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Weibel-Palade bodies (WPB) are secretory organelles of endothelial cells that undergo evoked exoyctosis following intracellular Ca2+ or cAMP elevation, thereby supplying the vasculature with factors controlling haemostasis. Several cytosolic and membrane-associated proteins, including the Rab family members Rab3, Rab15 and Rab27a, have been implicated in regulating the acute exocytosis of WPB. Here, we carried out a genome-wide screen to identify Rab pathways affecting WPB exocytosis. Overexpression of a specific subset of Rab GTPase-activating proteins (RabGAPs) inhibited histamine-evoked, Ca2+-dependent WPB exocytosis, presumably by inactivating the target RabGTPases. Among the inhibitory RabGAPs, we concentrated on TBC1D10A and showed that the inhibitory effect depends on its GAP activity. We confirmed Rab35 was a target Rab of TBC1D10A in human endothelial cells; Rab35 interacted with TBC1D10A, and expression of the GAP insensitive Rab35(Q67A) mutant rescued the inhibitory effect of TBC1D10A overexpression on WPB exocytosis. Furthermore, knockdown of Rab35 and expression of a dominant-negative Rab35 mutant both inhibited histamine-evoked secretion of the WPB cargos von-Willebrand factor (VWF) and P-selectin. Pulldown and co-immunoprecipitation experiments identified the ArfGAP with coiled-coil, Ank repeat and PH domain-containing protein ACAP2 as Rab35 effector in endothelial cells, and depletion as well as overexpression approaches revealed that ACAP2 acts as a negative regulator of WPB exocytosis. Interestingly, a known ACAP2 target, the small GTPase Arf6 supported histamine-evoked WPB exocytosis as shown by knockdown and overexpression of a dominant-negative Arf6 mutant. Our data identify Rab35 as a novel regulator of WPB exocytosis, most likely acting through the downstream effectors ACAP2 and Arf6.

The superfamily of G-protein-coupled receptors (GPCR) conveys signals in response to various endogenous and exogenous stimuli. Consequently, GPCRs are the most important drug targets. CCR10, the receptor for the chemokines CCL27/CTACK and CCL28/MEC, belongs to the chemokine receptor subfamily of GPCRs and is thought to function in immune responses and tumour progression. However, there is only limited information on the intracellular regulation of CCR10. We find that S100A10, a member of the S100 family of Ca2+ binding proteins, binds directly to the C-terminal cytoplasmic tail of CCR10 and that this interaction regulates the CCR10 cell surface presentation. This identifies S100A10 as a novel interaction partner and regulator of CCR10 that might serve as a target for therapeutic intervention.

Platelets have an essential role in primary haemostasis to arrest bleeding at a site of vessel wall disruption. Insufficient platelet activation or a reduced platelet number give rise to bleeding syndromes. On the other hand, high platelet activation contributes to thrombosis, which often starts with platelet deposition on a damaged atherosclerotic plaque. The formation of vaso-occlusive platelet thrombi or aggregates, usually with repeated embolization, is a major cause of arterial thrombosis, as occurring in the heart and brains, and resulting in myocardial infarction and so-called cerebrovascular accidents (stroke). Although there is little doubt that coronary and cerebral artery diseases are multifactorial disorders, where also abnormal coagulation, fibrinolysis, vessel wall function and blood flow dynamics can play a role, intervention studies show that in particular antiplatelet drugs provide a risk reduction of one out of every four cases.keywordsplatelet activationhuman plateletplatelet adhesioncalcium responsecollagen receptorthese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II-p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTPgammaS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.

The (annexin II-p11)(2) tetramer has been proposed to participate in exocytosis and several other members of the annexin superfamily have been reported to aggregate liposomes in vitro. In this context, the Ca2+-dependent binding of several annexins to chromaffin granules and liposomes was investigated by cryo-electron microscopy. The Ca2+-dependent aggregation of lipid membranes by (annexin II-p11)(2) results from the spontaneous self-organization of the protein into two-dimensional plaques, which are visualized in projection as characteristic junctions. The junctions have a constant thickness of 210(+/-10) Angstrom and present a symmetrical distribution of electron-dense material arranged into seven stripes. They were observed over a wide range of Ca2+ concentrations, down to 2 mu M. The molecular components corresponding to the seven electron-dense stripes were assigned as follows: the two associated membranes give rise to two outer stripes each and the three central stripes correspond to the (annexin II-p11)(2) tetramer. Each annexin II molecule interacts with the outer lipid leaflet of one membrane, giving rise to one stripe, while the central stripe is due to the (p11)(2) dimer with which both annexin II molecules interact. Both annexin II and annexin I also induced the Ca2+-dependent aggregation of liposomes via junctions that lack the central (p11)(2) moiety and present only six high-density stripes. As expected, both annexin V and annexin III bind to liposomes without inducing their aggregation. (C) 1997 Academic Press Limited.

Nanogram quantities of the beta-galactoside-specific lectin from mistletoe (ML-I) that is composed of two different types of subunits exhibit immunomodulatory potency and enhance cytokine secretion in vitro and in vivo. Partial sequence analysis of the carbohydrate-binding B chain revealed a ragged N-terminus and overall homologies to the B subunit of Ricin D and Ricin E. Two evolutionarily neutral substitutions were apparent in the otherwise identical N-terminal sequences of the two toxic chains within the lectin preparation. On the basis of the influence of chemical modification by group-specific reagents on ligand binding, the lectin was biotinylated with biotinyl-N-hydroxysuccinimide ester to allow monitoring of cell binding. Monocytic leukemia cells (THP-1) specifically bound the lectin with positive cooperativity at low lectin concentrations. Radiolabelled lectin could be found in several organs and in an experimental solid tumor in biodistribution in mice. Its presence in a notable amount in spleens is especially noteworthy with respect to the already reported immunomodulation. To determine intracellular responses that precede the lectin-dependent augmentation of cytokine secretion, phosphorylation of proteins and phospholipids as well as Ca(2+)-mobilization were assessed in THP-1 cells. Quantitative increases of [32P]-phosphate incorporation were determined for a 28 kDa protein and for phosphatidylinositol-4,5-biphosphate. Similarly, the fluorescence activity of the intracellular Ca(2+)-indicator fluo-3 is elevated by approximately 25% after lectin treatment. Apparently, cell binding of the lectin is followed by modulation of biosignalling processes.