Mutations in transmembrane domains 5 and 7 of the human excitatory amino acid transporter 1 affect the substrate-activated anion channel

L-Glutamate is the predominant excitatory neurotransmitter in the brain, and its extracellular concentration is tightly controlled by the excitatory amino acid transporters (EAATs). The transport of 1 glutamate molecule is coupled to the cotransport of 3 Na+ and 1 H+ and the countertransport of 1 K+. In addition to substrate transport, the binding of glutamate and Na+ activates an anion current which is thermodynamically uncoupled from the transport process. We have identified three amino acid residues in EAAT1 (D272 in TM5, K384 and R385 in TM7) that influence the amplitude of the anion channel current relative to the transport current. Transporters containing the mutations R268A, D272A, D272K, K384A, K384D, R385A, and R385D were expressed in Xenopus laevis oocytes and their transport and anion channel functions measured using the two-electrode voltage clamp techniques. The D272, K384, and R385 mutant transporters showed no change in transport properties but have increased levels of anion channel activity compared to wild-type transporters. These results identify additional residues of the EAAT1 transporter that may contribute to the gating mechanism of the anion channel of glutamate transporters and also provide hints as to how substrate binding leads to channel activation.     

Biochemical evidence for a 170-kilodalton, AF-2-dependent vitamin D receptor/retinoid X receptor coactivator that is highly expressed in osteoblasts

Human vitamin D receptor (hVDR) fused to glutathione S-transferase was utilized to detect a VDR-interacting protein (VIP) of approximately 170 kDa. VIP(170) is expressed in osteoblast-like ROS 17/2.8 cells and, to a lesser extent, in COS-7 and HeLa cells. VIP(170) may be a coactivator because it interacts only with 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) ligand-bound hVDR and because a mutation (E420A) in the activation function-2 (AF-2) of hVDR abolishes both receptor-mediated transactivation and VIP(170) binding. Unlike L254G hVDR, a heterodimerization mutant with an intact AF-2, the E420A mutant is only partially attenuated in its association with the retinoid X receptor (RXR) DNA-binding partner. Finally, the ability of overexpressed hVDR to squelch glucocorticoid receptor-mediated transactivation is lost in both the L254G and E420A mutants. These results suggest that several protein-protein interactions, including VDR association with RXR and VIP(170), are required for stabilization of a multimeric complex that transduces the signal for 1,25(OH)(2)D(3)-elicited transactivation.     

Overexpression of the human vitamin D3 receptor in mammalian cells using recombinant adenovirus vectors.

The human vitamin D3 receptor (hVDR) cDNA was cloned into the E1 region of the adenovirus genome to generate recombinant viruses which were used to infect 293 (adenovirus-transformed human fetal kidney) cells. High salt extracts from cells infected with the recombinant viruses were subjected to immunoblot analysis using a monoclonal antibody to chicken VDR and were shown to contain large quantities of a protein of approximately 50 kDa with a migration identical to that of the hVDR in T47D (human mammary adenocarcinoma) cells. Scatchard analysis showed that the infected cells express approximately 100-fold more receptor than T47D cells and that this receptor binds 1,25-dihydroxyvitamin D3 with high affinity. The overexpressed hVDR also binds to DNA-cellulose and is eluted with a KCl concentration similar to that determined for fully active endogenous VDR. Nuclear extracts from cells infected with the hVDR-expressing adenoviruses contain an activity that specifically binds an oligonucleotide with sequences from the rat osteocalcin vitamin D3 response element, as determined by gel mobility shift. This interaction can be inhibited by the presence of an anti-VDR antibody, but not by nonspecific immunoglobulins. We conclude, therefore, that the overexpressed receptor has the ligand- and DNA-binding characteristics defined for endogenous VDR and that adenoviruses can be used to efficiently express large quantities of functional hVDR in a human cell line. Finally, a second binding activity, specific for the vitamin D response element, but distinct from the VDR, has been identified in extracts from uninfected cells.     

The association of Duffy binding protein region II polymorphisms and its antigenicity in Plasmodium vivax isolates from Thailand

Plasmodium vivax Duffy binding protein II (DBPII) plays an important role in reticulocyte invasion and is a potential vaccine candidate against vivax malaria. However, polymorphisms in DBPII are a challenge for the successful design of a broadly protective vaccine. In this study, the genetic diversity of DBPII among Thai isolates was analyzed from Plasmodium vivax-infected blood samples and polymorphism characters were defined with the MEGA4 program. Sequence analysis identified 12 variant residues that are common among Thai DBPII haplotypes with variant residues L333F, L424I, W437R and I503K having the highest frequency. Variant residue D384K occurs in combination with either E385K or K386N/Q. Additionally, variant residue L424I occurs in conjunction with W437R in most Thai DBPII alleles and these variants frequently occur in combination with the I503K variant. The polymorphic patterns of Thai isolates were defined into 9 haplotypes (Thai DBL-1, -2, -3, etc.…). Thai DBL-2, -5, -6 haplotypes are the most common DBPII variants in Thai residents. To study the association of these Thai DBPII polymorphisms with antigenic character, the functional inhibition of anti-DBPII monoclonal antibodies against a panel of Thai DBL variants was characterized by an in vitro erythrocyte binding inhibition assay. The functional inhibition of anti-DBPII monoclonal antibodies 3C9, 2D10 and 2C6 against Thai variants was significantly different, suggesting that polymorphisms of Thai DBPII variants alter the antigenic character of the target epitopes. In contrast, anti-DBPII monoclonal antibody 2H2 inhibited all Thai DBPII variants equally well. Our results suggest that the immune efficacy of a DBPII vaccine will depend on the specificity of the anti-DBPII antibodies induced and that it is preferable to optimize responses to conserved epitopes for broadly neutralizing protection against P. vivax.     

The importance of nuclear import in protection of the vitamin D receptor from polyubiquitination and proteasome‐mediated degradation

Others and we previously showed that the vitamin D receptor (VDR) is subject to degradation by the 26S proteasome and that treatment with 1,25‐dihydroxyvitamin D₃ (1,25D₃) inhibited this degradation. In the present study, we found that in osteoblasts, but not in intestinal epithelial cells, the VDR was susceptible to degradation by the 26S proteasome. The subcellular site for degradation of the VDR in osteoblasts is the cytoplasm and the site for ligand‐dependent protection of the VDR from the 26S proteasome is the chromatin. These direct relationships between nuclear localization and protection of the VDR from 26S proteasome degradation led us to hypothesize that the unoccupied cytoplasmic VDR is a substrate for polyubiquitination, which targets VDR for degradation by the 26S proteasome, and that nuclear localization has the ability to protect the VDR from polyubiquitination and degradation. To test these hypotheses, we used Cos‐1 cells transfected with human VDR and histidine‐tagged ubiquitin expression vectors. We found that unoccupied VDR was polyubiquitinated and that 1,25D₃ inhibited this modification. Mutations in the nuclear localization signal of VDR (R49W/R50G and K53Q/R54G/K55E) or in the dimerization interface of VDR with retinoid X receptor (M383G/Q385A) abolished the ability of 1,25D₃ to protect the VDR from polyubiquitination, although these mutations had no effect on the ligand‐binding activity of VDR. Therefore, we concluded that in some cellular environments unoccupied cytoplasmic VDR is susceptible to polyubiquitination and proteasome degradation and that ligand‐dependent heterodimerization and nuclear localization protect the VDR from these modifications. J. Cell. Biochem. 110: 926–934, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular mechanism of the vitamin D antagonistic actions of (23S)-25-dehydro-1alpha-hydroxyvitamin D3-26,23-lactone depends on the primary structure of the carboxyl-terminal region of the vitamin d receptor

We reported that (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) antagonizes vitamin D receptor (VDR)-mediated genomic actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] in human cells but is agonistic in rodent cells. Human and rat VDR ligand-binding domains are similar, but differences in the C-terminal region are important for ligand binding and transactivation and might determine the agonistic/antagonistic effects of TEI-9647. We tested TEI-9647 on 1alpha,25(OH)(2)D(3) transactivation using SaOS-2 cells (human osteosarcoma) or ROS 24/1 cells (rat osteosarcoma) cotransfected with human or rodent VDR and a reporter. In both cell lines, TEI-9647 was antagonistic with wild-type human (h)VDR, but agonistic with overexpressed wild-type rat (r)VDR. VDR chimeras substituting the hVDR C-terminal region (activation function 2 domain) with corresponding rVDR residues diminished antagonism and increased agonism of TEI-9647. However, substitution of 25 C-terminal rVDR residues with corresponding hVDR residues diminished agonism and increased antagonism of TEI-9647. hVDR mutants (C403S, C410N) demonstrated that Cys403 and/or 410 was necessary for TEI-9647 antagonism of 1alpha,25(OH)(2)D(3) transactivation. These results suggest that species specificity of VDR, especially in the C-terminal region, determines the agonistic/antagonistic effects of TEI-9647 that determine, in part, VDR interactions with coactivators and emphasize the critical interaction between TEI-9647 and the two C-terminal hVDR Cys residues to mediate the antagonistic effect of TEI-9647.     

Uncoupling the ATPase activity of the N-ethylmaleimide sensitive factor (NSF) from 20S complex disassembly.

The N-ethylmaleimide sensitive factor (NSF) plays a critical role in intracellular trafficking by disassembling soluble NSF attachment protein receptor (SNARE ) complexes. The NSF protomer consists of three domains (NSF-N, NSF-D1, and NSF-D2). Two domains (NSF-D1 and NSF-D2) contain a conserved approximately 230 amino acid cassette, which includes a distinctive motif termed the second region of homology (SRH) common to all ATPases associated with various cellular activities (AAA). In hexameric NSF, several SRH residues become trans elements of the ATP binding pocket. Mutation of two conserved arginine residues in the NSF-D1 SRH (R385A and R388A) did not effect basal or soluble NSF attachment protein (SNAP)-stimulated ATPase activity; however, neither mutant underwent ATP-dependent release from SNAP-SNARE complexes. A trans element of the NSF-D2 ATP binding site (K631) has been proposed to limit the ATPase activity of NSF-D2, but a K631D mutant retained wild-type activity. A mutation of the equivalent residue in NSF-D1 (D359K) also did not affect nucleotide hydrolysis activity but did limit NSF release from SNAP-SNARE complexes. These trans elements of the NSF-D1 ATP binding site (R385, R388, and D359) are not required for nucleotide hydrolysis but are important as nucleotide-state sensors. NSF-N mediates binding to the SNAP-SNARE complex. To identify the structural features required for binding, three conserved residues (R67, S73, and Q76) on the surface of NSF-N were mutated. R67E completely eliminated binding, while S73R and Q76E showed limited effect. This suggests that the surface important for SNAP binding site lies in the cleft between the NSF-N subdomains adjacent to a conserved, positively charged surface.     

Charged amino-acid residues in transmembrane domains of the plastidic ATP/ADP transporter from arabidopsis are important for transport efficiency, substrate specificity, and counter exchange properties.

Structure-function relationships of the plastidic ATP/ADP transporter from Arabidopsis thaliana have been determined using site-directed mutants at positions K155, E245, E385, and K527. These charged residues are found within highly conserved domains of homologous transport proteins from plants and bacteria and are located in predicted transmembrane regions. Mutants of K155 to K155E, K155R, or K155Q reduced ATP transport to values between 4 and 16% of wild-type uptake, whereas ADP transport was always less then 3% of the wild-type value. Site-directed mutations in which glutamate at positions 245 or 385 was replaced with lysine, abolished transport. However, conservative (E245D, E385D) or neutral (E245Q, E385Q) replacement at these two positions allowed transport. The fourth reciprocal exchange, K527E, also abolished uptake of both adenylates. K527R and K527Q were unable to transport ATP, but ADP transport remained at 35 and 27%, respectively, of the wild-type activity. There was a 70-fold decreased apparent affinity of K527R for ATP, but only a twofold decrease for ADP. The efflux of ATP, but not ADP, was also greatly reduced in K527R. These observations show strikingly that K527 plays a role in substrate specificity that is manifest in both the influx and efflux components of this antiporter.     

Familial hypertrophic cardiomyopathy mutations in the regulatory light chains of myosin affect their structure, Ca2+ binding, and phosphorylation

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca(2+) binding site, had dramatically altered Ca(2+) binding properties. The K(Ca) value for E22K was decreased by approximately 17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca(2+). Interestingly, Ca(2+) binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the alpha-helical content of phosphorylated R58Q greatly increased with Ca(2+) binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K(Ca) than wild-type light chain, whereas phosphorylation of this mutant increased the Ca(2+) affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca(2+) affinity, the opposite was true for A13T. The alpha-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca(2+) binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.     

Irx4 forms an inhibitory complex with the vitamin D and retinoic X receptors to regulate cardiac chamber-specific slow MyHC3 expression

The slow myosin heavy chain 3 gene (slow MyHC3) is restricted in its expression to the atrial chambers of the heart. Understanding its regulation provides a basis for determination of the mechanisms controlling chamber-specific gene expression in heart development. The observed chamber distribution results from repression of slow MyHC3 gene expression in the ventricles. A binding site, the vitamin D response element (VDRE), for a heterodimer of vitamin D receptor (VDR) and retinoic X receptor alpha (RXR alpha) within the slow MyHC3 promoter mediates chamber-specific expression of the gene. Irx4, an Iroquois family homeobox gene whose expression is restricted to the ventricular chambers at all stages of development, inhibits AMHC1, the chick homolog of quail slow MyHC3, gene expression within developing ventricles. Repression of the slow MyHC3 gene in ventricular cardiomyocytes by Irx4 requires the VDRE. Unlike VDR and RXR alpha, Irx4 does not bind directly to the VDRE. Instead two-hybrid and co-immunoprecipitation assays show that Irx4 interacts with the RXR alpha component of the VDR/RXR alpha heterodimer and that the amino terminus of the Irx4 protein is required for its inhibitory action. These observations indicate that the mechanism of atrial chamber-specific expression requires the formation of an inhibitory protein complex composed of VDR, RXR alpha, and Irx4 that binds at the VDRE inhibiting slow MyHC3 expression in the ventricles.     

Essential role of a single arginine of photosystem I in stabilizing the electron transfer complex with ferredoxin

PsaE is one of the photosystem I subunits involved in ferredoxin binding. The central role of arginine 39 of this 8-kDa peripheral polypeptide has been established by a series of mutations. The neutral substitution R39Q leads to a 250-fold increase of the dissociation constant K(d) of the photosystem I-ferredoxin complex, as large as the increase induced by PsaE deletion. At pH 8.0, this K(d) value strongly depends on the charge of the residue substituting Arg-39: 0.22 microM for wild type, 1.5 microM for R39K, 56 microM for R39Q, and more than 100 microM for R39D. The consequences of arginine 39 substitution for the titratable histidine were analyzed as a function of pH. The K(d) value of R39H is increased 140 times at pH 8.0 but only 5 times at pH 5.8, which is assigned to the protonation of histidine at low pH. In the mutant R39Q, the association rate of ferredoxin was decreased 3-fold compared with wild type, whereas an 80-fold increase is calculated for the dissociation rate. We propose that a major contribution of PsaE is to provide a prominent positive charge at position 39 for controlling the electrostatic interaction and lifetime of the complex with ferredoxin.     

Role of vitamin D3 receptor in the synergistic differentiation of WEHI-3B leukemia cells by vitamin D3 and retinoic acid.

WEHI-3B D- cells differentiate in response to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) but not to all-trans-retinoic acid (RA) or other inducing agents. Combinations of RA with 1,25-(OH)2D3 interact to produce synergistic differentiation of WEHI-3B D- cells. To determine factors involved in the synergistic interaction, expression of the 1,25-(OH)2D3 receptor (VDR) and retinoid receptors, RARalpha and RXRalpha, was measured. No VDR was detected in untreated WEHI-3B D- cells; however, RA and 1,25-(OH)2D3 when used as single agents caused a slight induction of the VDR and in combination produced a marked increase in the VDR. In contrast, no changes in RARalpha and RXRalpha were initiated by these compounds. An RAR-selective agonist combined with 1,25-(OH)2D3 produced synergistic differentiation of WEHI-3B D- cells, whereas an RXR-selective agonist did not. To gain information on the role of the VDR in the synergistic interaction, the VDR gene was transferred into WEHI-3B D+ cells, in which no VDR was detected and no synergism was produced. Expression of the VDR conferred differentiation responsiveness to 1,25-(OH)2D3 in WEHI-3B D+ cells. These findings suggest that (a) induction of VDR expression is a key component in the synergistic differentiation induced by 1,25-(OH)2D3 and RA and (b) RAR and not RXR must be activated for enhanced induction of the VDR and for the synergistic differentiation produced by RA and 1, 25-(OH)2D3.