Human Alpha-subunit Research Articles

FcεRIα gene –18483A>C polymorphism affects transcriptional activity through YY1 binding

Three frequent genetic polymorphisms in the human high-affinity IgE receptor alpha-subunit (FcepsilonRIalpha) were shown to be associated with allergic disorders and/or total serum IgE levels in allergic patients. Two of these were previously demonstrated to affect FcepsilonRIalpha expression while the third -18483A>C (rs2494262) has not yet been subjected to functional studies. We hypothesized that the -18483A>C variant affects transcriptional activity of the FcepsilonRIalpha distal promoter in monocytes in which FcepsilonRIalpha transcription is driven through that regulatory region. Indeed, we confirmed preferential binding of the YY1 transcription factor to the -18483C allele, resulting in lower transcriptional activity when compared with the -18483A allele.

Effect of a Synthetic Peptide Corresponding to Residues 313 to 320 of the αIIbSubunit of the Human Platelet Integrin αIIbβ3on Carotid Artery Thrombosis in Rabbits

The platelet integrin receptor alpha(IIb)beta(3) plays a critical role in thrombosis. We have shown previously that the octapeptide YMESRADR, corresponding to sequences 313 to 320 of the human alpha(IIb) subunit, inhibits human platelet activation and fibrinogen binding to alpha(IIb)beta(3), possibly interacting with the ligand. We investigated the effect of YMESRADR on electrically induced carotid artery thrombosis in New Zealand white rabbits. Peptide was administered via the femoral vein, starting 60 min before and continuing for 90 min after the electrical stimulation. Carotid blood flow was monitored for 90 min after the electrical stimulation. The peptide effects on platelet aggregation, in vitro and ex vivo, and on various coagulation, bleeding, and hemostatic parameters were evaluated. YMESRADR significantly inhibited rabbit platelet aggregation in vitro in a dose-dependent manner. It is important that peptide administration in vivo, at doses ranging from 3 to 15 mg/kg, prolonged the duration of the patency of the carotid artery, and no artery occlusion was observed until the end of the study (90 min after electrical stimulation). Furthermore, YMESRADR administration reduced platelet aggregation ex vivo and thrombus weight; however, these reductions reached statistical significance, compared with the control group, at the peptide doses of 12 and 15 mg/kg. YMESRADR did not affect any coagulation parameter studied and the hemostatic response observed in control animals. Thus, YMESRADR represents a novel antiplatelet agent that can inhibit thrombus formation effectively and carotid artery occlusion without causing hemorrhagic complications in a rabbit model of arterial thrombosis.

Numerical Characterization of Protein Sequences and Application to Voltage-Gated Sodium Channel α Subunit Phylogeny

We propose a new method to compare sequences of protein families by generating numerical characterizations through a 20D representation. Using a walk along the axes representing the amino acids we generate a vector for each sequence whose components can be used to derive distance matrices between sequences and whose magnitudes can be used to compare the similarities/dissimilarities between the different sequences. The distance matrices enable creation of phylogenetic trees without need for multiple alignments or any other model dependencies. In this paper we test this technique with human globin gene sequences and then apply the method to a contemporary issue of evolutionary relationships of rat and human voltage-gated sodium channel alpha subunits and compare with published literature. The close match of the results demonstrates the reliability and ease of use of this method.

Structure of the Integrin αIIb Transmembrane Segment

Integrin cell-adhesion receptors transduce signals bidirectionally across the plasma membrane via the single-pass transmembrane segments of each alpha and beta subunit. While the beta3 transmembrane segment consists of a linear 29-residue alpha-helix, the structure of the alphaIIb transmembrane segment reveals a linear 24-residue alpha-helix (Ile-966 -Lys-989) followed by a backbone reversal that packs Phe-992-Phe-993 against the transmembrane helix. The length of the alphaIIb transmembrane helix implies the absence of a significant transmembrane helix tilt in contrast to its partnering beta3 subunit. Sequence alignment shows Gly-991-Phe-993 to be fully conserved among all 18 human integrin alpha subunits, suggesting that their unusual structural motif is prototypical for integrin alpha subunits. The alphaIIb transmembrane structure demonstrates a level of complexity within the membrane that is beyond simple transmembrane helices and forms the structural basis for assessing the extent of structural and topological rearrangements upon alphaIIb-beta3 association, i.e. integrin transmembrane signaling.

Structural characterization of human general transcription factor TFIIF in solution

Human general transcription factor IIF (TFIIF), a component of the transcription pre-initiation complex (PIC) associated with RNA polymerase II (Pol II), was characterized by size-exclusion chromatography (SEC), electrospray ionization mass spectrometry (ESI-MS), and chemical cross-linking. Recombinant TFIIF, composed of an equimolar ratio of alpha and beta subunits, was bacterially expressed, purified to homogeneity, and found to have a transcription activity similar to a natural one in the human in vitro transcription system. SEC of purified TFIIF, as previously reported, suggested that this protein has a size >200 kDa. In contrast, ESI-MS of the purified sample gave a molecular size of 87 kDa, indicating that TFIIF is an alphabeta heterodimer, which was confirmed by matrix-assisted laser desorption/ionization (MALDI) MS of the cross-linked TFIIF components. Recent electron microscopy (EM) and photo-cross-linking studies showed that the yeast TFIIF homolog containing Tfg1 and Tfg2, corresponding to the human alpha and beta subunits, exists as a heterodimer in the PIC, so the human TFIIF is also likely to exist as a heterodimer even in the PIC. In the yeast PIC, EM and photo-cross-linking studies showed different results for the mutual location of TFIIE and TFIIF along DNA. We have examined the direct interaction between human TFIIF and TFIIE by ESI-MS, SEC, and chemical cross-linking; however, no direct interaction was observed, at least in solution. This is consistent with the previous photo-cross-linking observation that TFIIF and TFIIE flank DNA separately on both sides of the Pol II central cleft in the yeast PIC.

A New Mouse Model of Autoimmune Ocular Myasthenia Gravis

To establish a novel model of autoimmune ocular myasthenia gravis (oMG) in mice and study the pathogenic mechanisms of oMG. oMG was induced in HLA-DQ8 transgenic, HLA-DR3 transgenic, major histocompatibility complex (MHC) class II-deficient, C57BL/6, and C57BL/10 mice by immunization with an Escherichia coli plasmid expressing the recombinant human acetylcholine receptor (AChR) alpha subunit. All strains of immunized mice developed ocular myasthenia gravis with varying disease incidence and severity. HLA-DQ8 transgenic mice were highly susceptible to oMG. Mice with oMG had serum autoantibodies to the mouse extraocular AChR, pathologic deposits of IgG, C3, and C5b-C9 in their extraocular and limb neuromuscular junctions, and droopiness of eyelids. HLA-DR3 transgenic and MHC class II-deficient mice were relatively resistant to oMG induced by AChR alpha subunit immunization and had minimal ocular abnormalities. These findings suggest that oMG pathogenesis could be triggered by immunity to the human AChR alpha subunit and that MHC class II molecule is required for human AChR alpha subunit presentation and CD4 cell-mediated anti-AChR antibody class switching. Differential oMG susceptibility observed in DQ8 and DR3 transgenic mice correlated with the intensity of lymphocytes to respond to the human AChR alpha subunit. This new model of oMG will be a valuable tool for studying the mechanism of oMG and gMG pathogenesis in humans and for preclinical therapeutic analysis.

Association of Human SCN5A Polymorphisms With Idiopathic Ventricular Arrhythmia in a Chinese Han Cohort

Mutations of human cardiac sodium channel alpha subunit gene (SCN5A) have been implicated in risk of various cardiac arrhythmias. The present study aimed to investigate whether SCN5A polymorphism was related to the occurrence of idiopathic ventricular arrhythmia (IVA) in Chinese Han patients. Genotyping for polymorphic variance of SCN5A was performed by polymerase chain reaction and automated gene scan sequencing analysis in 31 unrelated Chinese patients with IVA (IVA group) and 103 healthy controls (control group). The allele frequencies of c.87G>A (p.Ala29Ala) and c.1673A>G (p.His558Arg) were higher in men than in women for control group (p=0.023 and p=0.027, respectively). For women, patients with IVA had higher allele frequency of c.87G>A than controls (0.455 vs 0.198, p=0.013, odds ratio (OR) 3.382), whereas for men, minor allele c.1673A>G was significantly less prevalent in IVA patients compared with controls (0.025 vs 0.175, p=0.017, OR 0.121). Significant difference existed in occurrence of ventricular arrhythmia between female patients with 87GA/AA and those with 87GG genotypes (0.348 vs 0.097, p=0.039, OR 4.978) and between male patients with 1673AA and those with 1673AG/GG genotypes (0.311 vs 0.053, p=0.031, OR 8.143). The frequency of haplotype GG (87G-1673G) was significantly lower in IVA patients than in controls (0.016 vs 0.128, p=0.009, OR 0.109). The present study indicates that Chinese Han people have significant difference in distribution of 2 single nucleotide polymorphisms (c.87G>A and c.1673A>G) between healthy male and females. The 87A allele might be associated with an increased risk of IVA in females, whereas 1673G allele might have been positively selected because of its protective effects against ventricular arrhythmia in males.

Variola virus IL-18 binding protein interacts with three human IL-18 residues that are part of a binding site for human IL-18 receptor alpha subunit

Interleukin-18 (IL-18) plays an important role in host defense against microbial pathogens. Many poxviruses encode homologous IL-18 binding proteins (IL-18BP) that neutralize IL-18 activity. Here, we examined whether IL-18BP neutralizes IL-18 activity by binding to the same region of IL-18 where IL-18 receptor (IL-18R) binds. We introduced alanine substitutions to known receptor binding sites of human IL-18 and found that only the substitution of Leu5 reduced the binding affinity of IL-18 with IL-18BP of variola virus (varvIL-18BP) by more than 4-fold. The substitutions of Lys53 and Ser55, which were not previously known to be part of the receptor binding site but that are spatially adjacent to Leu5, reduced the binding affinity to varvIL-18BP by approximately 100- and 7-fold, respectively. These two substitutions also reduced the binding affinity with human IL-18R alpha subunit (hIL-18Rα) by 4- and 2-fold, respectively. Altogether, our data show that varvIL-18BP prevents IL-18 from binding to IL-18R by interacting with three residues that are part of the binding site for hIL-18Rα.

C-terminal modulator controls Ca2+-dependent gating of Cav1.4 L-type Ca2+ channels

Tonic neurotransmitter release at sensory cell ribbon synapses is mediated by calcium (Ca2+) influx through L-type voltage-gated Ca2+ channels. This tonic release requires the channels to inactivate slower than in other tissues. Ca(v)1.4 L-type voltage-gated Ca2+ channels (LTCCs) are found at high densities in photoreceptor terminals, and alpha1 subunit mutations cause human congenital stationary night blindness type-2 (CSNB2). Ca(v)1.4 voltage-dependent inactivation is slow and Ca2+-dependent inactivation (CDI) is absent. We show that removal of the last 55 or 122 (C122) C-terminal amino acid residues of the human alpha1 subunit restores calmodulin-dependent CDI and shifts voltage of half-maximal activation to more negative potentials. The C terminus must therefore form part of a mechanism that prevents calmodulin-dependent CDI of Ca(v)1.4 and controls voltage-dependent activation. Fluorescence resonance energy transfer experiments in living cells revealed binding of C122 to C-terminal motifs mediating CDI in other Ca2+ channels. The absence of this modulatory mechanism in the CSNB2 truncation mutant K1591X underlines its importance for normal retinal function in humans.

Biological Properties of a Novel Follicle-Stimulating Hormone/Human Chorionic Gonadotropin Chimeric Gonadotropin

A chimeric recombinant human gonadotropin, termed C3, demonstrates both follitropic and lutropic bioactivities. The alpha-subunit construct for C3 is comprised of the recombinant wild-type human glycoprotein hormone alpha-subunit. The beta-subunit DNA construct for C3 encodes residues 1-145 from human chorionic gonadotropin (hCG)-beta with the exceptions that FSH beta amino acid 88 (D) is substituted for hCG beta amino acid 94 (R) and FSH beta amino acids 95-108 (TVRGLGPSYCSFGE) are substituted for hCG beta amino acids 101-114 (GGPKDHPLTCDDPR). C3 is a potent FSH and LH agonist able to bind and to signal through FSH and LH receptors in vitro. In in vivo bioassays optimized to quantify each type of activity, C3 was found to have lutropin and follitropin potencies at levels similar to those of recombinant human LH and recombinant human FSH, respectively. In immature rats, C3 was sufficient to support the maturation of normal ovarian follicles. Moreover, a significant portion of follicles matured by C3 ruptured in response to an ovulatory hCG stimulus and gave rise to morphologically normal oocytes. Furthermore, a low dose of C3 promoted weight gain in the rodent uterus, suggesting it also supported preparation for implantation without histological evidence of excessive luteinization of the ovary. In summary, the biological properties of C3 indicate that its chimeric nature has resulted in a fully functional, dual-acting human gonadotropin.

Semi‐purification of the immunoglobulin E‐sweat antigen acting on mast cells and basophils in atopic dermatitis

Sweating aggravates the symptoms of atopic dermatitis (AD). We have recently reported positive skin reactions and histamine release from basophils in response to autologous sweat in patients with AD. To characterize the biochemical and immunological properties of the substance in sweat that evokes histamine release and to study the usability of the basophil-histamine release test with the sweat antigen for AD. Sweat collected from healthy volunteers was purified using chromatographies. Serum immunoglobulin (Ig)E of four patients with AD were purified using an affinity-chromatography column with anti-IgE antibodies. The amount of semi-purified sweat antigen (138 ng protein/ml) that induced a half-maximum reaction of basophils of a patient with AD was utilized for the basophil histamine release test. The involvement of specific IgE and high-affinity IgE receptor (FcepsilonRI) in the reactions was examined using basophils of healthy volunteers, a human mast cell line (LAD2), and a rat basophilic leukemia cell line transfected with human alpha-subunit of FcepsilonRI (RBL-48). The semi-purified sweat antigen induced histamine release from the basophils of 47 of 61 (74.6%) patients with AD and four of 46 (8.7%) healthy controls. Both basophils and mast cells sensitized with the patient-derived IgE showed degranulation upon stimulation with the sweat antigen. However, no reaction was observed when cells were sensitized with myeloma IgE or the antigen was treated with proteases. The semi-purified standardized sweat antigen consists of a protein that induces degranulation of basophils and mast cells via antigen-specific IgE and FcepsilonRI in patients with AD.

Inefficiency in GM2 Ganglioside Elimination by Human Lysosomal .BETA.-Hexosaminidase .BETA.-Subunit Gene Transfer to Fibroblastic Cell Line Derived from Sandhoff Disease Model Mice

Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

Mast Cell-Specific Gene Targeting.

For years mast cell (MC) studies have relied on MC lines or bone marrow-derived MC. Regular knockout mice have some disadvantages such as early lethality and developmental defects. Also, the phenotype in MCs from these animals could not be a primary defect but secondary to other cell lines affected by the mutation. The Cre/loxP system allows us to target genetic deletions to specific tissues or cells. Cre, a recombinase from bacteriophage P1, recognizes a DNA sequence motif of 34 bases (loxP). If a DNA segment is flanked by two loxP sites in the same orientation, Cre excises that segment activating or silencing a gene. Many genes have been “floxed” (flanked by two loxP sites), but no MC-specific Cre mouse has been created. We are using promoter regions of MC-specific proteins to drive expression of Cre-recombinase. We produced transgenic mice using the promoter for human alpha-subunit and murine beta-subunit of the high-affinity receptor for IgE (hFcεRIα and mFcεRIβ), both expressed in MCs and basophils. We also used the promoter of the murine mast cell protease mMCP-5, a member of a family of cargo proteins expressed only in MCs. For even more controlled expression, we knocked-in Cre cDNA into the mMCP-5, mMCP-6 and Ras guanine releasing protein 4 (RasGRP4) loci, all of them proteins expressed exclusively in MCs, while knocking-out the respective gene. Heterozygotes will express Cre only in MCs, and homozygotes for mMCP-5, mMCP-6 or RasGRP4 deletions will be regular knockouts that will allow us to study the role of these proteins in mast cells. These three transgenic and three knock-out/knock-in lines have been created and are being crossed with reporter mice. We decided to use a double reporter mouse, the Bgeo/GFP (Jackson Laboratory). This mouse has “floxed” lacZ that is constitutively expressed under the control of the CMV enhancer/chicken actin promoter, and when crossed with our Cre recombinase-expressing mouse, lacZ is excised and enhanced green fluorescent protein (EGFP) is expressed in cells expressing Cre. EGFP can be easily detected in MCs from peritoneal lavage by flow-cytometry, using antibodies against c-kit (CD117) and FcεRI to label the MCs. Results of this screening will be presented at the meeting. After documenting the presence of functional Cre-recombinase in our reporter mice, we will cross them with our “floxed” mice to obtain conditional mast cell-specific knockouts.

Transcript Profiling of Immediate Early Genes Reveals a Unique Role for Activating Transcription Factor 3 in Mediating Activation of the Glycoprotein Hormone α-Subunit Promoter by Gonadotropin-Releasing Hormone

Recent studies profiling immediate early gene responses to GnRH in the LbetaT2 gonadotrope cell model revealed increased expression of numerous genes including activating transcription factor (ATF) 3. The present studies demonstrate similar results with GnRH administration in vivo in ovariectomized mice. In this model, ATF3 mRNA was markedly up-regulated at 20, 40, and 60 min after in vivo administration of a GnRH analog. In alphaT3-1 gonadotrope cells, ATF3 mRNA and protein were induced by GnRH in a manner consistent with in vivo observations. Pharmacological studies implicated a combined role for the activities of protein kinase C isozymes, ERK and c-Jun N-terminal kinase, in modulating ATF3 expression. The role of ATF3 was further investigated in the activation of the human glycoprotein hormone alpha-subunit gene promoter. GnRH induced the alpha-subunit promoter-luciferase reporter approximately 16-fold, and this induction was completely abolished with mutations in the dual cAMP response elements (CREs) or the combined inhibition of GnRH-induced ERK and c-Jun N-terminal kinase. GnRH induced recruitment of ATF3, c-Jun, and c-Fos to the dual CREs. Overexpression and specific knockdown of ATF3 by small inhibitory RNA implicate a functional role for ATF3 in mediating activation of the alpha-subunit gene promoter. These studies provide clear evidence that ATF3 is a key immediate early gene induced by GnRH administration in vivo and in the alphaT3-1 gonadotrope cell model. These studies support the conclusion that the dual CREs of the human alpha-subunit promoter are the target of GnRH-induced MAPK regulation through ATF3.

Revising the Genomic Structure of the Human GM-CSF Receptor Alpha (GMRα) Subunit: Characterization of a Novel Membrane-Spanning GMRα Isoform Which Contains a Previously Undescribed Exon.

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key player in the processes of hematopoiesis and inflammation. The functional effects of GM-CSF are mediated by binding of the cytokine to the cell surface GM-CSF receptor, which is comprised of a ligand-binding alpha subunit (GMRα) and a signal-transducing beta subunit (GMRβ). In addition, there are at least 2 soluble GMRα isoforms (sGMRα)-one generated by alternative splicing, and a second produced by proteolytic release of the GMRα extracellular domain from the cell surface.While examining expression of the spliced soluble and transmembrane (tmGMRα) isoforms of GMRα in neutrophils by RT-PCR, we detected a third, higher molecular weight, GMRα transcript. This was surprising since the tmGMRα transcript is comprised of all 13 exons of the published GMRα gene structure (Nakagawa et al., 1994). We therefore hypothesized that we had discovered a previously undescribed exon of the GMRα gene.The novel GMRα (nGMRα) transcript was subsequently cloned from human neutrophils. Sequencing of nGMRα indicated inclusion of a 102 nucleotide sequence between exons 10 and 11 of the GMRα gene. Interestingly, the novel exon and surrounding intronic sequence appear to represent an Alu-repeat element, indicating that this exon is likely primate-specific. Segments of these repetitive DNA elements become inserted into mature mRNAs by way of splicing in a process termed “exonization”. In fact, it now seems that more than 5% of the alternatively spliced exons in the human genome are Alu-derived. At the protein level, this novel transcript is predicted to contain all 400 amino acids of the tmGMRα protein as well as an additional 34 amino acids within the membrane-proximal region of the extracellular domain. We have expressed the novel GMRα clone, as well as a soluble version of novel GMRα (sol-nGMRα), in the murine factor-dependent cell line Ba/F3. The sol-nGMRα protein is secreted from these cells into conditioned media, as expected. We have purified sol-nGMRα protein and used it to determine the affinity of nGMRα for GM-CSF. By flow cytometry, we were unable to detect full-length nGMRα on the surface of Ba/F3 cells, but we were able to detect nGMRα in conditioned media from nGMRα-expressing Ba/F3 cells. In addition, while tmGMRα-expressing Ba/F3 cells are able to proliferate in response to human GM-CSF in the absence of murine growth factors, the nGMRα-expressing Ba/F3 cells do not respond to GM-CSF. Our results indicate that the full-length nGMRα protein is proteolytically cleaved from the cell surface or alternatively, that its subcellular localization is otherwise disrupted.As the nGMRα transcript was first detected in neutrophils isolated from a human donor, we sought to determine whether nGMRα would be expressed by other donors and in other hematopoietic cell types. Our results indicate that all donors tested express nGMRα, and that nGMRα is present in all hematopoietic cell types expressing the tmGMRα transcript, although tmGMRα nonetheless represents the predominant transcript.It is becoming increasingly clear that GM-CSF signaling is more complex than was previously thought. In order to understand the actions of GM-CSF in the clinical setting, it is critical that we first define all components of the GM-CSF signaling axis.

Relationship between imbalanced synthesis of human chorionic gonadotropin alpha and beta subunits and pregnancy-induced hypertension

To study the relationship between imbalanced synthesis of human chorionic gonadotropin (hCG) alpha and beta subunits and the pathology of pregnancy-induced hypertension (PIH). Total hCG, free alphahCG and betahCG were measured in serum samples collected from 60 cases of PIH and 30 normal gravid women by radioimmunoassay. The ratio of total hCG/betahCG, betahCG/FalphahCG, (FalphahCG+betahCG)/betahCG were calculated. Immunohistochemical staining was performed in 13 placenta from each group. In PIH group the levels of FalphahCG, total hCG and betahCG were significantly higher than those of normal group (FalphahCG: 528 +/-421 IU/L compared with 222 +/-129 IU/L; betahCG: 39396 +/-6412 IU/L compared with 24265 +/-5575 IU/L; total hCG: 66597 +/-9294 IU/L compared with 36078 +/-4767 IU/L, all P<0.001). The betahCG/FalphahCG ratio in PIH was lower than that of normal group (91.23 +/-53.38 Compared with 119.4 +/-80.1, P<0.05); (FalphahCG+betahCG)/betahCG ratio and total hCG/betahCG ratio in two groups were (1.022 +/-0.026 compared with 1.015 +/-0.011; 1.802 +/-0.339 compared with 1.807 +/-0.258, respectively P>0.05). The immunohistochemical intensity of betahCG and FalphahCG in syncytiotrophoblast was significantly increased in 13 PIH compared with the control. These data suggested that the imbalanced synthesis of hCG alpha and beta subunits may cause hypertension.

Isoform specificity of human Na(+), K(+)-ATPase localization and aldosterone regulation in mouse kidney cells.

Short-term aldosterone coordinately regulates the cell-surface expression of luminal epithelial sodium channels (ENaC) and of basolateral Na(+) pumps (Na(+), K(+)-ATPase alpha1-beta1) in aldosterone-sensitive distal nephron (ASDN) cells. To address the question of whether the subcellular localization of the Na(+), K(+)-ATPase and its regulation by aldosterone depend on subunit isoform-specific structures, we expressed the cardiotonic steroid-sensitive human alpha isoforms 1-3 by retroviral transduction in mouse collecting duct mpkCCD(c14) cells. Each of the three exogenous human isoforms could be detected by Western blotting. Immunofluorescence indicated that the exogenous alpha1 subunit to a large extent localizes to the basolateral membrane or close to it, whereas much of the alpha2 subunit remains intracellular. An ouabain-sensitive current carried by exogenous pumps could be detected in apically amphotericin B-permeabilized epithelia expressing human alpha1 and alpha2 subunits, but not the alpha3 subunit. This current displayed a higher apparent Na(+) affinity in pumps containing human alpha2 subunits (10 mM) than in pumps containing human alpha1 (33.2 mM) or endogenous (cardiotonic steroid-resistant) mouse alpha1 subunits (mean: 16.3 mM). A very low mRNA level of the Na(+), K(+)-ATPase gamma subunit (FXYD2) in mpkCCD(c14) cells suggested that this ancillary gene product is not responsible for the relatively low apparent Na(+) affinity measured for a1 subunit-containing pumps. Aldosterone increased the pump current carried by endogenous pumps and by pumps containing the human alpha1 subunit. In contrast, the current carried by pumps with a human alpha2 subunit was not stimulated by the same treatment. In summary, quantitative basolateral localization of the Na(+), K(+)-ATPase and its responsiveness to aldosterone require alpha1 subunit-specific sequences that differentiate this isoform from the alpha2 and alpha3 subunit isoforms.

Crystallization of the proline-rich-peptide binding domain of human type I collagen prolyl 4-hydroxylase.

Collagen prolyl 4-hydroxylases catalyze the hydroxylation of -X-Pro-Gly- sequences and play an essential role in the synthesis of all collagens. They require Fe(2+), 2-oxoglutarate, molecular oxygen and ascorbate, and all vertebrate collagen prolyl 4-hydroxylases are alpha(2)beta(2) tetramers. The alpha-subunits contain separate catalytic and peptide substrate-binding domains. Here, the crystallization of the peptide substrate-binding domain consisting of residues 144-244 of the 517-residue human alpha(I) subunit is described. The crystals are well ordered and diffract to at least 3 A. The space group is P3(1) or P3(2) and the asymmetric unit most probably contains a dimer.

CDNA cloning, biochemical and phylogenetic characterization of beta- and beta'-subunits of Candida albicans protein kinase CK2.

We have previously reported that Candida albicans protein kinase CK2 is composed of two distinct catalytic (alpha- and alpha'-) and two distinct regulatory (beta- and beta'-) subunits. We report here the isolation of two cDNAs clones, CaCKB1 and CaCKB2, encoding C. albicans beta- and beta'-subunits, respectively. The predicted beta- and beta'-proteins have calculated molecular masses of 34 kDa and 31 kDa and show all major features conserved in beta-subunits of other organisms, including the N-terminal autophosphorylation site, the internal acidic region and a potential metal-binding motif. The deduced amino acid sequence of C. albicans beta-subunit displays 48% identity with that of Saccharomyces cerevisiae and has an unusually long C-terminal acidic region containing a putative autophosphorylation site. C. albicans beta' shows 54% sequence identity with its S. cerevisiae homologue. Semi-quantitative RT-PCR analyses indicate that the mRNAs corresponding to both subunits are present in similar amounts in the yeast and hyphal forms of the fungus. To evaluate the biochemical properties of C. albicans beta- and beta'-subunits, both proteins were expressed in Escherichia coli and purified. Experiments performed in vitro indicate that both recombinant subunits reconstitute a fully functional holoenzyme when incubated with stoichiometric amounts of human recombinant alpha-subunit, as judged by their ability to abolish basal phosphorylation of calmodulin by human recombinant alpha-subunit and the reversion of the inhibitory effect by polylysine. In addition, both regulatory subunits can be phosphorylated by human recombinant alpha subunit. Phylogenetic analysis of beta- and beta'-proteins of C. albicans and other organisms shows that the CKB gene duplication occurred before the split of the ascomycete and basidiomycete lineages.

A Functional Domain of the α1 Subunit of Soluble Guanylyl Cyclase Is Necessary for Activation of the Enzyme by Nitric Oxide and YC-1 but Is Not Involved in Heme Binding

Soluble guanylyl cyclase is a heterodimeric enzyme consisting of an alpha(1) and a beta(1) subunit and is an important target for endogenous nitric oxide and the guanylyl cyclase modulator YC-1. The activation of the enzyme by both substances is dependent on the presence of a prosthetic heme group. It has been unclear whether this prosthetic heme group is sandwiched between the alpha(1) and beta(1) subunits or whether it exclusively binds to the beta(1) subunit. Here we analyze progressive amino-terminal deletion mutants of the human alpha(1) subunit after co-expression with the human beta(1) subunit in the baculovirus/Sf9 system. Spectral, biochemical, and pharmacological analysis shows that the first 259 amino acids of the alpha(1) subunit can be deleted without loss of sensitivity to nitric oxide (NO) or YC-1 or loss of heme binding of the respective enzyme complex with the beta(1) subunit. This is in contrast to previous data indicating that NO sensitivity and a functional heme binding site requires full-length amino termini of bovine alpha(1) and beta(1) subunits. Further deletion of the first 364 amino acids of the alpha(1) subunit leads to an enzyme complex with preserved heme binding but loss of sensitivity to NO or YC-1 despite induction of the typical spectral shift by NO binding to the prosthetic heme group. We conclude that 1) the amino-terminal part of the alpha(1) subunit is not involved in heme binding and 2) amino acids 259-364 of the alpha(1) subunit represent an important functional domain for the transduction of the NO activation signal and likely represent the target for NO-sensitizing substances like YC-1.