Either the carboxyl- or the amino-terminal region of the human ecto-ATPase (E-NTPDase 2) confers detergent and temperature sensitivity to the chicken ecto-ATP-diphosphohydrolase (E-NTPDase 8)

Human ecto-ATPase (E-NTPDase 2) and chicken ecto-ATP-diphosphohydrolase (E-NTPDase 8) are cell surface nucleotidases with two transmembranous domains, one each at the N- and C-termini. Hydrolysis of substrates occurs in active sites residing in their extracellular domains. Human ecto-ATPase activity is decreased by NP-40 and at temperatures higher than 37 degrees C. Reduction of activity is abolished by prior cross-linking of the ecto-ATPase by lectin and chemical cross-linking agents [Knowles, A. F., and Chiang, W.-C. (2003) Arch. Biochem. Biophys. 418, 217-227]. In contrast, the chicken ecto-ATP-diphosphohydrolase is not inhibited by NP-40, and activity is approximately 2-fold higher at 55 degrees C. To determine if the transmembranous domains of the two E-NTPDases mediate their respective responses to detergents and high temperature, we first constructed a chimera (ck-hu ACR5) in which the C-terminus of the chicken ecto-ATP-diphosphohydrolase is substituted by the corresponding region of the human ecto-ATPase. While this chimera displays many similar enzymatic characteristics as the parental chicken ecto-ATP-diphosphohydrolase, its inhibition by NP-40, high temperature, and substrate resemble that of the human ecto-ATPase, which donates the C-terminus including the C-terminal transmembranous domain. Additionally, comparison of the effects of ConA, disuccinimidyl suberate, and glutaraldehyde on the parental enzymes and the chimera indicated that catalysis which occurs in the extracellular domains of the two E-NTPDases responds differently to conformational constraints. Enzyme activity of a second chimera (ck-hu ACR1) in which the N-terminus of the chicken ecto-ATP-diphosphohydrolase is substituted by the corresponding region of the human ecto-ATPase is also inhibited by NP-40 and is less active at 55 degrees C; however, its temperature dependence differs from that of ck-hu ACR5. These results indicate that (1) the C- and N-termini of the two E-NTPDases encompassing the two transmembranous domains are important elements in determining the sensitivity of the human ecto-ATPase to NP-40 and high temperatures; (2) incorporation of either the C- or N-terminus of the human ecto-ATPase alone in the chicken ecto-ATP-diphosphohydrolase is sufficient to impart negative regulation on ATP hydrolysis due to membrane perturbation; and (3) interactions of the two sets of heterologous transmembranous domains are not equivalent, which are most likely related to their different amino acid sequences.     

Dengue virus nonstructural protein NS5 induces interleukin-8 transcription and secretion

Elevated circulating levels of chemokines have been reported in patients with dengue fever and are proposed to contribute to the pathogenesis of dengue disease. To establish in vitro models for chemokine induction by dengue 2 virus (DEN2V), we studied a variety of human cell lines and primary cells. DEN2V infection of HepG2 and primary dendritic cells induced the production of interleukin-8 (IL-8), RANTES, MIP-1alpha, and MIP-1beta, whereas only IL-8 and RANTES were induced following dengue virus infection of HEK293 cells. Chemokine secretion was accompanied by an increase in steady-state mRNA levels. No chemokine induction was observed in HEK293 cells treated with poly(I:C) or alpha interferon, suggesting a direct effect of virus infection. To determine the mechanism(s) involved in the induction of chemokine production by DEN2V, individual dengue virus genes were cloned into plasmids and expressed in HEK293 cells. Transfection of a plasmid expressing NS5 or a dengue virus replicon induced IL-8 gene expression and secretion. RANTES expression was not induced under these conditions, however. Reporter assays showed that IL-8 induction by NS5 was principally through CAAT/enhancer binding protein, whereas DEN2V infection also induced NF-kappaB. These results indicate a role for the dengue virus NS5 protein in the induction of IL-8 by DEN2V infection. Recruitment and activation of potential target cells to sites of DEN2V replication by virus-induced chemokine production may contribute to viral replication as well as to the inflammatory components of dengue virus disease.     

Characterisation of the zebrafish serotonin transporter functionally links TM10 to the ligand binding site

The selective serotonin reuptake inhibitors and tricyclic antidepressants act by inhibiting pre-synaptic reuptake of serotonin (5-HT) leading to elevated synaptic 5-HT concentrations. However, despite extensive efforts little is known about the protein-ligand interactions of serotonin transporter (SERT) and inhibitors. To identify domains and individual amino acids important for ligand binding, we cloned the serotonin transporter from zebrafish, Danio rerio , (drSERT) and compared its pharmacological profile to that of the human serotonin transporter (hSERT) with respect to inhibition of [³H]5-HT uptake and [³H]-escitalopram binding in transiently transfected human embryonic kidney cells; HEK293-MSR. Residues responsible for altered affinities inhibitors were pinpointed by generating cross-species chimeras and subsequent point mutations by site directed mutagenesis. drSERT has a higher affinity towards compounds of the imipramine class, desipramine in particular, exhibiting a 35-fold increased affinity compared to hSERT. drSERT has a 15–30-fold lower affinity towards cocaine and cocaine analogues. The differences in ligand recognition are shown to be primarily caused by interspecies differences in TM10 and were tracked down to three residues (Ala⁵⁰⁵, Leu⁵⁰⁶ and Ile⁵⁰⁷).     

Expression of type XXIII collagen mRNA and protein

Collagen XXIII is a member of the transmembranous subfamily of collagens containing a cytoplasmic domain, a membrane-spanning hydrophobic domain, and three extracellular triple helical collagenous domains interspersed with non-collagenous domains. We cloned mouse, chicken, and humanalpha1(XXIII) collagen cDNAs and showed that this non-abundant collagen has a limited tissue distribution in non-tumor tissues. Lung, cornea, brain, skin, tendon, and kidney are the major sites of expression. In contrast, five transformed cell lines were tested for collagen XXIII expression, and all expressed the mRNA. In vivo the alpha1(XXIII) mRNA is found in mature and developing organs, the latter demonstrated using stages of embryonic chick cornea and mouse embryos. Polyclonal antibodies were generated in guinea pig and rabbit and showed that collagen XXIII has a transmembranous form and a shed form. Comparison of collagen XXIII with its closest relatives in the transmembranous subfamily of collagens, types XIII and XXV, which have the same number of triple helical and non-collagenous regions, showed that there is a discontinuity in the alignment of domains but that striking similarities remain despite this.     

The preparation of HEK293 alpha1A or HEK293 alpha1B cell membrane stationary phase and the chromatographic affinity study of ligands of alpha1 adrenoceptor

As a novel bioaffinity chromatographic technique, cell membrane chromatography (CMC) originated in 1996. The cell membrane stationary phase (CMSP) consists of porous silica coated with active cell membranes. By immersing silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes. The present study repeatedly investigated the interaction between ligands and receptors by employing the system of CMC and especially evaluated the accuracy and feasibility of the CMC model in the study of subtype receptors. The cDNA encoding alpha1A or alpha1B adrenergic receptors (ARs) was transfected into human embryonic kidney 293 (HEK293) cell lines; cell lines stably overexpressing subtype receptors were obtained. HEK293 alpha1A or HEK293 alpha1B ARs CMSP were prepared by immobilizing relevant cell membranes on silica. In the described chromatography-based CMSP, the retention times of nine alpha1 adrenoceptor ligands and calculated capacity factors, as chromatographic parameters, were recorded carefully. These results showed a good correlation with the affinity of the same compounds for the corresponding cloned alpha1 adrenoceptor subtype. The rank order of capacity factors was consistent with the affinity rank order obtained from radioligand binding assays. The immobilized subtype-selective CMSP was stable and reproducible. The study demonstrates that the HEK293 alpha1A and HEK293 alpha1B CMSP can be utilized for initial screening of drug candidates.     

Characterization of a novel sphingosine 1-phosphate receptor, Edg-8

Three G protein-coupled receptors (Edg-1, Edg-3, and Edg-5) for the lysolipid phosphoric acid mediator sphingosine 1-phosphate have been described by molecular cloning. Using a similar sequence that we found in the expressed sequence tag data base, we cloned and characterized of a fourth, high affinity, rat brain sphingosine 1-phosphate receptor, Edg-8. When HEK293T cells were co-transfected with Edg-8 and G protein DNAs, prepared membranes showed sphingosine 1- phosphate-dependent increases in [(35)S]guanosine 5'-(3-O-thio)triphosphate binding with an EC(50) of 90 nm. In a rat hepatoma Rh7777 cell line that exhibits modest endogenous responses to sphingosine 1-phosphate, this lipid mediator inhibited forskolin-driven rises in cAMP by greater than 90% when the cells were transfected with Edg-8 DNA (IC(50) 0.7 nm). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Furthermore, Xenopus oocytes exhibit a calcium response to sphingosine 1-phosphate after injection of Edg-8 mRNA, but only when oocytes are co-injected with chimeric G(q/i)alpha protein mRNA. Membranes from HEK293T and Rh7777 cell cultures expressing Edg-8 exhibited high affinity (K(D) = 2 nm) binding for radiolabeled sphingosine 1-phosphate. Rat Edg-8 RNA is expressed in spleen and throughout adult rat brain where in situ hybridization revealed it to be associated with white matter. Together our data demonstrate that Edg-8 is a high affinity sphingosine 1-phosphate receptor that couples to G(i/o)alpha proteins and is expressed predominantly by oligodendrocytes and/or fibrous astrocytes in the rat brain.     

Transmembrane domain interactions affect the stability of the extracellular domain of the human NTPDase 2

Human NTPDase2 and chicken NTPDase8 are cell surface nucleotidases that contain two transmembrane domains (TMD) and five apyrase conserved regions (ACRs). ACR1 is located near the N-terminal TMD whereas ACR5 is located near the C-terminal TMD. The human NTPDase2 activity is decreased by low concentration of NP-40 and at temperatures higher than 37 °C, and undergoes substrate inactivation, whereas the chicken NTPDase8 activity is not. When freed from membrane anchorage, the soluble human NTPDase2 is no longer inactivated by detergents, high temperature, and substrate. These characteristics are retained in the hu-ck ACR1,5 chimera in which the extracellular domain is anchored to the membrane by the two TMDs of the chicken NTPDase8. The hu-ck ACR1,5 chimera is the first chimeric NTPDase reported that shows a resistance to membrane perturbation and substrate inactivation. Our results indicate that the strengths of interaction of the respective TMD pairs of the human NTPDase2 and chicken NTPDase8 determine their different responses to membrane perturbation and substrate.     

cDNA cloning and functional expression of the dolphin retinal rod Na-Ca+K exchanger NCKX1: comparison with the functionally silent bovine NCKX1.

cDNAs of human and bovine retinal rod Na+-Ca2++K+ exchanger (NCKX1) have previously been cloned, but potassium-dependent Na-Ca exchange activity upon heterologous expression has not been demonstrated. We have cloned NCKX1 cDNA from dolphin, examined function upon transfection in HEK293 cells, and compared the dolphin sequence encoded by the cDNA with those of human and bovine. The dolphin NCKX1 cDNA encodes 1013 amino acid residues. Comparison to bovine and human NCKX1 revealed strong conservation in the transmembrane domains (>95%), but relatively low conservation in the large extracellular ( approximately 50%) and cytosolic (<50%) domains. The dolphin cytosolic domain differs from the bovine sequence by the absence of a stretch of 114 amino acids. HEK293 cells transfected with dolphin NCKX1 cDNA showed K+-dependent Na-Ca exchange in >95% of the experiments, whereas transfection with bovine NCKX1 yielded no function. The bovine NCKX1 phenotype was imparted on dolphin NCKX1 when the dolphin cytosolic loop was replaced by that from bovine. Conversely, deletion of 114 amino acids from the bovine sequence to match the dolphin sequence resulted in a mutant bovine NCKX1 which performed K+-dependent Na-Ca exchange. These results suggest that domains within the large cytosolic loop of NCKX1 control functional activity when expressed in heterologous systems.     

克隆、表达及纯化核糖体蛋白S6用于体外激酶活性检测

目的:构建40S核糖体蛋白S6的原核表达载体,表达并纯化S6蛋白,将其作为底物用于S6激酶(S6K)的体外活性测定。方法:采用RT-PCR方法从人胚肾细胞HEK293中获取S6 cDNA,将扩增产物克隆至大肠杆菌表达载体中,进行酶切及测序鉴定;IPTG诱导GST-S6融合蛋白在大肠杆菌中表达,用谷胱甘肽亲和层析纯化GST-S6,免疫沉淀法检测该蛋白是否可作为底物用于S6K的体外激酶活性测定。结果:酶切及测序鉴定表明构建了S6原核表达载体,并表达及纯化出GST-S6融合蛋白,相对分子质量为55×103。该蛋白可用于S6K的体外激酶活性测定,特异性强。结论:S6蛋白的克隆、表达与纯化成功,可用于S6K的体外激酶活性测定,为研究S6K的功能奠定了基础。     

Participation of the extracellular domain in (pro)renin receptor dimerization

The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin–angiotensin system. (P)RR is known to form a homodimer, but the region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.     

Identification of urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14

Urotensin II (UII) is a neuropeptide with potent cardiovascular effects. Its sequence is strongly conserved among different species and has structural similarity to somatostatin. No receptor for UII has been molecularly identified from any species so far. GPR14 was cloned as an orphan G protein-coupled receptor with similarity to members of the somatostatin/opioid receptor family. We have now demonstrated that GPR14 is a high affinity receptor for UII and designate it UII-R1a. HEK293 cells and COS-7 cells transfected with rat GPR14 showed strong, dose-dependent calcium mobilization in response to fish, frog, and human UII. Radioligand binding analysis showed high affinity binding of UII to membrane preparations isolated from HEK293 cells stably expressing rat GPR14. In situ hybridization analysis showed that GPR14 was expressed in motor neurons of the spinal cord, smooth muscle cells of the bladder, and muscle cells of the heart. The identification of the first receptor for UII will allow better understanding of the physiological and pharmacological roles of UII.     

Complex inhibition of tyrosinase by thiol-composed Cu2+ chelators: a clue for designing whitening agents

The inhibition of tyrosinase has attracted considerable attention for potential medicinal and cosmetic applications, as well as in agriculture. This study investigated the inhibition effects of thiol-associated Cu(2+) chelators and deduced a strategy for designing and/or selecting tyrosinase inhibitors. Among the several compounds tested, dithioglycerine (DTGC) was selected for further experiments on the inhibition kinetics on tyrosinase. Different types of tyrosinases derived from mushroom and from the transient overexpression in HEK293 cells were tested individually. The results showed that DTGC significantly inhibited human tyrosinase in a complex manner (slope-parabolic mixed-type inhibition), which was comparable to mushroom tyrosinase. The affinity of DTGC affinity to human tyrosinase was evaluated by setting up a K(i slope) equation. The results suggest that a Cu(2+) chelator modified with thiol groups has potential as a whitening agent. In addition, a strategy for designing and/or selecting tyrosinase inhibitors that target the active enzyme site was also suggested.     

Lack of direct interaction between enalaprilat and the kinin B1 receptors

It has been recently proposed that the second extracellular loop of the human bradykinin (BK) B1 receptor (B1R) contains a conserved HExxH motif also present in peptidases possessing a Zn2+ prosthetic group, such as angiotensin converting enzyme (ACE), and that ACE inhibitors directly activate B1R signaling in endothelial cells. However, the binding of ACE inhibitors to the B1Rs has never been directly evaluated. Information about binding of a radiolabeled inhibitor to natural or recombinant ACE in intact cells (physiologic ionic composition) was also collected. We used the tritiated form of an ACE inhibitor previously proposed to activate the B1R, enalaprilat, to address these questions using recombinant human B1Rs and naturally expressed or recombinant ACE. [3H]Lys-des-Arg9-BK bound to the human recombinant B1Rs with high affinity (KD 0.35 nM) in HEK 293a cells. [3H]Enalaprilat (0.25-10 nM) did not bind to cells expressing recombinant human B1R, but bound with a subnanomolar affinity to recombinant ACE or to naturally expressed ACE in human umbilical vein endothelial cells. The radioligand was further validated using a binding competition assay that involved unlabeled ACE inhibitors or their prodrug forms in endothelial cells. Membranes of HEK 293a cells that expressed B1Rs did not hydrolyze hippuryl-glycylglycine (an ACE substrate). Enalaprilat did not stimulate calcium signaling in HEK 293a cells that expressed B1Rs. A typical ACE inhibitor did not bind to nor stimulate the human B1Rs; nevertheless, several other indirect mechanisms could connect ACE inhibition to B1R stimulation in vivo.     

Kinetic analysis of L1 homophilic interaction: role of the first four immunoglobulin domains and implications on binding mechanism

L1 is a cell adhesion molecule of the immunoglobulin (Ig) superfamily, critical for central nervous system development, and involved in several neuronal biological events. It is a type I membrane glycoprotein. The L1 ectodomain, composed of six Ig-like and five fibronectin (Fn) type-III domains, is involved in homophilic binding. Here, co-immunoprecipitation studies between recombinant truncated forms of human L1 expressed and purified from insect Spodoptera frugiperda Sf9 cells, and endogenous full-length L1 from human NT2N neurons, showed that the L1 ectodomain (L1/ECD) and L1/Ig1-4 interacted homophilically in trans, contrary to mutants L1/Ig1-3 and L1/Ig2-Fn5. All mutants were correctly folded as evaluated by combination of far-UV CD and fluorescence spectroscopy. Surface plasmon resonance analysis showed comparable dissociation constants of 116 +/- 2 and 130 +/- 6 nm for L1/ECD-L1/ECD and L1/ECD-L1/Ig1-4, respectively, whereas deletion mutants for Ig1 or Ig4 did not interact. Accordingly, in vivo, Sf9 cells stably expressing L1 were found to adhere only to L1/ECD- and L1/Ig1-4-coated surfaces. Furthermore, only these mutants bound to HEK293 cells overexpressing L1 at the cell surface. Enhancement of neurite outgrowth, which is the consequence of signaling events caused by L1 homophilic binding, was comparable between L1/ECD and L1/Ig1-4. Altogether, these results showed that domains Ig1 to Ig4 are necessary and sufficient for L1 homophilic binding in trans, and that the rest of the molecule does not contribute to the affinity under the conditions of the current study. Furthermore, they are compatible with a cooperative interaction between modules Ig1-Ig4 in a horseshoe conformation.     

Soluble endoglin specifically binds bone morphogenetic proteins 9 and 10 via its orphan domain, inhibits blood vessel formation, and suppresses tumor growth

Endoglin (CD105), a transmembrane protein of the transforming growth factor β superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.     

Specific high affinity interactions of monomeric endotoxin.protein complexes with Toll-like receptor 4 ectodomain

Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligand-receptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin.protein complexes of high specific radioactivity. The binding of endotoxin.MD-2 to the TLR4 ectodomain (TLR4ECD) and transfer of endotoxin from CD14 to MD-2/TLR4ECD were demonstrated using HEK293T-conditioned medium containing TLR4ECD+/-MD-2. These interactions are specific, of high affinity (KD<300 pm), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a Mr approximately 190,000 complex composed of endotoxin, MD-2, and TLR4ECD. CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin.MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin.protein complexes with TLR4ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.     

The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.     

Role of PRL-3, a human muscle-specific tyrosine phosphatase, in angiotensin-II signaling

Action of protein kinases and phosphatases contributes to myocardial hypertrophy. PRL-3, a protein tyrosine phosphatase, was identified in a cDNA library from an explanted human heart obtained from a patient with idiopathic cardiomyopathy. PRL-3 is expressed in heart and skeletal muscle, exhibiting approximately 76% identity to the ubiquitous tyrosine phosphatase PRL-1, which was reported to increase cell proliferation. PRL-3 was cloned into E. coli and purified using affinity chromatography. PRL-3 activity was determined using the substrate 6,8-difluoro-4-methylumbelliferyl phosphate, and was inhibited by vanadate and analogs. HEK293 cells expressing PRL-3 demonstrated increased growth rates versus nontransfected cells or cells transfected with the catalytically inactive C104S PRL-3 mutant. The tyrosine phosphatase inhibitor, potassium bisperoxo (bipyridine) oxovanadate V, normalizes the growth rate of PRL-3 expressing cells to that of parental HEK293 cells in a concentration-dependent manner. Using FLIPR analysis, parental HEK293 cells mobilize calcium when stimulated with angiotensin-II (AngII). However, calcium mobilization is inhibited in cells expressing wild-type PRL-3 when stimulated with AngII, while cells expressing the inactive mutant of PRL-3 mobilize calcium to the same extent as parental HEK293 cells. Western blots comparing PRL-3 transfected cells to parental HEK293 cells showed dephosphorylation of p130(cas) in response to AngII. These data suggest a role for PRL-3 in the modulation of intracellular calcium transients induced by AngII.     

Purification, characterization, cloning, and expression of the chicken liver ecto-ATP-diphosphohydrolase.

We previously demonstrated that the major ecto-nucleoside triphosphate phosphohydrolase in the chicken liver membranes is an ecto-ATP-diphosphohydrolase (ecto- ATPDase) [Caldwell, C., Davis, M.D. & Knowles, A.F. (1999) Arch. Biochem. Biophys. 362, 46-58]. Enzymatic properties of the liver membrane ecto-ATPDase are similar to those of the chicken oviduct ecto-ATPDase that we have previously purified and cloned. Using antibody developed against the latter, we have purified the chicken liver ecto-ATPDase to homogeneity. The purified enzyme is a glycoprotein with a molecular mass of 85 kDa and a specific activity of approximately 1000 U.mg protein-1. Although slightly larger than the 80-kDa oviduct enzyme, the two ecto-ATPDases are nearly identical with respect to their enzymatic properties and mass of the deglycosylated proteins. The primary sequence of the liver ecto-ATPDase deduced from its cDNA obtained by RT-PCR cloning also shows only minor differences from that of the oviduct ecto-ATPDase. Immunochemical staining demonstrates the distribution of the ecto-ATPDase in the bile canaliculi of the chicken liver. HeLa cells transfected with the chicken liver ecto-ATPDase cDNA express an ecto-nucleotidase activity with characteristics similar to the enzyme in its native membranes, most significant of these is stimulation of the ATPDase activity by detergents, which inhibits other members of the ecto- nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. The stimulation of the expressed liver ecto-ATPDase by detergents indicates that this property is intrinsic to the enzyme protein, and cannot be attributed to the lipid environment of the native membranes. The molecular identification and expression of a liver ecto-ATPDase, reported here for the first time, will facilitate future investigations into the differences between structure and function of the different E-NTPDases, existence of liver ecto-ATPDase isoforms in different species, its alteration in pathogenic conditions, and its physiological function.     

Tumor necrosis factor (TNF) receptor superfamily member TACI is a high affinity receptor for TNF family members APRIL and BLyS

An expression cloning approach was employed to identify the receptor for B-lymphocyte stimulator (BLyS) and identified the tumor necrosis factor receptor superfamily member TACI as a BLyS-binding protein. Expression of TACI in HEK293T cells confers on the cells the ability to bind BLyS with subnanomolar affinity. Furthermore, a TACI-Fc fusion protein recognizes both the cleaved, soluble form of BLyS as well as the membrane BLyS present on the cell surface of a recombinant cell line. TACI mRNA is found predominantly in B-cells and correlates with BLyS binding in a panel of B-cell lines. We also demonstrate that TACI interacts with nanomolar affinity with the BLyS-related tumor necrosis factor homologue APRIL for which no clear in vivo role has been described. BLyS and APRIL are capable of signaling through TACI to mediate NF-kappaB responses in HEK293 cells. We conclude that TACI is a receptor for BLyS and APRIL and discuss the implications for B-cell biology.