common molecular pathways underlying APL. The t(5;17) variant of APL fuses the N-terminal 117 amino acids of nucleophosmin (NPM) with the C-terminal 402 amino acids of the retinoic acid receptor alpha (RAR). NPM plays a role in several important pathways within cells beyond its originally described function in the transport of ribonucleoproteins. It serves a chaperone function in transport of proteins such as p53, Tat, Rb, YY1, IRF-1, and Arf. NPM binds to and becomes a target of CDK2/cyclin E to play a role in regulation of centrosomal duplication. The importance of NPM in myeloid leukemias has been highlighted by the recent finding that mutation of the NPM C-terminus is the most common genetic event in non-APL acute myeloid leukemia. We have previously shown that the RAR portion of NPM-RAR acts as a dominant-negative towards wild-type RAR. We now investigate the hypothesis that NPM-RAR serves as a dominant negative for NPM and partially contributes to the leukemic phenotype through disruption of NPM-dependent cellular functions. We have focused our studies on the effects of NPM-RAR on p53. It has previously been shown that NPM directly binds to p53 to increase p53 stability and p53-dependent transcription. The p53-interaction domain maps to a region of NPM that is not contained in NPM-RAR. We hypothesized that NPM-RAR might bind directly with NPM, disrupt NPM association with p53, and thereby indirectly affect p53 activity. We first used pull-down assays of Maltose Binding Protein (MBP)-NPM or MBP-NPM-RAR fusions incubated with 35S-NPM to determine that the N-terminal domain of NPM-RAR is sufficient to interact with NPM. To investigate whether NPM-RAR alters NPM interaction with p53, we transfected COS cells with expression vectors encoding NPM, p53 and either NPM-RAR or control plasmid (pBluescript). Complexes were precipitated with an anti-NPM antibody (that recognizes epitopes not contained in NPM-RAR) and immunoblotted with an anti-p53 antibody. We found a decrease in the p53 signal captured by NPM in the lysate expressing NPM-RAR, compared to the control lysate. To further investigate the effects of NPM-RAR on p53, we made use of a transcriptional assay utilizing a reporter gene under control of a p53 response element. We co-transfected COS cells with a mix containing NPM-RAR, p53, and NPM expression plasmids, along with a p53-luciferase reporter (containing a 20 base pair p53 response element derived from the p21 promoter), and a ß-gal transfection control plasmid, or a similar mix in which NPM-RAR was replaced by an equivalent amount of carrier DNA (pBluescript). We found that the co-expression of NPM-RAR led to a decrease in the luciferase activity. These results support our hypothesis that NPM-RAR binding to NPM interferes with NPM’s ability to regulate p53.

scholarly journals Functional characterization of alternatively spliced human SERCA3 transcripts

1998 ◽  
Vol 275 (6) ◽  
pp. C1449-C1458 ◽  
Author(s):  
Esteban Poch ◽  
Stephen Leach ◽  
Susan Snape ◽  
Tasha Cacic ◽  
David H. MacLennan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Functional Characterization ◽  
Lymphoid Tissues ◽  
Gene Products ◽  
Alternatively Spliced ◽  
Lower Capacity

The sarcoplasmic (or endoplasmic) reticulum Ca2+-ATPase (SERCA)-3 has been implicated in the possible dysregulation of Ca2+ homeostasis that accompanies the pathology of hypertension and diabetes. We report the molecular cloning of two alternatively spliced transcripts from the human SERCA3 gene, ATP2A3, that encode proteins that differ at their carboxy termini by 36 amino acids. SERCA3 transcripts were most abundantly expressed in lymphoid tissues, intestine, pancreas, and prostate. The two human SERCA3 proteins encoded by alternatively spliced transcripts were recognized by the monoclonal antibody PL/IM430 and demonstrated Ca2+ uptake and ATPase activity with an apparent Ca2+ affinity 0.5 pCa unit lower than that of other SERCA gene products. The subcellular distribution of SERCA3 protein was indistinguishable from that of SERCA2b, with expression in the nuclear envelope and in the endoplasmic reticulum throughout the cell. Two variant SERCA3 constructs, huS3-I and huS3-II, were isolated that encode proteins with three amino acid differences: Ala-673 (in huS3-I) substituted for Thr (in huS3-II), Ile-817 substituted for Met, and an insertion of Glu-994. huS3-I displayed a 10-fold lower capacity to transport Ca2+ than huS3-II.

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