Biochemical and pharmacological study of N-linked glycosylation of the human serotonin 5-HT₇a receptor

The 5-hydroxytryptamine (5-HT)(7(a)) receptor is a G-protein-coupled receptor critically involved in human psychiatric and neurological disorders. In the present study, we evaluate the presence and the functional role of N-glycosylation of the human 5-HT(7) receptor. Western blot analysis of HEK293T cells transiently expressing the 5-HT(7(a)) receptor in the presence of tunicamycin gave rise to a band shift, indicating the existence of an N-glycosylated form of the 5-HT(7(a)) receptor. To further investigate this, we mutated the two predicted N-glycosylation sites (N5Q and N66Q) and compared the molecular mass of the immunoreactive bands with those of the wild-type receptor, indicating that both asparagines were N-glycosylated. The mutant receptors had the same binding affinity for [(3) H]5-CT and the same potency and efficacy with regard to 5-HT-induced activation of adenylyl cyclase. However, there was a reduction in maximal ligand binding for the single and double mutants compared to the wild-type receptor. Next, membrane labelling and immunocytochemical studies demonstrated that the N-glycosylation mutants were expressed at the cell surface. We conclude that N-glycosylation is not important for cell surface expression of the 5-HT(7) receptor.     

Identification and functional significance of N-glycosylation of the 5-ht5A receptor

The presence and roles of N-glycosylation of the human (h) 5-ht(5A) receptor were investigated using a heterologous expression system. Following transient transfection of COS-7 cells with h5-ht(5A) receptor cDNA, SDS-PAGE/Western blot analysis of immunoreactivity demonstrated two protein species; a predominant species with a molecular weight of approximately 35-45 kDa and a minor species of approximately 45-55 kDa. Transfected cells grown in the presence of the N-glycosylation inhibitor tunicamycin, failed to express the minor immunoreactive species indicating this represented the N-glycosylated form of the h5-ht(5A) receptor. Comparison of the molecular weights of immunoreactive bands arising from the wild-type and each of the mutant 5-ht(5A) receptors with disruption of the predicted N-glycosylation sites (N6S and N21S) demonstrated that both identified asparagines were N-glycosylated. Immunocytochemical and ELISA studies demonstrated that the [N6S]h5-ht(5A) receptor mutation, but not the [N21S]h5-ht(5A) receptor mutation, reduced protein expression in the cell membrane, indicating that N-glycosylation of the N6 residue is important for the membrane expression of this neurotransmitter receptor; a requirement for receptor function.     

人P2X7真核表达载体的构建及稳定转染细胞株的建立

目的：构建人P2X7基因的真核表达载体,并通过转染获得稳定表达P2X7分子的HEK293细胞株。方法：以人脑组织P2X7cDNA为模板扩增出P2X7基因,插入到真核表达载体pEGFP-N1中,构建重组质粒pEGFP-N1/P2X7。用X-fect试剂盒将重组质粒转染HEK293细胞,通过G418辅助荧光筛选建立稳定表达P2X7-EGFP细胞株。经流式细胞仪、Western blot和激光共聚焦显微镜检测,了解人P2X7在HEK293细胞中的表达水平及细胞内定位。结果：重组质粒pEGFP-N1/P2X7构建正确,建立了稳定表达人P2X7的HEK293细胞系。Western blot和流式细胞仪检测证实,P2X7在HEK293细胞系中成功表达,激光共聚焦显微镜检测显示P2X7-EGFP定位在细胞膜上。结论：重组载体pEGFP-N1/P2X7构建成功并建立了稳定表达人P2X7的HEK293细胞系,为进一步研究P2X7离子通道结构和功能奠定基础。     

缺氧对稳定表达人淀粉样前体蛋白HEK293细胞的存活及阿尔茨海默病相关蛋白表达的影响

目的：探讨缺氧对稳定表达人淀粉样前体蛋白的HEK293细胞（HEK293-APP695）存活及相关蛋白表达的影响,为深入研究缺氧对阿尔茨海默病的调节作用提供稳定的细胞模型。方法：利用缺氧手套箱（0.3% O₂）处理HEK293-APP695细胞,CCK-8法检测细胞的存活情况;Western blot检测缺氧条件下阿尔茨海默病（AD）相关蛋白APP、APP-CTFs和BACE1的表达变化。结果：缺氧处理后,HEK293-APP695细胞的存活率明显下降,APP表达降低,其剪切体APP-CTFs表达升高。结论：缺氧导致APP剪切的增多,抑制细胞的存活,提示缺氧可能通过影响BACE1的活性在AD的发病进程中起重要的调节作用。     

Impact of Cell Type and Epitope Tagging on Heterologous Expression of G Protein-Coupled Receptor: A Systematic Study on Angiotensin Type II Receptor

Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2) receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.     

RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT3A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits

Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A–E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT₃ receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT₃ receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca²⁺ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT₃A receptors but leads to differential increases of 5-HT-induced maximum response (E_max) on cells expressing different subunits. Increases of E_max were due to analogously enhanced B_max values for binding of the 5-HT₃ receptor antagonist [³H]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT₃A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT₃ receptor composition in vivo.     

Endogenous expression and protein kinase A-dependent phosphorylation of the guanine nucleotide exchange factor Ras-GRF1 in human embryonic kidney 293 cells

We have previously reported the Ras-dependent activation of the mitogen-activated protein kinases p44 and p42, also termed extracellular signal-regulated kinases (ERK)1 and 2 (ERK1/2), mediated through Gs-coupled serotonin receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Whereas Gi- and Gq-coupled receptors have been shown to activate Ras through the guanine nucleotide exchange factor (GEF) called Ras-GRF1 (CDC25Mm) by binding of Ca2+/calmodulin to its N-terminal IQ domain, the mechanism of Ras activation through Gs-coupled receptors is not fully understood. We report the endogenous expression of Ras-GRF1 in HEK293 cells. Serotonin stimulation of HEK293 cells transiently expressing Gs-coupled 5-HT7 receptors induced protein kinase A-dependent phosphorylation of the endogenous human Ras-GRF1 on Ser927 and of transfected mouse Ras-GRF1 on Ser916. Ras-GRF1 overexpression increased basal and serotonin-stimulated ERK1/2 phosphorylation. Mutations of Ser916 inhibiting (Ser916Ala) or mimicking (Ser916Asp/Glu) phosphorylation did not alter these effects. However, the deletion of amino acids 1-225, including the Ca2+/calmodulin-binding IQ domain, from Ras-GRF1 reduced both basal and serotonin-stimulated ERK1/2 phosphorylation. Furthermore, serotonin treatment of HEK293 cells stably expressing 5-HT7 receptors increased [Ca2+]i, and the serotonin-induced ERK1/2 phosphorylation was Ca2+-dependent. Therefore, both cAMP and Ca2+ may contribute to the Ras-dependent ERK1/2 activation after 5-HT7 receptor stimulation, through activation of a guanine nucleotide exchange factor with activity towards Ras.     

Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

Two different mutated forms of BRI2 protein are linked with familial British and Danish dementias, which present neuropathological similarities with Alzheimer's disease. BRI2 is a type II transmembrane protein that is trafficked through the secretory pathway to the cell surface and is processed by furin and ADAM10 (a disintegrin and metalloproteinase domain 10) to release secreted fragments of unknown function. Its apparent molecular mass (42-44 kDa) is significantly higher than that predicted by the number and composition of amino acids (30 kDa) suggesting that BRI2 is glycosylated. In support, bioinformatics analysis indicated that BRI2 bears the consensus sequence Asn-Thr-Ser (residues 170-173) and could be N-glycosylated at Asn170. Given that N-glycosylation is considered essential for protein folding, processing and trafficking, we examined whether BRI2 is N-glycosylated. Treatment of HEK293 (human embryonic kidney) cells expressing BRI2 with the N-glycosylation inhibitor tunicamycin or mutation of Asn170 to alanine reduced its molecular mass by ~2 kDa. These data indicate that BRI2 is N-glycosylated at Asn170. To examine the effect of N-glycosylation on BRI2 trafficking at the cell surface, we performed biotinylation and (35)S methionine pulse-chase experiments. These experiments showed that mutation of Asn170 to alanine reduced BRI2 trafficking at the cell surface and its steady state levels at the plasma membrane. Furthermore, we obtained data indicating that this mutation did not affect cleavage of BRI2 by furin or ADAM10. Our results confirm the theoretical predictions that BRI2 is N-glycosylated at Asn170 and show that this post-translational modification is essential for its expression at the cell surface but not for its proteolytic processing.     

Co-expression of the 5-HT3B serotonin receptor subunit alters the biophysics of the 5-HT3 receptor

Homomeric complexes of 5-HT(3A) receptor subunits form a ligand-gated ion channel. This assembly does not fully reproduce the biophysical and pharmacological properties of native 5-HT(3) receptors which might contain the recently cloned 5-HT(3B) receptor subunit. In the present study, heteromeric assemblies containing human 5-HT(3A) and 5-HT(3B) subunits were expressed in HEK 293 cells to detail the functional diversity of 5-HT(3) receptors. We designed patch-clamp experiments with homomeric (5-HT(3A)) and heteromeric (5-HT(3AB)) receptors to emphasize the kinetics of channel activation and desensitization. Co-expression of the 5-HT(3B) receptor subunit reduced the sensitivity for 5-HT (5-HT(3A) receptor: EC(50) 3 micro M, Hill coefficient 1.8; 5-HT(3AB) receptor: EC(50) 25 micro M, Hill coefficient 0.9) and markedly altered receptor desensitization. Kinetic modeling suggested that homomeric receptors, but not heteromeric receptors, desensitize via an agonist-induced open-channel block. Furthermore, heteromeric 5-HT(3AB) receptor assemblies recovered much faster from desensitization than homomeric 5-HT(3A) receptor assemblies. Unexpectedly, the specific 5-HT(3) receptor agonist mCPBG induced an open-channel block at both homomeric and heteromeric receptors. Because receptor desensitization and resensitization massively affect amplitude, duration, and frequency of synaptic signaling, these findings are evidence in favor of a pivotal role of subunit composition of 5-HT(3) receptors in serotonergic transmission.     

High-level expression and purification of the human bradykinin B(2) receptor in a tetracycline-inducible stable HEK293S cell line

The B(2) bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B(2) receptor for future biophysical studies. Different tagged B(2) receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B(2) receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B(2) receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B(2) receptor with tetracycline and sodium butyrate led to a level of 100pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-beta-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B(2) receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor.     

Sequestration of dopamine D2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5.

We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride-binding activity from the cell surface and by transfer of [3H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway.     

Novel interaction between the human 5-HT7 receptor isoforms and PLAC-24/eIF3k

Three 5-HT(7) receptor isoforms are expressed in rat and man, which differ in the amino acid sequence of their C-terminus. Thus far, no changes have been observed in the pharmacological profile of all three isoforms. To further elucidate the signal transduction pathway specific for these receptor variants, we screened for possible interacting proteins of the C-terminus of the h5-HT(7(a)) variant in a human foetal brain cDNA library. Using a yeast two-hybrid assay, we isolated PLAC-24/eIF3k as a possible interacting candidate. The association of PLAC-24 with all three receptor variants was observed and further reconfirmed in vivo by co-immunoprecipitation of PLAC-24 with the full-length receptor isoforms in transfected COS-7 cells. Studies with different deletion mutants of the receptor showed that the interaction between PLAC-24 and the receptor is not restricted to the C-terminus of the receptor. PLAC-24/eIF3k consists of 3 domains: an N-terminal HAM domain, a central WH domain and a C-terminal tail. We generated different domain constructs of PLAC-24, which indicated that the HAM and WH domain both interact with the 5-HT(7(a)) receptor. Overexpression of PLAC-24 in HEK293 cells, stably expressing the h5-HT(7(a)) receptor, caused a threefold augmentation in the expression levels of the receptor. Co-localisation studies in COS-7 cells showed that PLAC-24 relocates from the nucleus and perinuclear sites towards the plasma membrane upon co-expression with the receptor. On the other hand, the expression of domain variants of PLAC-24 seems to block the translocation of the receptor towards the membrane. These observations suggest that PLAC-24 may play a role in the transport and the stabilisation of newly synthesised 5-HT(7) receptor towards the plasma membrane.     

Human ABCA3, a product of a responsible gene for abca3 for fatal surfactant deficiency in newborns, exhibits unique ATP hydrolysis activity and generates intracellular multilamellar vesicles

ABCA3 is highly expressed at the membrane of lamellar bodies in alveolar type II cells, in which pulmonary surfactant is stored. ABCA3 gene mutations cause fatal surfactant deficiency in newborns. We established HEK293 cells stably expressing human ABCA3 and analyzed the function. Exogenously expressed ABCA3 is glycosylated and localized at the intracellular vesicle membrane. ABCA3 is efficiently photoaffinity labeled by 8-azido-[alpha(32)P]ATP, but not by 8-azido-[gamma(32)P]ATP, when the membrane fraction is incubated in the presence of orthovanadate. Photoaffinity labeling of ABCA3 shows unique metal ion-dependence and is largely reduced by membrane pretreatment with 5% methyl-beta-cyclodextrin, which depletes cholesterol. Electron micrographs show that HEK293/hABCA3 cells contain multivesicular, lamellar body-like structures, which do not exist in HEK293 host cells. Some fuzzy components such as lipids accumulate in the vesicles. These results suggest that ABCA3 shows ATPase activity, which is induced by lipids, and may be involved in the biogenesis of lamellar body-like structures.     

Ric-3 promotes functional expression of the nicotinic acetylcholine receptor alpha7 subunit in mammalian cells

Expression of functional, recombinant alpha7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor alpha7 subunit. In addition to confirming the previously reported effect on alpha7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional alpha7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface alpha-bungarotoxin binding. Upon application of 1 mm nicotine, currents were undetectable in HEK293 cells expressing only the alpha7 subunit. In contrast, co-expression of alpha7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous alpha7 receptors. Immunoprecipitation studies demonstrate that alpha7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of alpha7 protein on the plasma membrane of cells lacking ric-3, but surface alpha-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of alpha7 receptors in HEK293 cells.     

Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6

The molecular assembly of the epithelial Ca(2+) channels (TRPV5 and TRPV6) was investigated to determine the subunit stoichiometry and composition. Immunoblot analysis of Xenopus laevis oocytes expressing TRPV5 and TRPV6 revealed two specific bands of 75 and 85-100 kDa, corresponding to the core and glycosylated proteins, respectively, for each channel. Subsequently, membranes of these oocytes were sedimented on sucrose gradients. Immuno blotting revealed that TRPV5 and TRPV6 complexes migrate with a mol. wt of 400 kDa, in line with a tetrameric structure. The tetrameric stoichiometry was confirmed in an electrophysiological analysis of HEK293 cells co-expressing concatemeric channels together with a TRPV5 pore mutant that reduced Cd(2+) sensitivity and voltage-dependent gating. Immuno precipitations using membrane fractions from oocytes co-expressing TRPV5 and TRPV6 demonstrated that both channels can form heteromeric complexes. Expression of all possible heterotetrameric TRPV5/6 complexes in HEK293 cells resulted in Ca(2+) channels that varied with respect to Ca(2+)-dependent inactivation, Ba(2+) selectivity and pharmacological block. Thus, Ca(2+)-transporting epithelia co-expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca(2+) transport kinetics.     

Calcium-activated K+ channels increase cell proliferation independent of K+ conductance

The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K(+) ions nor promotes Ca(2+) entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca(2+) entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K(+) efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca(2+) entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.     

Evidence for the role of glycosylation in accessibility of the extracellular domains of human MRP1 (ABCC1)

To enable cell surface localization of the human multidrug resistance protein (MRP1, ABCC1) and to assess the role of the extracellular domains of this transporter, the FLAG epitope tag was introduced into different extracellular loops of the three membrane-spanning domains (MSDs) of the transporter. We constructed and expressed various partially and fully glycosylation-deficient, FLAG-tagged MRP1 proteins in a Vaccinia virus-based transient expression system, and the cell surface expression level of MRP1 on intact cells was followed by flow cytometry, using the FLAG tag specific monoclonal antibody M2. We also expressed the wild-type MRP1 protein and some of the FLAG-tagged mutants in stably transfected HEK293 cells, and followed the cell surface expression and the transport function of MRP1 both by monitoring the efflux of fluorescent substrate and by their ability to confer resistance to HEK293 transfectants to anticancer agents such as daunorubicin and etoposide. When we inserted the FLAG epitope in extracellular loops of the MSD1 or MSD3, the tag was accessible upon removal of N-glycosylation sites (N --> Q at positions 17, 23, and 1006, respectively), whereas the FLAG epitope placed in the MSD2 was not accessible even after removal of all three N-glycosylation sites, indicating that MSD2 region is deeply buried in the plasma membrane. However, all FLAG tagged MRP1 mutants were expressed at the cell surface to the same extent as the wild-type protein and also exhibited normal transport function. Our results demonstrate that the accessibility of the external FLAG epitope is strongly dependent on the position of the tag and the glycosylation state of the different FLAG-tagged MRP1s, and the conformation of extracellular loops in MSD1 and MDS3 does not appear to contribute to the functional status of MRP1.     

Cooperation of NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferases reduces menadione cytotoxicity in HEK293 cells

Previous studies have shown that NAD(P)H:quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of menadione (2-methyl-1,4-naphthoquinone, also known as vitamin K3). However, menadiol (2-methyl-1,4-naphthalenediol) formed from menadione by NQO1-mediated reduction continues to be an unstable substance, which undergoes the reformation of menadione with concomitant formation of reactive oxygen species (ROS). Hence, we focused on the roles of phase II enzymes, with particular attention to UDP-glucuronosyltransferases (UGTs), in the detoxification process of menadione. In this study, we established an HEK293 cell line stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with doxycycline (DOX)-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and UGT1A10 (HEK293/NQO1/UGT1A10), and evaluated the role of NQO1 and UGTs against menadione-induced cytotoxicity. Our results differed from those of previous studies. HEK293/NQO1 was the most sensitive cell line to menadione cytotoxicity among cell lines established in this study. These phenomena were also observed in HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells in which the expression of UGT was suppressed by DOX treatment. On the contrary, HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells without DOX treatment were resistant to menadione-induced cytotoxicity. These results demonstrated that NQO1 is not a detoxification enzyme for menadione and that UGT-mediated glucuronidation of menadiol is the most important detoxification process.     

Active PIKfyve associates with and promotes the membrane attachment of the late endosome-to-trans-Golgi network transport factor Rab9 effector p40

PIKfyve, a kinase that displays specificity for phosphatidylinositol (PtdIns), PtdIns 3-phosphate (3-P), and proteins, is important in multivesicular body/late endocytic function. Enzymatically inactive PIKfyve mutants elicit enormous dilation of late endocytic structures, suggesting a role for PIKfyve in endosome-to-trans-Golgi network (TGN) membrane retrieval. Here we report that p40, a Rab9 effector reported previously to bind Rab9-GTP and stimulate endosome-to-TGN transport, interacts with PIKfyve as determined by yeast two-hybrid assays, glutathione S-transferase (GST) pull-down assays, and co-immunoprecipitation in doubly transfected HEK293 cells. The interaction engages the PIKfyve chaperonin domain and four out of the six C-terminally positioned kelch repeats in p40. Differential centrifugation in a HEK293 cell line, stably expressing PIKfyveWT, showed the membrane-associated immunoreactive p40 co-sedimenting with PIKfyve in the high speed pellet (HSP) fraction. Remarkably, similar analysis in a HEK293 cell line stably expressing dominant-negative kinase-deficient PIKfyveK1831E demonstrated a marked depletion of p40 from the HSP fraction. GST-p40 failed to specifically associate with the PIKfyve lipid products PtdIns 5-P and PtdIns 3,5-P2 in a liposome binding assay but was found to be an in vitro substrate of the PIKfyve serine kinase activity. A band with the p40 electrophoretic mobility was found to react with a phosphoserine-specific antibody mainly in the PIKfyveWT-containing fractions obtained by density gradient sedimentation of total membranes from PIKfyveWT-expressing HEK293 cells. Together these results identify the Rab9 effector p40 as a PIKfyve partner and suggest that p40-PIKfyve interaction and the subsequent PIKfyve-catalyzed p40 phosphorylation anchor p40 to discrete membranes facilitating late endosome-to-TGN transport.