scholarly journals RIAM-VASP Module Relays Integrin Complement Receptors in Outside-In Signaling Driving Particle Engulfment

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1166
Author(s):  
Alvaro Torres-Gomez ◽  
Jose Luis Sanchez-Trincado ◽  
Víctor Toribio ◽  
Raul Torres-Ruiz ◽  
Sandra Rodríguez-Perales ◽  
...  
Keyword(s):  
Cell Surface ◽  
Complement Receptors ◽  
Binding Partner ◽  
Downstream Signaling ◽  
Contact Sites ◽  
Phagocytic Cups ◽  
Cytoskeletal Rearrangements ◽  
Inside Out ◽  
Particle Internalization ◽  
Β2 Integrins

The phagocytic integrins and complement receptors αMβ2/CR3 and αXβ2/CR4 are classically associated with the phagocytosis of iC3b-opsonized particles. The activation of this receptor is dependent on signals derived from other receptors (inside-out signaling) with the crucial involvement of the Rap1-RIAM-Talin-1 pathway. Here, we analyze the implication of RIAM and its binding partner VASP in the signaling events occurring downstream of β2 integrins (outside-in) during complement-mediated phagocytosis. To this end, we used HL-60 promyelocytic cell lines deficient in RIAM or VASP or overexpressing EGFP-tagged VASP to determine VASP dynamics at phagocytic cups. Our results indicate that RIAM-deficient HL-60 cells presented impaired particle internalization and altered integrin downstream signaling during complement-dependent phagocytosis. Similarly, VASP deficiency completely blocked phagocytosis, while VASP overexpression increased the random movement of phagocytic particles at the cell surface, with reduced internalization. Moreover, the recruitment of VASP to particle contact sites, amount of pSer157-VASP and formation of actin-rich phagocytic cups were dependent on RIAM expression. Our results suggested that RIAM worked as a relay for integrin complement receptors in outside-in signaling, coordinating integrin activation and cytoskeletal rearrangements via its interaction with VASP.

scholarly journals Vav Family Proteins Couple to Diverse Cell Surface Receptors

2000 ◽  
Vol 20 (17) ◽  
pp. 6364-6373 ◽  
Author(s):  
Sheri L. Moores ◽  
Laura M. Selfors ◽  
Jessica Fredericks ◽  
Timo Breit ◽  
Keiko Fujikawa ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Receptor Activation ◽  
Cell Surface Receptors ◽  
Nucleotide Exchange ◽  
Surface Receptors ◽  
Downstream Signaling

ABSTRACT Vav proteins are guanine nucleotide exchange factors for Rho family GTPases which activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. Vav proteins contain several protein binding domains which can link cell surface receptors to downstream signaling proteins. Vav1 is expressed exclusively in hematopoietic cells and tyrosine phosphorylated in response to activation of multiple cell surface receptors. However, it is not known whether the recently identified isoforms Vav2 and Vav3, which are broadly expressed, can couple with similar classes of receptors, nor is it known whether all Vav isoforms possess identical functional activities. We expressed Vav1, Vav2, and Vav3 at equivalent levels to directly compare the responses of the Vav proteins to receptor activation. Although each Vav isoform was tyrosine phosphorylated upon activation of representative receptor tyrosine kinases, integrin, and lymphocyte antigen receptors, we found unique aspects of Vav protein coupling in each receptor pathway. Each Vav protein coprecipitated with activated epidermal growth factor and platelet-derived growth factor (PDGF) receptors, and multiple phosphorylated tyrosine residues on the PDGF receptor were able to mediate Vav2 tyrosine phosphorylation. Integrin-induced tyrosine phosphorylation of Vav proteins was not detected in nonhematopoietic cells unless the protein tyrosine kinase Syk was also expressed, suggesting that integrin activation of Vav proteins may be restricted to cell types that express particular tyrosine kinases. In addition, we found that Vav1, but not Vav2 or Vav3, can efficiently cooperate with T-cell receptor signaling to enhance NFAT-dependent transcription, while Vav1 and Vav3, but not Vav2, can enhance NFκB-dependent transcription. Thus, although each Vav isoform can respond to similar cell surface receptors, there are isoform-specific differences in their activation of downstream signaling pathways.

The effects of inhibition of protein glycosylation on the aggregation of Dictyostelium discoideum

Development ◽  
1983 ◽  
Vol 78 (1) ◽  
pp. 229-248
Author(s):  
Charles John McDonald ◽  
Jeffrey Sampson
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Total Protein ◽  
Subsequent Development ◽  
Protein Glycosylation ◽  
Normal Process ◽  
Contact Sites ◽  
Cyclic Amp Phosphodiesterase

At concentrations greater than 10 µg ml−1 tunicamycin inhibited the incorporation of [3H]mannose into glycoproteins during the early phase of development in Dictyostelium discoideum, however, total protein synthesis was unaffected. Tunicamycin also interfered with the normal process of aggregation. In its presence small aggregates were observed at the time of normal aggregation, but amoebae failed to aggregate completely and subsequent development was inhibited. Inhibition of normal aggregation by tunicamycin was found to be reversible. The appearance of cell-associated and secreted cyclic AMP phosphodiesterase and cell-surface contact sites A was prevented by tunicamycin but cell surface cyclic AMP receptor activity developed normally in its presence. Tunicamycin also prevented amoebae from acquiring the ability to chemotact toward cyclic AMP. Addition of exogenous cyclic AMP phosphodiesterase restored the ability of amoebae to chemotact toward cyclic AMP in the presence of tunicamycin. Our data suggest that the primary block in aggregation caused by tunicamycin results from the inhibition of expression of active cyclic AMP phosphodiesterase.

Abstract 497: Cholesterol and Toll-like Receptor Signaling Are Important Regulators of Macrophage Interactions with Atherosclerotic Lipoprotein Aggregates

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Harvey F Chin ◽  
Abigail Haka ◽  
Frederick R Maxfield
Keyword(s):  
Molecular Mechanisms ◽  
Atherosclerotic Plaques ◽  
Cholesteryl Esters ◽  
Cellular Mechanisms ◽  
Downstream Signaling ◽  
Cellular Processes ◽  
Atherosclerotic Plaque Formation ◽  
Subendothelial Space ◽  
Morphological Responses ◽  
Cytoskeletal Rearrangements

Macrophages encounter deposits of aggregated low-density lipoproteins (agLDL) in the subendothelial space of blood vessels during the first stages of atherosclerotic plaque formation. Notably, current models for the mechanism of macrophage internalization of cholesterol in early atherosclerotic plaques are incomplete due to the lack of attention paid to the unique cellular mechanisms that are required for macrophages to degrade aggregates of LDL in particular, which can comprise >90% of the LDL in atherosclerotic plaques. In fact, internalization of cholesterol from cholesteryl esters in agLDL involves the development of intriguing cellular processes in which extracellular acidic compartments, lysosomal synapses (LSs), are formed whereby agLDL is partially degraded prior to internalization. This process requires extensive cytoskeletal rearrangements and secretion of lysosomal enzymes responsible for hydrolysis of cholesteryl esters from the agLDL. Subsequent delivery of free cholesterol from agLDL to the macrophage plasma membrane is central for development of the LS. Nonetheless, the molecular mechanism underlying initiation and propagation of the LS are currently largely unknown. This research proposal aims to elucidate the molecular mechanisms of LS formation and the role that cholesterol plays in eliciting these morphological responses to agLDL. Fluorescence microscopy assays were used to identify activation of TLR4 and downstream signaling involving PI3K and Akt as important events leading to LS formation. Furthermore, morphological responses of macrophages to cholesterol overloading require overlapping signaling pathways, indicating the role of interplay of cholesterol and TLR4 signaling in development of this novel macrophage interaction with aggregated LDL found in plaques. Identification of specific molecular pathways involved in this process will not only contribute to the basic understanding of one of the primary cellular processes contributing to atherosclerosis, one of the primary causes of heart disease, but also provide tangible molecular targets for the ultimate development of therapies.

Neutrophil activation via β2 integrins (CD11/CD18): Molecular mechanisms and clinical implications

2007 ◽  
Vol 98 (08) ◽  
pp. 262-273 ◽  
Author(s):  
Jürgen Schymeinsky ◽  
Attila Mócsai ◽  
Barbara Walzog
Keyword(s):  
Receptor Tyrosine Kinase ◽  
Acute Inflammation ◽  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Polymorphonuclear Neutrophils ◽  
Downstream Signaling ◽  
Β2 Integrin ◽  
Pmn Functions ◽  
Integrin Family ◽  
Β2 Integrins

SummaryPolymorphonuclear neutrophils (PMN) are key components of the innate immunity and their efficient recruitment to the sites of lesion is a prerequisite for acute inflammation. Signaling via adhesion molecules of the β2 integrin family (CD11/CD18) plays an essential role for PMN recruitment and activation during inflammation. In this review, we will focus on the non-receptor tyrosine kinase Syk, an important downstream signaling component of β2 integrins that is required for the control of different PMN functions including adhesion,migration and phagocytosis. The exploration of β2 integrin-mediated Syk activation provided not only novel insights into the control of PMN functions but also led to the identification of Syk as a new molecular target for therapeutic intervention during inflammatory diseases.

scholarly journals Binding of Kingella kingae RtxA Toxin Depends on Cell Surface Oligosaccharides, but Not on β2 Integrins

2020 ◽  
Vol 21 (23) ◽  
pp. 9092
Author(s):  
Waheed Ur Rahman ◽  
Adriana Osickova ◽  
Nela Klimova ◽  
Jinery Lora ◽  
Nataliya Balashova ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Mammalian Species ◽  
Cell Types ◽  
Surface Structures ◽  
Target Cells ◽  
Kingella Kingae ◽  
Toxin Binding ◽  
Cell Surface Structures ◽  
Β2 Integrins

The Gram-negative coccobacillus Kingella kingae is increasingly recognized as an important invasive pediatric pathogen that causes mostly bacteremia and skeletal system infections. K. kingae secretes an RtxA toxin that belongs to a broad family of the RTX (Repeats in ToXin) cytotoxins produced by bacterial pathogens. Recently, we demonstrated that membrane cholesterol facilitates interaction of RtxA with target cells, but other cell surface structures potentially involved in toxin binding to cells remain unknown. We show that deglycosylation of cell surface structures by glycosidase treatment, or inhibition of protein N- and O-glycosylation by chemical inhibitors substantially reduces RtxA binding to target cells. Consequently, the deglycosylated cells were more resistant to cytotoxic activity of RtxA. Moreover, experiments on cells expressing or lacking cell surface integrins of the β2 family revealed that, unlike some other cytotoxins of the RTX family, K. kingae RtxA does not bind target cells via the β2 integrins. Our results, hence, show that RtxA binds cell surface oligosaccharides present on all mammalian cells but not the leukocyte-restricted β2 integrins. This explains the previously observed interaction of the toxin with a broad range of cell types of various mammalian species and reveals that RtxA belongs to the group of broadly cytolytic RTX hemolysins.

scholarly journals CD44-mediated phagocytosis induces inside-out activation of complement receptor-3 in murine macrophages

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4492-4502 ◽  
Author(s):  
Eric Vachon ◽  
Raiza Martin ◽  
Vivian Kwok ◽  
Vera Cherepanov ◽  
Chung-Wai Chow ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Immunofluorescence Microscopy ◽  
Complement Receptor ◽  
Fcγ Receptors ◽  
Intracellular Signaling Pathways ◽  
Large Particles ◽  
Complement Receptor 3 ◽  
Inside Out ◽  
Β2 Integrins

Diverse receptors, including Fcγ receptors and β2 integrins (complement receptor-3 [CR3], CD11b/CD18), have been implicated in phagocytosis, but their distinct roles and interactions with other receptors in particle engulfment are not well defined. CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, may have additional functions in regulation of inflammation and phagocytosis. We have recently reported that CD44 is a fully competent phagocytic receptor that is able to trigger ingestion of large particles by macrophages. Here, we investigated the role of coreceptors and intracellular signaling pathways in modulation of CD44-mediated phagocytosis. Using biotinylated erythrocytes coated with specific antibodies (anti-CD44–coated erythrocytes [Ebabs]) as the phagocytic prey, we determined that CD44-mediated phagocytosis is reduced by 45% by a blocking CD11b antibody. Further, CD44-mediated phagocytosis was substantially (42%) reduced in CD18-null mice. Immunofluorescence microscopy revealed that CD11b is recruited to the phagocytic cup. The mechanism of integrin activation and mobilization involved activation of the GTPase Rap1. CD44-mediated phagocytosis was also sensitive to the extracellular concentration of the divalent cation Mg2+ but not Ca2+. In addition, buffering of intracellular Ca2+ did not affect CD44-mediated phagocytosis. Taken together, these data suggest that CD44 stimulation induces inside-out activation of CR3 through the GTPase Rap1.

scholarly journals Internalization of B Cell Receptors in Human EU12μHC+Immature B Cells Specifically Alters Downstream Signaling Events

2013 ◽  
Vol 2013 ◽  
pp. 1-9
Author(s):  
Jing Liu ◽  
Wanqin Xie ◽  
Miles D. Lange ◽  
Sang Yong Hong ◽  
Kaihong Su ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Surface ◽  
B Cell ◽  
Cell Fate ◽  
Cell Activation ◽  
Underlying Mechanism ◽  
Cell Fate Decisions ◽  
Downstream Signaling ◽  
Signaling Events ◽  
Immature B Cells

It has been recognized for a long time that engagement of B cell antigen receptors (BCRs) on immature B cells or mature B cells leads to completely opposite cell fate decisions. The underlying mechanism remains unclear. Here, we show that crosslinking of BCRs on human EU12μHC+immature B cells resulted in complete internalization of cell surface BCRs. After loss of cell surface BCRs, restimulation of EU12μHC+cells showed impaired Ca2+flux, delayed SYK phosphorylation, and decreased CD19 and FOXO1 phosphorylation, which differ from those in mature Daudi or Ramos B cells with partial internalization of BCRs. In contrast, sustained phosphorylation and reactivation of ERK upon restimulation were observed in the EU12μHC+cells after BCR internalization. Taken together, these results show that complete internalization of cell surface BCRs in EU12μHC+cells specifically alters the downstream signaling events, which may favor receptor editing versus cell activation.

scholarly journals RacF1, a Novel Member of the Rho Protein Family inDictyostelium discoideum, Associates Transiently with Cell Contact Areas, Macropinosomes, and Phagosomes

1999 ◽  
Vol 10 (4) ◽  
pp. 1205-1219 ◽  
Author(s):  
Francisco Rivero ◽  
Richard Albrecht ◽  
Heidrun Dislich ◽  
Enrico Bracco ◽  
Laura Graciotti ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Confocal Laser Scanning Microscope ◽  
Confocal Laser ◽  
Cell Contact ◽  
Rho Family ◽  
Phagocytic Cups ◽  
Dynamic Structures ◽  
Green Fluorescent ◽  
Cytoskeletal Rearrangements

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. TheracF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyosteliumlife cycle. Highest identity (94%) was found to a RacF2 isoform, toDictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64–69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP–RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP–RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP–RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncatedracF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.

scholarly journals Tissue Transglutaminase Is an Integrin-Binding Adhesion Coreceptor for Fibronectin

2000 ◽  
Vol 148 (4) ◽  
pp. 825-838 ◽  
Author(s):  
Sergey S. Akimov ◽  
Dmitry Krylov ◽  
Laurie F. Fleischman ◽  
Alexey M. Belkin
Keyword(s):  
Cell Surface ◽  
Focal Adhesion ◽  
Factor Xiii ◽  
Tissue Transglutaminase ◽  
Focal Adhesions ◽  
Cross Linking ◽  
Binding Motifs ◽  
High Affinity ◽  
Β2 Integrins

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of β1 and β3 subfamilies, but not with β2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

P-selectin binds to the D′-D3 domains of von Willebrand factor in Weibel-Palade bodies

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3922-3924 ◽  
Author(s):  
Grégoire Michaux ◽  
Timothy J. Pullen ◽  
Sandra L. Haberichter ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Membrane Protein ◽  
Cell Surface ◽  
Von Willebrand Factor ◽  
Integral Membrane Protein ◽  
Hek293 Cells ◽  
Binding Partner ◽  
Lumenal Domain ◽  
Von Willebrand ◽  
Willebrand Factor

It has recently been shown that the ultralarge platelet–recruiting von Willebrand factor (VWF) strings formed immediately at exocytosis from endothelial cells may be anchored to the cell surface by interaction with the integral membrane protein P-selectin. This finding of a new binding partner for VWF immediately prompts the question which domains of VWF bind to P-selectin. We have exploited the fact that VWF expression in HEK293 cells triggers the formation of Weibel-Palade body–like structures that can recruit P-selectin. A suitably modified version of this assay using coexpressed truncations of VWF, together with P-selectin variants in HEK293 cells, allowed us to determine which domains of VWF would recruit P-selectin within a physiologically appropriate intracellular environment. Confirming the results of such a cellular assay by conventional coimmunoprecipitation, we concluded that the lumenal domain of P-selectin interacts with the D′-D3 domains of VWF.

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