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H T Haigler

Publications and source records attributed to H T Haigler.

At least 19 recordsLinked to original sources
PubMed2004

Diffusion of single cardiac ryanodine receptors in lipid bilayers is decreased by annexin 12.

Diffusion of cardiac ryanodine receptors (RyR2) in lipid bilayers was characterized. RyR2 location was monitored by imaging fluo-3 fluorescence due to Ca2+ flux through RyR2 channels or fluorescence from RyR2 conjugated with Alexa 488 or containing green fluorescent protein. Single channel currents were recorded to ensure that functional channels were studied. RyR2 exhibited an apparent diffusion coefficient (DRyR) of 1.2 x 10(-8) cm2 s(-1) and a mean path length of 5.0 microm. Optimal use of optical methods for analysis of RyR2 channel function requires that RyR2 diffusion be limited. Therefore, we tested the effect of annexin 12, which interacts with anionic phospholipids in a Ca2+-dependent manner. Addition of annexin 12 (0.25-4.0 microM) to the trans side of bilayers containing an 80:20 ratio of phosphatidylethanolamine/phosphatidylserine decreased RyR2 diffusion in a concentration-dependent manner. Annexin 12 (2 microM) decreased the apparent DRyR 683-fold from 1.2-10(-8) to 1.8 x 10(-11) cm2 s(-1) and the mean path length 10-fold from 5.0 to 0.5 micro m without obvious changes in the conductance of the native bilayer or in activation of RyR2 channels by Ca2+ or suramin. Thus, annexin 12 may provide a useful tool for optimizing optical analysis of RyR2 channels in lipid bilayers.

Animals↗
PubMed2003

Structure and dynamics of annexin 12 bound to a planar lipid bilayer.

Site directed spin labeling is used to investigate the protein annexin 12 absorbed on a single planar phospholipid bilayer of approximately 2-3 cm(2). Electron paramagnetic resonance spectra of nitroxide side chain at several topological sites reveal a conserved tertiary fold of the protein in the absorbed state, in agreement with earlier diffraction results. The angular dependent spectra of the two-dimensional microcrystals are shown to provide information on the degree of ordering of spin labels in a alpha-helix and in turn on the orientation of the alpha-helix with respect to the surface.

Annexins↗
PubMed2001

Calcium-dependent binding of annexin 12 to phospholipid bilayers: stoichiometry and implications.

Annexins (ANXs) are a superfamily of proteins whose functional hallmark is Ca2+-dependent binding to anionic phospholipids. Their core domains are usually composed of a 4-fold repeat of a conserved amino acid sequence, with each repeat containing a type II Ca2+ binding site that is generally thought to mediate Ca2+-dependent binding to the membrane. We now report that ANX12 binding to phospholipid vesicles is highly cooperative with respect to Ca2+ concentration (Hill constant approximately 7), thereby suggesting that more than the four well-characterized type II Ca2+ binding sites are involved in phospholipid binding. Two independent approaches, a novel 45Ca2+ copelleting assay and isothermal titration calorimetry, indicate a stoichiometry of approximately 12 mol of Ca2+/mol of ANX12 for binding to phospholipid vesicles. On the basis of the "low-affinity" Ca2+-binding sites in a number of ANX X-ray crystal structures, we propose a model for ANX12 bilayer binding that involves three types of Ca2+ sites in each of the four repeats. In this model, there is a complementarity between the spacing of the ANX12 Ca2+ binding sites and the spacing of the phospholipid headgroups in bilayers. We tested the implications of the model by manipulating the physical state of vesicles composed of phospholipids with saturated acyl chains with temperature and measuring its influence on ANX12 binding. ANX12 bound to vesicles in a Ca2+-dependent manner when the vesicles were in the liquid crystal phase but not when the phospholipid was in the gel phase. Furthermore, ANX12 bound initially to fluid bilayers remained bound when cooled to 4 degrees C, a temperature that should induce the gel phase transition. Overall, these studies suggest that ANX12 is well suited to being a Ca2+ sensor for rapid all-or-none intercellular membrane-related events.

Animals↗
PubMed2000

Annexins V and XII insert into bilayers at mildly acidic pH and form ion channels.

The functional hallmark of annexins is the ability to bind to the surface of phospholipid membranes in a reversible, Ca(2+)-dependent manner. We now report that human annexin V and hydra annexin XII reversibly bound to phospholipid vesicles in the absence of Ca(2+) at low pH; half-maximal vesicle association occurred at pH 5.3 and 5. 8, respectively. The following biochemical data support the hypothesis that these annexins insert into bilayers at mildly acidic pH. First, a photoactivatable reagent (3-trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine) which selectively labels proteins exposed to the hydrophobic domain of bilayers reacted with these annexins at pH 5.0 and below but not at neutral pH. Second, in a Triton X-114 partitioning assay, annexins V and XII act as integral membrane proteins at low pH and as hydrophilic proteins at neutral pH; in the presence of phospholipids half-maximal partitioning into detergent occurred at pH approximately 5.0. Finally, annexin V or XII formed single channels in phospholipid bilayers at low pH but not at neutral pH. A model is discussed in which the concentrations of H(+) and Ca(2+) regulate the reversible conversion of three forms of annexins-soluble, peripheral membrane, and transmembrane.

Alkanesulfonic Acids↗
PubMed2000

Annexin XII E105K crystal structure: identification of a pH-dependent switch for mutant hexamerization.

Annexins are a family of calcium- and phospholipid-binding proteins involved with numerous cellular processes including membrane fusion, ion channel activity, and heterocomplex formation with other proteins. The annexin XII (ANXB12) crystal structure presented evidence that calcium mediates the formation of a hexamer through a novel intermolecular calcium-binding site [Luecke et al. (1995) Nature 378, 512-515]. In an attempt to disrupt hexamerization, we mutated a conserved key ligand in the intermolecular calcium-binding site, Glu105, to lysine. Despite its occurrence in a new spacegroup, the 1.93 A resolution structure reveals a hexamer with the Lys105 epsilon-amino group nearly superimposable with the original intermolecular calcium position. Our analysis shows that the mutation is directly involved in stabilizing the hexamer. The local residues are reoriented to retain affinity between the two trimers via a pH-dependent switch residue, Glu76, which is now protonated, allowing it to form tandem hydrogen bonds with the backbone carbonyl and nitrogen atoms of Thr103 located across the trimer interface. The loss of the intermolecular calcium-binding site is recuperated by extensive hydrogen bonding favoring hexamer stabilization. The presence of this mutant structure provides further evidence for hexameric annexin XII, and possible in vivo roles are discussed.

Animals↗
PubMed2000

Annexins V and XII alter the properties of planar lipid bilayers seen by conductance probes.

Annexins are proteins that bind lipids in the presence of calcium. Though multiple functions have been proposed for annexins, there is no general agreement on what annexins do or how they do it. We have used the well-studied conductance probes nonactin, alamethicin, and tetraphenylborate to investigate how annexins alter the functional properties of planar lipid bilayers. We found that annexin XII reduces the nonactin-induced conductance to approximately 30% of its original value. Both negative lipid and approximately 30 microM Ca(2+) are required for the conductance reduction. The mutant annexin XIIs, E105K and E105K/K68A, do not reduce the nonactin conductance even though both bind to the membrane just as wild-type does. Thus, subtle changes in the interaction of annexins with the membrane seem to be important. Annexin V also reduces nonactin conductance in nearly the same manner as annexin XII. Pronase in the absence of annexin had no effect on the nonactin conductance. But when added to the side of the bilayer opposite that to which annexin was added, pronase increased the nonactin-induced conductance toward its pre-annexin value. Annexins also dramatically alter the conductance induced by a radically different probe, alamethicin. When added to the same side of the bilayer as alamethicin, annexin has virtually no effect, but when added trans to the alamethicin, annexin dramatically reduces the asymmetry of the I-V curve and greatly slows the kinetics of one branch of the curve without altering those of the other. Annexin also reduces the rate at which the hydrophobic anion, tetraphenylborate, crosses the bilayer. These results suggest that annexin greatly reduces the ability of small molecules to cross the membrane without altering the surface potential and that at least some fraction of the active annexin is accessible to pronase digestion from the opposite side of the membrane.

Alamethicin↗
PubMed1999

Annexin 5 mediates a peroxide-induced Ca(2+) influx in B cells.

Annexin 5 is a Ca(2+)-binding protein, the function of which is poorly understood. Structural and electrophysiological studies have shown that annexin 5 can mediate Ca(2+) fluxes across phospholipid membranes in vitro [1]. There is, however, no direct evidence for the existence of annexin 5 Ca(2+) channels in living cells. Here, we show that annexin 5 inserts into phospholipid vesicle membranes at neutral pH in the presence of peroxide. We then used targeted gene disruption to explore the role of annexin 5 in peroxide-induced Ca(2+) signaling in DT40 pre-B cells. DT40 clones lacking annexin 5 exhibited normal Ca(2+) responses to both thapsigargin and B-cell receptor stimulation, but lacked the sustained phase of the response to peroxide. This late phase was due to Ca(2+) influx from the extracellular space, demonstrating that annexin 5 mediates a peroxide-induced Ca(2+) influx. Thus, peroxide induces annexin 5 membrane insertion in vitro, and peroxide-induced Ca(2+) entry in vivo in DT40 cells requires annexin 5. Our results are consistent with a role for annexin 5 either as a Ca(2+) channel, or as a signaling intermediate in the peroxide-induced Ca(2+)-influx pathway.

Animals↗
PubMed1998

A transmembrane form of annexin XII detected by site-directed spin labeling.

Previous studies of the annexin family of Ca2+ binding proteins identified a soluble monomer in the absence of Ca2+ and a trimer adsorbed on the membrane surface in the presence of Ca2+. On the basis of site-directed spin-labeling studies of annexin XII at low pH, we now report a membrane-inserted form of the protein with a dramatically different structure. The data suggest that upon insertion a continuous transmembrane alpha-helix is reversibly formed from a helix-loop-helix motif in the solution structure. Other regions with similar membrane-insertion potential were identified in the amino acid sequence, and we propose that the corresponding helices come together to form an aqueous pore that mediates the ion channel activity reported for several annexins.

Amino Acid Sequence↗
PubMed1998

Membrane-mediated assembly of annexins studied by site-directed spin labeling.

Annexins are soluble proteins that bind to membranes in the presence of Ca2+. Crystal structures have been determined for some soluble forms, but little is known about the important membrane-bound state. We employed site-directed spin labeling to demonstrate that 1) annexin XII assumes a trimer configuration similar to the crystal structure when bound to bilayers under physiological conditions; 2) trimer assembly on bilayers is remarkably rapid, occurring on a millisecond time scale, whereas subunit exchange requires hours; and 3) different annexins can mix to form heterotrimers. The rapid assembly and heterotrimer formation have important implications concerning the cellular functions of annexins.

Annexins↗
PubMed1998

CCG1/TAF(II)250 regulates epidermal growth factor receptor gene transcription in cell cycle mutant ts13.

The epidermal growth factor receptor (EGFR) plays a critical role in normal growth and its overexpression is associated with several types of cancer. To learn more about regulation of the expression of this important receptor, we investigated the role of the TAF(II)250 subunit of transcription factor IID in the transcription of the EGFR gene. The EGFR gene has a TATA-less promoter and TAF(II)250 has previously been shown to have an important regulatory role in such promoters. The study was performed in the ts13 hamster cell line which has a temperature-sensitive mutation in the CCG1 gene that encodes TAF(II)250. At the nonpermissive temperature, the transcription of a few cell cycle-dependent genes is depressed in ts13 cells while global RNA synthesis is unaffected. Using this model system, we found that EGFR promoter-driven luciferase expression in transiently transfected ts13 cells decreased 8, 25, and 50-fold after 12, 24, and 48 hours, respectively, at the nonpermissive temperature. The decrease was partially rescued by cotransfection with the wild-type CCG1 gene. The expression of endogenous EGFR also appeared to be regulated by TAF(II)250--the maximum binding capacity of ts13 cells for 125I-labeled EGF decreased approximately twofold when incubated for 2 days at the nonpermissive temperature. Placing these studies in the context of the current understanding of the TFIID transcription complex, we speculate that selective stimulation of EGFR gene transcription may be mediated by TAF(II)250 interaction with enhancer-bound activators and the basal transcription machinery.

Animals↗
PubMed1997

Annexin XII forms calcium-dependent multimers in solution and on phospholipid bilayers: a chemical cross-linking study.

The annexins are a family of proteins that bind in a Ca2+-dependent manner to phospholipids that are preferentially located on the intracellular face of plasma membranes. Recent X-ray studies of hydra annexin XII showed that it crystallized as a homohexamer with an intermolecular Ca2+ binding site separate from the type II Ca2+-dependent phospholipid binding site. On the basis of this hexamer structure, a novel mechanism was proposed to explain how annexins interact with membranes. The first step toward evaluating this proposal is to determine whether the annexin XII hexamer exists when the protein is not in a crystalline form. We now report that annexin XII in solution can be cross-linked with dimethyl suberimidate into multimers with apparent Mr's corresponding to trimers and hexamers as determined by SDS--polyacrylamide gel electrophoresis--the trimer band may correspond to incompletely cross-linked hexamers. Multimer formation was dependent on Ca2+ and was enhanced when the protein first was bound to phospholipid vesicles. To evaluate the role of the intermolecular Ca2+ site in annexin XII hexamer formation, one of the residues used to coordinate Ca2+, glutamate 105, was replaced with lysine (E105K). In solution, the E105K mutation inhibited hexamer formation in the presence of moderate (3 mM) but not high (25 mM) Ca2+. No inhibition of E105K annexin XII hexamer formation was observed in the presence of phospholipid, thereby suggesting that either (i) other interactions are capable of stabilizing the hexamer when bound to bilayers or (ii) only trimers form on bilayers and the observed hexamer bands were due to cross-linking of closely packed trimers. In summary, this study shows for the first time that annexin XII can form hexamers in solution and implicates the intermolecular Ca2+ site in hexamer formation. This study also shows that multimers form on bilayers but does not clearly establish whether the multimers are trimers or hexamers.

Annexins↗
PubMed1996

Reduced epidermal growth factor receptor expression in hypohidrotic ectodermal dysplasia and Tabby mice.

Patients with hypohidrotic ectodermal dysplasia (HED) and Tabby (Ta) mice lack sweat glands and there is compelling evidence that these phenotypes are caused by mutations in the same highly conserved but unidentified X-linked gene. Previous studies showed that exogenous epidermal growth factor (EGF) reversed the Ta phenotype but the EGF status in HED patients has not been studied at all. Studies reported herein investigated the hypothesis that the EGF signaling pathway is involved in HED/Ta. Fibroblasts from HED patients had a two- to eightfold decrease in binding capacity for (125)I-labeled EGF, a decreased expression of the immunoreactive 170-kD EGF receptor (EGFR) protein, and a corresponding reduction in EGFR mRNA. Reduced expression of the EGFR also was observed in Ta fibroblasts and liver membranes. Other aspects of the EGF signaling pathway, including EGF concentration in urine and plasma, were normal in both HED patients and Ta mice. We propose that a decreased expression of the EGFR plays a causal role in the HED/Ta phenotype.

Animals↗
PubMed1996

Calcium-dependent binding of S100C to the N-terminal domain of annexin I.

The annexin family of proteins is characterized by a conserved core domain that binds to phospholipids in a Ca(2+)-dependent manner. Each annexin also has a structurally distinct N-terminal domain that may impart functional specificity. To search for cellular proteins that interact with the N-terminal domain of annexin I, we constructed a fusion protein consisting of glutathione S-transferase fused to amino acids 2-47 of human annexin I (GST-AINT; AINT = annexin I N-terminal). Extracts from metabolically labeled A431 cells contained a single protein (M(r) approximately 10,000) that bound to GST-AINT in a Ca(2+)-dependent manner. A synthetic peptide corresponding to amino acids 2-18 of annexin I inhibited the binding of the 10-kDa protein to GST-AINT with half-maximal inhibition occurring at approximately 15 microM peptide. In cellular extracts, endogenous annexin I and the 10-kDa protein associated in a reversible Ca(2+)-dependent manner. Experiments with other annexins and with N-terminal truncated forms of annexin I indicated that the 10-kDa protein bound specifically to a site within the first 12 amino acids of annexin I. The 10-kDa protein was purified from human placenta by hydrophobic and affinity chromatography. Amino acid sequence analysis indicated that the 10-kDa protein is the human homologue of S100C, a recently identified member of the S100 subfamily of EF-hand Ca(2+)-binding proteins.

Amino Acid Sequence↗
PubMed1995

Crystal structure of the annexin XII hexamer and implications for bilayer insertion.

Annexins are a family of calcium- and phospholipid-binding proteins implicated in a number of biological processes including membrane fusion and ion channel formation. The crystal structure of the annexin XII hexamer, refined at 2.8 A resolution, forms a concave disk with 3-2 symmetry, about 100 A in diameter and 70 A thick with a central hydrophilic pore. Six intermolecular Ca2+ ions are involved in hexamer formation. An additional 18 Ca2+ ions are located on the perimeter of the disk, accessible only from the side of the hexameric disk. On the basis of the hexamer structure we propose here a new mode of protein-phospholipid bilayer interaction that is distinct from the hydrophobic insertion of typical membrane proteins. This speculative model postulates the Ca(2+)-dependent insertion of the hydrophilic annexin XII hexamer into phospholipid bilayers with local reorientation of the bilayer phospholipids.

Animals↗
PubMed1992

Identification and characterization of columbid annexin Icp37. Insights into the evolution of annexin I phosphorylation sites.

Annexin I (AnxI) contains phosphorylation sites in its "hinge region" that have been implicated in the regulation of cell growth and/or differentiation. A pigeon (Columba livia) isoform of this protein, annexin Icp35 (cp35), has a very similar amino acid sequence overall but an unrelated sequence that lacks phosphorylation sites in the hinge region. We now report the identification and characterization of annexin Icp37 (cp37) from pigeon. Genomic cloning and Southern blot analysis demonstrated that cp37 and cp35 were encoded by separated genes. Prolactin induced the expression of cp35 mRNA but not cp37. The amino acid sequence of cp37 was deduced from a cDNA clone and found to share 93 and 75% sequence identity with cp35 and human AnxI, respectively. The amino acid sequence of cp37 bore similarities to both AnxI and cp35 in the critical hinge region. Like AnxI, cp37 contained consensus phosphorylation sites in its amino acid sequence and was phosphorylated on tyrosine by the EGF receptor/kinase and on serine by protein kinase C in vitro. Despite the functional similarities between cp37 and AnxI, the nucleotide sequence that encoded the hinge region of cp37 was very similar to the analogous region of cp35, but different from that of AnxI. We propose that certain features shared by cp37 and AnxI are the products of convergent evolution. The fact that evolution independently selected for two annexin I-like genes (cp37 and anxI) encoding analogous phosphorylation sites is strong evidence that phosphorylation is important for the regulation of the biological activity of these proteins.

Amino Acid Sequence↗
PubMed1992

Identification of a novel annexin in Hydra vulgaris. Characterization, cDNA cloning, and protein kinase C phosphorylation of annexin XII.

As a first step toward the elucidation of a simple animal model in which to investigate annexin function, we identified, isolated, and characterized a novel annexin from Hydra vulgaris, annexin XII. A hydra cDNA library was screened using a probe generated by polymerase chain reaction from primers based on the partial amino acid sequence of annexin XII. Annexin XII cDNA was cloned and the functional protein was expressed in high yields in Escherichia coli. The annexin XII cDNA sequence predicted a 316-amino acid protein that had between 44 and 54% sequence identity with the Ca2+-binding core domains of previously characterized vertebrate and Drosophila annexins. The amino-terminal domain of annexin XII did not have sequence similarity with other known annexins except at and around a site that resembled known protein kinase C (PKC) phosphorylation sites in other annexins. As anticipated from its sequence, annexin XII was a high affinity substrate for purified rat brain PKC; half-maximal phosphorylation occurred below 0.1 microM annexin XII, and incorporation of up to 0.8 mol of phosphate/mol of annexin XII was observed. A PKC-like activity in hydra extracts also phosphorylated annexin XII. In summary, hydra promises to be a valuable model system for investigating the biological function of annexins and for determining how this function is modulated by PKC phosphorylation.

Amino Acid Sequence↗
PubMed1992

Inhibition of protein kinase C by annexin V.

Annexin V is a protein of unknown biological function that undergoes Ca(2+)-dependent binding to phospholipids located on the cytosolic face of the plasma membrane. Preliminary results presented herein suggest that a biological function of annexin V is the inhibition of protein kinase C (PKC). In vitro assays showed that annexin V was a specific high-affinity inhibitor of PKC-mediated phosphorylation of annexin I and myosin light chain kinase substrates, with half-maximal inhibition occurring at approximately 0.4 microM. Annexin V did not inhibit epidermal growth factor receptor/kinase phosphorylation of annexin I or cAMP-dependent protein kinase phosphorylation of the Kemptide peptide substrate. Since annexin V purified from both human placenta and recombinant bacteria inhibited protein kinase C activity, it is not likely that the inhibitor activity was associated with a minor contaminant of the preparations. The following results indicated that the mechanism of inhibition did not involve annexin V sequestration of phospholipid that was required for protein kinase C activation: similar inhibition curves were observed as phospholipid concentration was varied from 0 to 800 micrograms/mL; the extent of inhibition was not significantly affected by the order of addition of phospholipid, substrate, or PKC, and the core domain of annexin I was not a high-affinity inhibitor of PKC even though it had similar Ca2+ and phospholipid binding properties as annexin V. These data indirectly indicate that inhibition occurred by direct interaction between annexin V and PKC. Since the concentration of annexin V in many cell types exceeds the amounts required to achieve PKC inhibition in vitro, it is possible that annexin V inhibits PKC in a biologically significant manner in intact cells.

Annexin A5↗