Biomedical subjects

Ursula Rescher

Publications and source records attributed to Ursula Rescher.

15 recordsLinked to original sources

PubMed2006

Requirement for annexin A1 in plasma membrane repair.

Ca2+ entering a cell through a torn or disrupted plasma membrane rapidly triggers a combination of homotypic and exocytotic membrane fusion events. These events serve to erect a reparative membrane patch and then anneal it to the defect site. Annexin A1 is a cytosolic protein that, when activated by micromolar Ca2+, binds to membrane phospholipids, promoting membrane aggregation and fusion. We demonstrate here that an annexin A1 function-blocking antibody, a small peptide competitor, and a dominant-negative annexin A1 mutant protein incapable of Ca2+ binding all inhibit resealing. Moreover, we show that, coincident with a resealing event, annexin A1 becomes concentrated at disruption sites. We propose that Ca2+ entering through a disruption locally induces annexin A1 binding to membranes, initiating emergency fusion events whenever and wherever required.

Annexin A1↗

PubMed2006

Proteolytic cleavage of annexin 1 by human leukocyte elastase.

Annexin 1 has been shown to participate through its unique N-terminal domain in the recruitment and activation of leukocytes at sites of inflammation. Peptides derived from this domain are true mimetics of the annexin 1 action in all inflammation models tested and most likely serve as the active entities generated at sites of inflammation. To elucidate mechanisms underlying peptide generation we used isolated blood leukocytes and endothelial cell monolayers. We show that following endothelial adhesion, annexin 1 was externalized from leukocytes and rapidly cleaved. Addition of purified annexin 1 to degranulating leukocytes resulted in the truncation of annexin 1, which seemed to depend on the proteolytic activity of human leukocyte elastase (HLE). The capacity of elastase to proteolytically cleave annexin 1 was confirmed using both purified annexin 1 and HLE. The identification of annexin 1 as a substrate for HLE supports the model in which annexin 1 participates in regulating leukocyte emigration into inflamed tissue through N-terminal peptides generated at inflammatory sites.

Annexin A1↗

PubMed2006

Annexins as intracellular calcium sensors.

The annexins are a multigene family of Ca(2+)- and charged phospholipid-binding proteins. Although they have been ascribed with diverse functions, there is no consensus about the role played by this family as a whole. We have mapped the Ca(2+)-induced translocations of four members of the annexin family and of two truncated annexins in live cells, and demonstrated that these proteins interact with the plasma membrane as well as with internal membrane systems in a highly coordinated manner. Annexin 2 was the most Ca(2+) sensitive of the studied proteins, followed by annexins 6, 4 and 1. The calcium sensitivity of annexin 2 increased further following co-expression with S100A10. Upon elevation of [Ca(2+)](i), annexins 2 and 6 translocated to the plasma membrane, whereas annexins 4 and 1 also became associated with intracellular membranes and the nuclear envelope. The NH(2)-terminus had a modulatory effect on plasma membrane binding: its truncation increased the Ca(2+) sensitivity of annexin 1, and decreased that of annexin 2. Given the fact that several annexins are present within any one cell, it is likely that they form a sophisticated [Ca(2+)] sensing system, with a regulatory influence on other signaling pathways.

Annexins↗

PubMed2006

Annexin A8 displays unique phospholipid and F-actin binding properties.

Annexin A8 is a poorly characterized member of the annexin family of Ca2+-regulated membrane binding proteins. Initially only identified at the cDNA level it had been tentatively linked to acute promyelocytic leukaemia (APL) due to its high and regulated expression in APL-derived cells. Here we identify unique properties of the annexin A8 protein. We show that it binds Ca2+-dependently and with high specificity to phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) and is also capable of interacting with F-actin. In line with these characteristics annexin A8 is recruited to F-actin-associated PtdIns(4,5)P2-rich membrane domains formed in HeLa cells upon infection with non-invading enteropathogenic Escherichia coli. These properties suggest a role of annexin A8 in the organization of certain actin-associated membrane domains.

Actins↗

PubMed2004

Specific association of annexin 1 with plasma membrane-resident and internalized EGF receptors mediated through the protein core domain.

Phosphorylation of the Ca2+ and membrane-binding protein annexin 1 by epidermal growth factor (EGF) receptor tyrosine kinase has been thought to be involved in regulation of the EGF receptor trafficking. To elucidate the interaction of annexin 1 during EGF receptor internalization, we followed the distribution of annexin 1-GFP fusion proteins at sites of internalizing EGF receptors. The observed association of annexin 1 with EGF receptors was confirmed by immunoprecipitation. We found that this interaction was independent of a functional phosphorylation site in the annexin 1 N-terminal domain but mediated through the Ca2+ binding core domain.

Annexin A1↗

PubMed2004

Annexin 2 is a phosphatidylinositol (4,5)-bisphosphate binding protein recruited to actin assembly sites at cellular membranes.

Annexin 2 is a Ca(2+)-regulated membrane protein and an F-actin-binding protein enriched at actin assembly sites both, on the plasma membrane and on endosomal vesicles. Here, we identify annexin 2 as a phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2))-interacting protein, thereby explaining this specific membrane association. Using the pleckstrin-homology (PH) domain of phospholipase Cdelta1 fused to yellow fluorescent protein as a marker for PtdIns(4,5)P(2), we show that annexin 2 and its ligand p11 (S100A10) are targeted to sites of PtdIns(4,5)P(2) enrichment where F-actin accumulates. At the plasma membrane, adhesion of pedestal-forming enteropathogenic Escherichia coli induces a recruitment of 1-phosphatidylinositol-4-phosphate 5-kinase (PtdIns4P 5-kinase) and an enrichment of PtdIns(4,5)P(2) and annexin 2-p11 at sites of bacterial adhesion. Induction of PtdIns(4,5)P(2)-enriched ruffles and PtdIns(4,5)P(2)-positive, actin-coated vacuoles by Arf6-mediated activation of PtdIns4P 5-kinase also leads to a concomitant accumulation of the annexin 2-p11 complex and the PH domain. Binding studies with immobilized phosphoinositides and phosphoinositide-containing liposomes reveal that the purified annexin 2-p11 complex directly and specifically binds to PtdIns(4,5)P(2) with an affinity comparable to that of the PH domain of phospholipase Cdelta1. Experiments using individual subunits identify annexin 2 as the PtdIns(4,5)P(2)-binding entity. Thus, the direct interaction of annexin 2 with PtdIns(4,5)P(2) is a means of specifically recruiting the annexin 2-p11 complex to sites of membrane-associated actin assembly.

ADP-Ribosylation Factor 6↗

PubMed2004

An annexin 1 N-terminal peptide activates leukocytes by triggering different members of the formyl peptide receptor family.

The human N-formyl peptide receptor (FPR) is a key modulator of chemotaxis directing granulocytes toward sites of bacterial infections. FPR is the founding member of a subfamily of G protein-coupled receptors thought to function in inflammatory processes. The other two members, FPR-like (FPRL)1 and FPRL2, have a greatly reduced affinity for bacterial peptides or do not bind them at all, with FPRL2 being considered an orphan receptor so far. In this study we show that a peptide derived from the N-terminal domain of the anti-inflammatory protein annexin 1 (lipocortin 1) can activate all three FPR family members at similar concentrations. The annexin 1 peptide initiates chemotactic responses in human monocytes that express all three FPR family members and also desensitizes the cells toward subsequent stimulation with bacterial peptide agonists. Experiments using HEK 293 cells stably expressing a single FPR family member reveal that all three receptors can be activated and desensitized by the N-terminal annexin 1 peptide. These observations identify the annexin 1 peptide as the first endogenous ligand of FPRL2 and indicate that annexin 1 participates in regulating leukocyte emigration into inflamed tissue by activating and desensitizing different receptors of the FPR family.

Annexin A1↗

PubMed2004

Annexins--unique membrane binding proteins with diverse functions.

Annexins are a well-known multigene family of Ca(2+)-regulated phospholipid-binding and membrane-binding proteins. Recent work employing annexin-knockdown or - knockout models has provided new insights into the biological functions of different annexin proteins. Transient annexin depletion by RNA interference and the expression of dominant-negative mutant proteins has revealed roles for the proteins in membrane processes ranging from the control of membrane structure to certain membrane transport phenomena. Although such functions correlate well with the ability of annexins to interact with cellular membranes in a reversible and regulated manner, some activities are membrane independent, probably because annexins can also engage in specific protein-protein interactions. Among other things, this is evident in annexin A1- and A2-knockout mice, which show impaired regulation of neutrophil extravasation and defects in plasmin generation, respectively.

Animals↗

PubMed2004

Annexin-actin interactions.

The actin cytoskeleton is a malleable framework of polymerised actin monomers that may be rapidly restructured to enable diverse cellular activities such as motility, endocytosis and cytokinesis. The regulation of actin dynamics involves the coordinated activity of numerous proteins, among which members of the annexin family of Ca2+- and phospholipid-binding proteins play an important role. Although the roles of annexins in actin dynamics are not understood at a mechanistic level, annexins have the requisite properties to integrate Ca2+-signaling with actin dynamics at membrane contact sites. In this review we discuss the current state of knowledge on this topic, and consider how and where annexins may fit into the complex molecular machinery that regulates the actin cytoskeleton.

Actins↗

PubMed2003

Annexin II is required for apical transport in polarized epithelial cells.

The sorting of apical proteins comprises an initial recognition step in the trans Golgi network and a final partitioning of the apical pool of proteins into at least two different types of vesicular carriers. One criteria of these carriers is the association or non-association of the protein content with lipid rafts. We have previously characterized a population containing the raft-associated sucrase-isomaltase-carrying vesicles (SAVs) and another one, the non-raft-associated lactase-phlorizin hydrolase-carrrying vesicles (LAVs) that are targeted separately to the apical membrane. Here, we demonstrate biochemically and by employing confocal laser microscopy that the annexin II-S100A10 complex is a component of SAVs and is absent from LAVs. The unequivocal role of annexin II in the apical targeting of SI is clearly demonstrated when down-regulation of this protein by annexin II-specific small interfering RNA drastically decreases the apical delivery of SI in the epithelial cell line Madin-Darby canine kidney. The annexin II-S100A10 complex plays therefore a crucial role in routing SAVs to the apical membrane of epithelial cells.

Animals↗

PubMed2003

The annexin 2/S100A10 complex controls the distribution of transferrin receptor-containing recycling endosomes.

The Ca2+- and lipid-binding protein annexin 2, which resides in a tight heterotetrameric complex with the S100 protein S100A10 (p11), has been implicated in the structural organization and dynamics of endosomal membranes. To elucidate the function of annexin 2 and S100A10 in endosome organization and trafficking, we used RNA-mediated interference to specifically suppress annexin 2 and S100A10 expression. Down-regulation of both proteins perturbed the distribution of transferrin receptor- and rab11-positive recycling endosomes but did not affect uptake into sorting endosomes. The phenotype was highly specific and could be rescued by reexpression of the N-terminal annexin 2 domain or S100A10 in annexin 2- or S100A10-depleted cells, respectively. Whole-mount immunoelectron microscopy of the aberrantly localized recycling endosomes in annexin 2/S100A10 down-regulated cells revealed extensively bent tubules and an increased number of endosome-associated clathrin-positive buds. Despite these morphological alterations, the kinetics of transferrin uptake and recycling was not affected to a significant extent, indicating that the proper positioning of recycling endosomes is not a rate-limiting step in transferrin recycling. The phenotype generated by this transient loss-of-protein approach shows for the first time that the annexin 2/S100A10 complex functions in the intracellular positioning of recycling endosomes and that both subunits are required for this activity.

Annexin A2↗

PubMed2003

Atypical properties displayed by annexin A9, a novel member of the annexin family of Ca(2+) and lipid binding proteins.

Annexin A9 is a novel member of the annexin family of Ca(2+) and phospholipid binding proteins which has so far only been identified in EST data bases and whose deduced protein sequence shows mutations in residues considered crucial for Ca(2+) coordination in other annexins. To elucidate whether the annexin A9 protein is expressed as such and to characterize its biochemical properties we probed cell extracts with specific anti-annexin A9 antibodies and developed a recombinant expression system. We show that the protein is found in HepG2 hepatoma cell lysates and that a green fluorescent protein-tagged form is abundantly expressed in the cytosol of HeLa cells. Recombinant expression in bacteria yields a soluble protein that can be enriched by conventional chromatographic procedures. The protein is capable of binding phosphatidylserine containing liposomes albeit only at Ca(2+) concentrations exceeding 2 mM. Moreover and in contrast to other annexins this binding appears to be irreversible as the liposome-bound annexin A9 cannot be released by Ca(2+) chelation. These results indicate that annexin A9 is a unique member of the annexin family whose intracellular activity is not subject to Ca(2+) regulation.

Annexins↗

PubMed2003

Functional expression of the epithelial Ca(2+) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex.

TRPV5 and TRPV6 constitute the Ca(2+) influx pathway in a variety of epithelial cells. Here, we identified S100A10 as the first auxiliary protein of these epithelial Ca(2+) channels using yeast two-hybrid and GST pull-down assays. This S100 protein forms a heterotetrameric complex with annexin 2 and associates specifically with the conserved sequence VATTV located in the C-terminal tail of TRPV5 and TRPV6. Of these five amino acids, the first threonine plays a crucial role since the corresponding mutants (TRPV5 T599A and TRPV6 T600A) exhibited a diminished capacity to bind S100A10, were redistributed to a subplasma membrane area and did not display channel activity. Using GST pull-down and co-immunoprecipitation assays we demonstrated that annexin 2 is part of the TRPV5-S100A10 complex. Furthermore, the S100A10-annexin 2 pair colocalizes with the Ca(2+) channels in TRPV5-expressing renal tubules and TRPV6-expressing duodenal cells. Importantly, downregulation of annexin 2 using annexin 2-specific small interfering RNA inhibited TRPV5 and TRPV6-mediated currents in transfected HEK293 cells. In conclusion, the S100A10-annexin 2 complex plays a crucial role in routing of TRPV5 and TRPV6 to plasma membrane.

Animals↗

PubMed2002

Functional activation of the formyl peptide receptor by a new endogenous ligand in human lung A549 cells.

The formyl peptide receptor (FPR), a heptahelical G protein-coupled receptor on phagocytic leukocytes, can be triggered by bacterially derived oligopeptides of the prototype fMLP. Although FPR expression and activation have been associated with cells of myeloid origin and bacterial inflammation, the receptor has recently been identified in nonmyeloid cells, thus suggesting additional physiological functions and the existence of an endogenous agonist. In this study, we demonstrate the presence and functional activation of the FPR in the human lung cell line A549, which represents an extrahepatic model for the regulation of acute-phase proteins. Activation of the FPR in A549 cells cannot only be triggered by fMLP, but also by an agonistic peptide of the recently identified endogenous FPR ligand, annexin 1. In addition to inducing changes in the F-actin content, annexin 1-mediated triggering of the FPR results in an increased expression of acute-phase proteins. Hence, activation of nonmyeloid FPR by its endogenous ligand annexin 1 could participate in the regulation of acute-phase responses, e.g., during inflammation and/or wound healing.

Actins↗

PubMed2002

Cell-surface attachment of pedestal-forming enteropathogenic E. coli induces a clustering of raft components and a recruitment of annexin 2.

Annexin 2 is a Ca2+-regulated membrane- and F-actin-binding protein implicated in the stabilization or regulation of membrane/cytoskeleton contacts, or both, at the plasma membrane and at early endosomal membranes. To analyze the dynamic nature of such action we investigated whether annexin 2 could be found at sites of localized actin rearrangements occurring at the plasma membrane of HeLa cells infected with noninvading enteropathogenic Escherichia coli (EPEC). We show that adherent EPEC microcolonies, which are known to induce the formation of actin-rich pedestals beneath them, specifically recruit annexin 2 to the sites of their attachment. Mutant EPEC (EPECtir), which lack a functional receptor for intimate attachment (Tir, translocated intimin receptor) and which fail to produce full pedestal formation, are still capable of recruiting annexin 2 to the bacterial contact sites. Accumulation of annexin 2 at sites of EPEC or EPECtir attachment is accompanied by a recruitment of the annexin 2 protein ligand S100A10. EPEC and EPECtir attachment also induces a concentration of cholesterol and glycosyl phosphatidylinositol-anchored proteins at sites of bacterial contact. This indicates that membrane components present in rafts or raft-like microdomains are clustered upon EPEC adherence and that annexin 2 is recruited to the cytoplasmic membrane surface of such clusters, possibly stabilizing raft patches and their linkage to the actin cytoskeleton beneath adhering EPEC.

Actins↗