Sustained effect of angiotensin II on tyrosine phosphorylation of annexin I in glomerular mesangial cells

By means of selective extraction in a Ca(2+)-chelating medium and immunoblotting, four annexins (I, II, V, and VI) were identified in both isolated rat renal glomeruli and rat glomerular mesangial cells. Upon 32P labeling of these cells in culture, annexin I was immunoprecipitated using a specific polyclonal antibody and was found to incorporate radioactivity in a constitutive manner. However, as with epidermal growth factor (200 ng/ml), addition of angiotensin II (10(-7) M), arginine-vasopressin (10(-7) M), or endothelin I (10(-7) M) resulted in a 2-3-fold stimulation of annexin I phosphorylation. The basal phosphorylation as well as the stimulating effect of angiotensin II were also detected by immunoblotting annexin extracts using an antiphosphotyrosine antibody. In addition, among various phosphotyrosyl proteins isolated from EGTA extracts by adsorption onto an anti-phosphotyrosine antibody, annexin I was specifically recognized by Western blotting using a monoclonal anti-annexin I antibody, and displayed the same increase upon cell stimulation with angiotensin II. Moreover, thin layer chromatographic analysis of phosphoamino acids present in immunoprecipitated [32P]annexin I showed an exclusive labeling of phosphotyrosine residue(s). Finally, the effect of angiotensin II was detectable after 10 min, maximal at 6 h, and present until 12 h of incubation. Using 12-h stimulation, tyrosine phosphorylation of annexin I displayed a maximum at 10(-7) to 10(-6) M angiotensin II. These data report for the first time the stimulation of annexin I tyrosine phosphorylation by biologically active peptides acting via receptors belonging to the superfamily of seven hydrophobic domain, G-protein-linked receptors, which lack an intrinsic protein tyrosine kinase. This suggests a possible role of annexin I in the mitogenic effect of angiotensin II, arginine-vasopressin, and endothelin I, which was previously observed on rat glomerular mesangial cells as well as on other cells.     

Voltage dependent binding of annexin V, annexin VI and annexin VII-core to acidic phospholipid membranes

Annexin V, VI and VII-core (delta1-107) are members of the annexin protein family and bind to acidic phospholipid membranes in a calcium dependent manner. They also show ion channel activity under certain conditions. As annexins bind peripherally to lipid membranes, ion channel formation must consist of at least two steps: An adsorption reaction regulating the binding of annexin to the membrane surface and the opening and closing of the active species controlling the channel activity. By using the baseline current through the patch clamp seal as a probe for unoccupied binding sites at the membrane, we show that the adsorption of annexins to membranes is not only calcium dependent but also strongly voltage dependent. Whereas the free transfer energies at low calcium concentrations are similar for all three annexins, the binding of annexin V becomes much tighter with higher calcium levels, compared to annexin VI and VII-core. This correlates with the finding that annexin VI and VII-core display channel activity much more often than annexin V if one assumes that a high coverage of the membrane surface with annexins stabilizes the bilayer. At higher protein concentrations weaker binding is observed in agreement with the previously reported anti-cooperativity of membrane binding.     

Manipulation of xenogeneic skin and/or renal graft survival in the rat-mouse concordant combination by portal vein pretransplant transfusion

In cell culture, human osteoblasts and the osteosarcoma cell line MG-63 express annexins I, II, IV, V and VI. Small proportions of annexins IV and V are lost from MG-63 cells into the culture medium in a sedimentable form. however, the bulk of these annexins is intracellular. In non-confluent cells 3 days after passaging, annexin IV and annexin V are strongly present throughout the nucleus and are also present in the cytoplasm. On elevation of the intracellular calcium concentration with the lonophore ionomycin, the intranuclear pools of annexin IV in 38 +/- 4% of cells and annexin V in 70 +/- 5% of cells show relocation to the nuclear membrane within 40 s. Extracellular ATP, which causes a transient increase in the cytosolic free calcium concentration by acting at P2-purinoceptors, also causes relocation of the intranuclear pool of annexin IV in 22 +/- 4% of cells and of annexin V in 38 +/- 8% of cells. After stimulation no significant reversal of the relocation is observed. Elevation of intracellular calcium with ionophore and ATP also causes relocation of the cytoplasmic pools of annexins IV and V. The results support a role for annexins at cellular membranes in response to elevation of cytosolic calcium levels.     

Glucocorticoid-induced annexin 1 secretion by monocytes and peritoneal leukocytes

1. We have studied the ability of the glucocorticoid, dexamethasone, to induce annexin 1 secretion by either human blood monocytes or rat peritoneal leukocytes. 2. The in vivo treatment of rats with dexamethasone (1.25 mg kg-1) selectively induced secretion of annexin 1 by peritoneal leukocytes, as assessed by incubating these cells in culture medium. Annexin 1 secretion was also induced in human cultured monocytes, in vitro, by 10(-6) M dexamethasone. 3. Annexin 1 secretion was inhibited in the presence of 20 mM NH4Cl or by conducting the experiments at 18 degrees C. In contrast, it was not inhibited by monensin, nocodazole or brefeldin A. 4. The time necessary for annexin 1 synthesis and secretion was less than 15 min. 5. These data indicate that glucocorticoids induce annexin 1 secretion by monocytes or peritoneal leukocytes. Because it is not inhibited by monensin, nocodazole or brefeldin A and it is rapid, annexin 1 secretion seems to occur by the secretory pathway similar to that used by several cytosolic proteins such as interleukin-1 beta.     

Glycosaminoglycan binding properties of annexin IV, V, and VI

We have previously demonstrated that annexin IV, one of the calcium/phospholipid-binding annexin family proteins, binds to glycosaminoglycans (GAGs) in a calcium-dependent manner (Kojima, K., Yamamoto, K., Irimura, T., Osawa, T., Ogawa, H., and Matsumoto, I. (1996) J. Biol. Chem. 271, 7679-7685). In this study, we investigated the GAG binding specificities of annexins IV, V, and VI by affinity chromatography and solid phase assays. Annexin IV was found to bind in a calcium-dependent manner to all the GAG columns tested. Annexin V bound to heparin and heparan sulfate columns but not to chondroitin sulfate columns. Annexin VI was adsorbed to heparin and heparan sulfate columns in a calcium-independent manner, and to chondroitin sulfate columns in a calcium-dependent manner. An N-terminal half fragment (A6NH) and a C-terminal half fragment (A6CH) of annexin VI, each containing four units, were prepared by digestion with V8 protease and examined for GAG binding activities. A6NH bound to heparin in the presence of calcium but not to chondroitin sulfate C, whereas A6CH bound to heparin calcium-independently and to chondroitin sulfate C calcium-dependently. The results showed that annexin IV, V, and VI have different GAG binding properties. Some annexins have been reported to be detected not only in the cytoplasm but also on the cell surface or in extracellular components. The findings suggest that the some annexins function as recognition elements for GAGs in extracellular space.     

Roles of individual domains of annexin I in its vesicle binding and vesicle aggregation: a comprehensive mutagenesis study

To understand the mechanism by which annexin I induces membrane aggregation, a comprehensive mutagenesis of all six Ca2+-binding sites was performed. When the cap residues of type II Ca2+-binding sites were systematically mutated to Ala, a type II site in domain II was shown to be essential for Ca2+-dependent vesicle binding of annexin I. Domain II was not, however, directly involved in vesicle aggregation. Instead, type II sites in domains III and IV, respectively, and type III sites in domains I and IV were involved in vesicle aggregation. When all type II sites were deactivated, three type III sites provided residual vesicle binding and aggregating activities. Their contributions to these activities in the presence of type II sites were, however, relatively insignificant. To further investigate the role of each domain harboring a type II site, a set of mutants containing only a specific type II site(s) were generated and their activities measured. These measurements again underscored the importance of domain II in vesicle binding of annexin I and the involvement of domains III and IV in vesicle aggregation. The roles of individual domains in vesicle binding and aggregation can be accounted for by the conformational change of membrane-bound annexin I involving modular rotation of domains (I/IV) following the initial membrane adsorption of domains (II/III). In conjunction with mutagenesis studies on other annexins, these results show that individual domains of annexins, although structurally homologous, have distinct functions and that different annexins might interact with membranes via different domains.     

Distribution of annexins I, II, and IV in bovine mammary gland

Annexins belong to a family of proteins that are characterized by their ability to bind phospholipids in a Ca(2+)-dependent manner that is thought to be involved in a variety of biological processes. The present study determined the localization of annexins in subcellular fractions, nuclei in particular, of cow mammary gland by immunoblot analysis using monoclonal antibodies to annexins I, II, IV, and VI. The analysis revealed that annexins I, II, and IV were present in cytosol, but VI was not. Annexins I and IV were found in the nuclear fraction, but annexin II was only faintly present. Annexin VI was also undetectable in this fraction. Cytosolic annexin I had a molecular mass of 36 kDa. The 36-kDa annexin I was also found in the nuclear fraction. A 38-kDa annexin I was additionally detected in nuclei. The cytosolic and nuclear 36-kDa annexin I and the nuclear 38-kDa annexin I showed different isoelectric points, as revealed by two-dimensional PAGE. Annexin IV from cytosolic and nuclear fractions had similar molecular masses and isoelectric points.     

Structure-function correlations of calcium binding and calcium channel activities based on 3-dimensional models of human annexins I, II, III, V and VII

The annexins are a family of calcium-dependent phospholipid-binding proteins which share a high degree of primary sequence similarity. Using a model of the crystal structure of annexin V as a template, 3-dimensional models of human annexins I, II, III and VII were constructed by homology modeling (J. Greer, J. Mol. Biol. 153, 1027-1042, 1981; J.M. Chen, G. Lee, R.B. Murphy, R.P. Carty, P.W. Brant-Rauf, E. Friedman and M.R. Pincus, J. Biomolec. Str. Dyn. 6, 859-87, 1989) for the 316 amino acid portions corresponding to the annexin V structure published by Huber et al. (J. Mol. Biol. 223, 683-704, 1992). These methods were used to study structure-function correlations for calcium ion binding and calcium channel activity. Published experimental data are specifically shown to be consistent with the annexin models. Possible intramolecular disulfide bridges were identified in annexin I (between Cys297 and Cys316) and in annexins II and VII (between Cys115 and Cys243). Each of the annexin models have 3 postulated calcium binding sites, usually via a Gly-Xxx-Gly-Thr loop with an acidic Glu or Asp residue 42 positions C-terminal to the first Gly. Despite a nonconserved binding site sequence, annexins I and II are able to coordinate calcium in domain 3 since the residue in the second loop position is directed toward the solvent away from the binding pocket. This finding also suggests a mechanism for a conformational change upon binding calcium. Highly conserved Arg and acidic sidechains stabilize the channel pore structure; annexin channels probably exist in a closed state normally. Arg271 may be involved in channel opening upon activation: basic residue 254 can stabilize Glu112, which allows Arg271 to interact with residue 95 instead of Glu112. Residue 267, found on the convex surface at the pore opening, may also be important in modifying channel activity.     

Differential effect of dexamethasone on interleukin 1beta- and cyclic AMP-triggered expression of GTP cyclohydrolase I in rat renal mesangial cells

1. Endogenous synthesis of tetrahydrobiopterin (BH4) is an essential requirement for cytokine-stimulated nitric oxide (NO) synthesis in rat mesangial cells. GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, is expressed in renal mesangial cells in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin (IL)-1beta and agents that elevate cellular levels of cyclic AMP. 2. We examined the action of the potent anti-inflammatory drug dexamethasone on GTP cyclohydrolase I induction in response to IL-1beta and a membrane-permeable cyclic AMP analogue, N6, O-2'-dibutyryladenosine 3'-5'-phosphate (Bt2cyclic AMP). 3. Nanomolar concentrations of dexamethasone markedly attenuated IL-1beta-induced GTP cyclohydrolase I mRNA steady state level as well as IL-1beta-induced GTP cyclohydrolase I protein expression and enzyme activity. In contrast, dexamethasone did not inhibit Bt2cyclic AMP-triggered increase in GTP cyclohydrolase I mRNA level and protein expression, and low (1 nM) or high (1 and 10 microM) doses of dexamethasone consistently increased Bt2cyclic AMP-induced GTP cyclohydrolase activity. 4. In summary, these results suggest that glucocorticoids act at several levels, critically dependent on the stimulus used, to control GTP cyclohydrolase I expression.     

Neurotrophic effects of annexin V on cultured neurons from embryonic rat brain

Recombinant human annexin V showed survival promoting activity in embryonic rat neocortical and mesencephalic neurons in vitro. The neurotrophic effect was observed from a relatively low dose and in a dose-dependent manner. The neurotrophic activity of annexin V was completely blocked by anti-annexin V antibody. Northern blot analysis demonstrated that annexin V mRNA is expressed in non-neuronal cells in the CNS. These results suggest that annexin V plays certain roles in the CNS as a paracrine-type neurotrophic factor.     

Cleavage of annexin I in human neutrophils is mediated by a membrane-localized metalloprotease

A truncated form of annexin I, formed during Ca2+-induced translocation to neutrophil specific granules and secretory vesicles/plasma membranes, is generated through the action of an endogenous membrane protease. The cleavage of annexin I is inhibited by the metalloprotease inhibitor 1,10-phenanthroline as well as by Triton X-100 and dithiothreitol, classifying the protease as a membrane-bound, thiol-dependent metalloprotease. The cleavage site is located close to the N-terminal of annexin I, leaving a truncated form of the molecule, des1-8 annexin I, that contains the Ca2+-binding sites, as well as a number of phosphorylation sites of importance for the function of the protein. When assessing binding capacity to different neutrophil organelles, full-length annexin I bound to azurophil granules, specific granules, and secretory vesicles/plasma membranes, while des1-8 annexin I only bound to specific granules and secretory vesicles/plasma membranes, but not to azurophil granules (C. Sj?lin, C. Dahlgren, Biochim. Biophys. Acta 1281 (1996) 227-234). This implies that there are different mechanisms of binding to neutrophil organelles of full-length annexin I and the truncated form, and that cleavage of annexin I might be of regulatory importance for the degranulation process.     

Distinct annexin subfamilies in plants and protists diverged prior to animal annexins and from a common ancestor

Annexin homologues in the kingdoms of Planta and Protista were characterized by molecular sequence analysis to determine their phylogenetic and structural relationship with annexins of Animalia. Sequence fragments from 19 plant annexins were identified in sequence databases and composite sequences were also assembled from expressed sequence tags for Arabidopsis thaliana. Length differences in protein aminotermini and evidence for unique exon splice sites indicated that plant annexins were distinct from those of animals. A third annexin gene of Giardia lamblia (Anx21-Gla) was identified as a distant relative to other protist annexins and to those of higher eukaryotes, thus providing a suitable outgroup for evolutionary reconstruction of the family tree. Rooted evolutionary trees portrayed protist, plant, and Dictyostelium annexins as early, monophyletic ramifications prior to the appearance of closely related animal annexin XIII. Molecular phylogenetic analyses of DNA and protein sequence alignments revealed at least seven separate plant subfamilies, represented by Anx18 (alfalfa, previously classified), Anx22 (thale cress), Anx23 (thale cress, cotton, rape and cabbage), Anx24 (bell pepper and tomato p34), Anx25 (strawberry, horseradish, pea, soybean, and castor bean), Anx26-Zma, and Anx27-Zma (maize). Other unique subfamilies may exist for rice, tomato p35, apple, and celery annexins. Consensus sequences compiled for each eukaryotic kingdom showed some breakdown of the "annexin-fold" motif in repeats 2 and 3 of protist and plant annexins and a conserved codon deletion in repeat 3 of plants. The characterization of distinct annexin genes in plants and protists reflects their comparable diversity among animal species and offers alternative models for the comparative study of structure-function relationships within this important gene family.     

Expression of rat homeobox gene, rHOX, in developing and adult tissues in mice and regulation of its mRNA expression in osteoblasts by bone morphogenetic protein 2 and parathyroid hormone-related protein

The rat homeobox gene, rHox, was cloned from a rat osteosarcoma cDNA library. Southwestern and gel mobility shift analyses showed that rHox binds to the promoter regions of collagen (alpha1)I and osteocalcin genes while transient transfection with rHox resulted in repression of their respective promoter activities. In situ hybridization studies showed that rHox mRNA was widely expressed in osteoblasts, chondrocytes, skeletal muscle, skin epidermis, and bronchial and intestinal epithelial cells, as well as cardiac muscle in embryonic and newborn mice. However in 3-month-old mice, rHox mRNA expression was restricted to osteoblasts, megakaryocytes, and myocardium. Bone morphogenetic protein 2, a growth factor that commits mesenchymal progenitor cells to differentiate into osteoblasts, down-regulated rHox mRNA expression by 40-50% in UMR 201, a rat preosteoblast cell line, in a time- and dose-dependent manner. In contrast, PTH-related protein (PTHrP), recently shown to be a negative regulator of chondrocyte differentiation, significantly enhanced rHox mRNA expression in UMR 106-06 osteoblastic cells by 3-fold at 24 h while at the same time down-regulating expression of pro-alpha1(I) collagen mRNA by 60%. Expression of rHox mRNA in calvarial osteoblasts derived from PTHrP -/- mice was approximately 15% of that observed in similar cells obtained from normal mice. In conclusion, current evidence suggests that rHox acts as a negative regulator of osteoblast differentiation. Furthermore, down-regulation of rHox mRNA by bone morphogenetic protein 2 and its up-regulation by PTHrP support a role of the homeodomain protein, rHox, in osteoblast differentiation.     

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.     

Differential expression and localization of annexin V in cardiac myocytes during growth and hypertrophy

Recently it was shown that annexin V is the most prominent member of the annexin family in the adult heart [1]. Amongst others, annexin V has been suggested to play a role in developmental processes. The aim of the present study was to explore whether in the heart annexin V content and localization change during maturational and hypertrophic growth, in order to obtain indications that annexin V is involved in cardiac growth processes. First, in the intact rat heart annexin V content and localization were studied during perinatal development. It was clearly demonstrated that annexin V content in total heart transiently increased in the first week after birth, from 0.79 +/- 0.06 microg/mg protein at 1 day before birth to a peak value of 1.24 +/- 0.08 microg/mg protein 6 days after birth, whereafter annexin V protein levels declined to a value of 0.70 +/- 0.06 microg/mg protein at 84 days after birth (p < 0.05). Differences in annexin V content were also observed between myocytes isolated from neonatal and adult hearts [0.81 +/- 0.09 and 0.17 +/- 0.08 microg/mg protein, respectively (p < 0.05)]. Moreover, during cardiac maturational growth the subcellular localization of annexin V might change from a cytoplasmic to a more prominent sarcolemmal localization. Second, in vivo hypertrophy induced by aortic coarctation resulted in a marked degree of hypertrophy (22% increase in ventricular weight), but was not associated with a change in annexin V localization or content. The quantitative results obtained with intact hypertrophic rat hearts are supported by findings in neonatal ventricular myocytes, in which hypertrophy was induced by phenylephrine (10(-5) M). In the latter model no changes in annexin V content could be observed either. In conclusion, the marked alterations in annexin V content during the maturational growth in the heart suggest a possible involvement of this protein in this process. In contrast, the absence of changes in annexin V content and localization in hypertrophied hearts compared to age matched control hearts suggests that annexin V does not play a crucial role in the maintenance of the hypertrophic phenotype of the cardiac muscle cell. This notion is supported by observations in phenylephrine-induced hypertrophied neonatal cardiomyocytes.     

Calcium-dependent interaction of annexin I with annexin II and mapping of the interaction sites

Annexins are multifunctional intracellular proteins with Ca2+- and phospholipid-binding properties. Their structures consist of four conserved repeat domains that form the core and a diverse N-terminal tail, from which their functional differences may arise. We searched for cellular proteins that interact with the N-terminal tail plus domain I of annexin I (ANX1) by using the yeast two-hybrid method. Screening of a HeLa cell cDNA library yielded annexin II (ANX2) cDNA. The interaction between ANX1 and ANX2 also occurred in vitro in a Ca2+-dependent manner. Mapping of the interaction sites revealed that interaction between domain I of ANX1 and domain IV of ANX2 was stronger than the other combinations.     

Effect of bicarbonate-based dialysis solutions on intracellular pH (pHi) and TNFalpha production by peritoneal macrophages

The first evidence that higher plants contain annexins was presented in 1989. Since that time, annexins have been purfied and characterized from a variety of plant sources. Analyses of the deduced proteins encoded by annexin cDNAs indicate that the majority of these plant annexins possess the characteristic four repeats of 70 to 75 amino acids and possess motifs proposed to be involved in Ca2+ binding. Like animal annexins, plant annexins bind Ca2+ and phospholipids and are abundant proteins, but there are indications that the number of distinct plant annexin genes may be considerably fewer than that found in animals. Regarding function, a number of studies show that various members of the annexin family of plants may play roles in secretion and/or fruit ripening, show interaction with the enzyme callose (1.3-beta-glucan) synthase, possess intrinsic nucleotide phosphodiesterase activity, bind to F-actin, and/or have peroxidase activity.     

The long amino-terminal tail domain of annexin XI is necessary for its nuclear localization

Annexin XI is a newly identified annexin which localizes mainly in the nucleus of rat embryonic fibroblasts. There are no typical nuclear localization signals (NLS) in the molecule. To define the region responsible for its nuclear localization, a series of mutants and chimeric cDNA were constructed. These were transiently expressed in COS-7 cells, and the subcellular distributions of the mutants and chimeric proteins were determined by indirect immunofluorescence microscopy. Wild-type annexin XI was located predominantly within the nucleus. Deletion of the N-terminal tail domain (residues 3-196) changed the distribution of the protein from the nucleus to the cytoplasm whereas deletion of the C-terminal core domain (residues 208-504) still kept the protein sorting to the nucleus. Three other mutants lacking 60-80 amino acids in the N-terminal region (residues 3-61, 61-115, and 115-197, respectively) no longer efficiently imported into the nucleus. Furthermore, Escherichia coli beta-galactosidase polypeptide was efficiently localized to the nucleus only when fused with the whole N-terminal region of annexin XI (residues 1-207), not with part of the N-terminal region. In primary cultured rat hepatocytes, annexin XI was distributed in the cytoplasm but not in the nucleus. These results suggest that the whole N-terminal tail domain of annexin XI is necessary and sufficient for its nuclear localization, and may function as NLS in a cell-type specific manner.     

Calcium-dependent neutrophil secretion: characterization and regulation by annexins

To gain direct access to the secretory machinery and study the regulation, mechanisms, and effectors of Ca2+-dependent neutrophil secretion, we developed an efficient and reproducible method of plasma membrane permeabilization using streptolysin O. We confirmed previous studies that permeabilized neutrophils secrete in response to calcium alone, but we also found that the Ca2+ dose-response is biphasic. Secretion is detectable at <1.0 microM Ca2+ and reaches a plateau between 1.0 and 60 to 80 microM. When stimulated with >80 microM Ca2+, secretion is two- to threefold greater than at lower [Ca2+], suggesting that two distinct mechanisms of Ca2+-dependent secretion that differ in their affinity for Ca2+ exist in neutrophils. Although permeabilization allows 100% leak of lactate dehydrogenase, maximum secretion from permeabilized cells is 80% that of f-met-leu-phe-stimulated intact cells, indicating that the essential components of the Ca2+-dependent secretory apparatus are predominantly, if not entirely, membrane bound. Permeabilization causes leakage of 100% of annexins V and VI, but 41% of annexin I and 12% of annexin III are retained. Immunofluorescence microscopy revealed that retained annexins I and III are associated with granule membranes. Addition of soluble annexins I and III to permeabilized cells increased Ca2+-induced secretion up to 15% and 90%, respectively, implying that both annexins participate in this secretory pathway. While annexin V is not required for secretion, it inhibits the low Ca2+-affinity mechanism of secretion.