Munc13-1 Promotes Secretory Granule Exocytosis Through Two Separate Calcium-Dependent Mechanisms

We screened the National Institutes of Health’s Molecular Libraries Small Molecule Repository for inhibitors of cytotoxic T lymphocyte (CTL) lytic granule exocytosis by measuring binding of an antibody in the extracellular solution to a lysosomal membrane protein (LAMP-1) that is transferred to the plasma membrane by exocytosis. We used TALL-104 human leukemic CTLs stimulated with soluble chemicals. Using high-throughput cluster cytometry to screen 364,202 compounds in a 1536-well plate format, we identified 2404 initial hits: 161 were confirmed on retesting, and dose–response measurements were performed. Seventy-five of those compounds were obtained, and 48 were confirmed active. Experiments were conducted to determine the molecular mechanism of action (MMOA) of the active compounds. Fifteen blocked increases in intracellular calcium >50%. Seven blocked phosphorylation of extracellular signal-regulated kinase (ERK) by upstream mitogen-activated protein kinase kinases >50%. One completely blocked the activity of the calcium-dependent phosphatase calcineurin. None blocked ERK catalytic activity. Eight blocked more than one pathway. For 8 compounds, we were unable to determine an MMOA. The activity of 1 of these compounds was confirmed from powder resupply. We conclude that a screen based on antibody binding to CTLs is a good means of identifying novel candidate immunosuppressants with either known or unknown MMOAs.

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‘Neurosecretion’ by aminergic synaptic terminals In vivo—a study of secretory granule exocytosis in the corpus cardiacum of the flying locust

Neuroscience ◽

10.1016/0306-4522(87)92989-7 ◽

1987 ◽

Vol 22 (3) ◽

pp. 1145-1149 ◽

Cited By ~ 19

Author(s):

D.V. Pow ◽

D.W. Golding

Keyword(s):

Secretory Granule ◽

Corpus Cardiacum ◽

Granule Exocytosis ◽

Synaptic Terminals

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Melanophilin accelerates insulin granule fusion without predocking to the plasma membrane

10.2337/figshare.12990773.v1 ◽

2020 ◽

Author(s):

Ada Admin ◽

Hao Wang ◽

Kouichi Mizuno ◽

Noriko Takahashi ◽

Eri Kobayashi ◽

...

Keyword(s):

Plasma Membrane ◽

Secretory Granule ◽

Β Cells ◽

Pancreatic Β Cells ◽

Granule Exocytosis ◽

Synaptic Vesicle Exocytosis ◽

Granule Membrane ◽

Myosin Va ◽

Vesicle Exocytosis ◽

Glucose Stimulated Insulin Secretion

Direct observation of fluorescence-labeled secretory granule exocytosis in living pancreatic β cells has revealed heterogeneous prefusion behaviors: some granules dwell beneath the plasma membrane before fusion, while others fuse immediately once they are recruited to the plasma membrane. Although the former mode seems to follow sequential docking-priming-fusion steps as found in synaptic vesicle exocytosis, the latter mode, which is unique to secretory granule exocytosis, has not been explored well. Here, we show that melanophilin, one of the effectors of the monomeric GTPase Rab27 on the granule membrane, is involved in such an accelerated mode of exocytosis. Both melanophilin-mutated leaden mouse and melanophilin-downregulated human pancreatic β cells exhibit impaired glucose-stimulated insulin secretion, with a specific reduction in fusion events that bypass stable docking to the plasma membrane. Upon stimulus-induced [Ca2+]i rise, melanophilin mediates this type of fusion by dissociating granules from myosin-Va and actin in the actin cortex and by associating them with a fusion-competent, open form of syntaxin-4 on the plasma membrane. These findings provide the hitherto unknown mechanism to support sustainable exocytosis by which granules are recruited from the cell interior and fuse promptly without stable predocking to the plasma membrane.

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Secretory Granule Exocytosis

Physiological Reviews ◽

10.1152/physrev.00031.2002 ◽

2003 ◽

Vol 83 (2) ◽

pp. 581-632 ◽

Cited By ~ 562

Author(s):

Robert D. Burgoyne ◽

Alan Morgan

Keyword(s):

Secretory Granule ◽

Secretory Granules ◽

Physiological Role ◽

Cell Types ◽

Control Mechanisms ◽

Granule Exocytosis ◽

Synaptic Vesicle Exocytosis ◽

Wide Range ◽

Vesicle Exocytosis ◽

Protein Components

Regulated exocytosis of secretory granules or dense-core granules has been examined in many well-characterized cell types including neurons, neuroendocrine, endocrine, exocrine, and hemopoietic cells and also in other less well-studied cell types. Secretory granule exocytosis occurs through mechanisms with many aspects in common with synaptic vesicle exocytosis and most likely uses the same basic protein components. Despite the widespread expression and conservation of a core exocytotic machinery, many variations occur in the control of secretory granule exocytosis that are related to the specialized physiological role of particular cell types. In this review we describe the wide range of cell types in which regulated secretory granule exocytosis occurs and assess the evidence for the expression of the conserved fusion machinery in these cells. The signals that trigger and regulate exocytosis are reviewed. Aspects of the control of exocytosis that are specific for secretory granules compared with synaptic vesicles or for particular cell types are described and compared to define the range of accessory control mechanisms that exert their effects on the core exocytotic machinery.

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Calcium-dependent exocytosis in an in vitro secretory granule plasma membrane preparation from sea urchin eggs and the effects of some inhibitors of cytoskeletal function

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1983.0047 ◽

1983 ◽

Vol 218 (1213) ◽

pp. 397-413 ◽

Cited By ~ 55

Keyword(s):

Plasma Membrane ◽

Secretory Granule ◽

Regulatory Protein ◽

Calcium Sensitivity ◽

Magnesium Concentration ◽

Cortical Granule ◽

Cortical Granules ◽

Calcium Dependent ◽

Cortical Granule Exocytosis

Egg cortical granules remain attached to the egg plasma membrane when the egg is ruptured. We present evidence that demonstrates that, when the cytoplasmic face of the egg plasma membrane is exposed to micromolar calcium concentrations, an exocytosis of the cortical granules occurs which corresponds to the cortical granule exocytosis seen when the egg is fertilized. The calcium sensitivity of the preparation is decreased by an increase in magnesium concentration and increased by a decrease in magnesium concentration. Exocytosis is inhibited by trifluoperazine (half inhibition at 6 μm), a drug that inhibits the action of the calciumdependent regulatory protein calmodulin. Colchicine, vinblastine, nocodazole, cytochalasin B, phalloidin N -ethylmaleimide-modified myosin subfragment 1, and antibody to actin are without effect on this in vitro exocytosis at concentrations that far exceed those required to disrupt microtubules and microfilaments. Conditions are such that penetration to the exocytotic site is optimal. It is unlikely, therefore, that either actin or tubulin participate intimately in exocytosis. Our data also exclude on quantitative grounds several other mechanisms postulated to account for the fusion of the secretory granule with the plasma membrane.

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Calcium-dependent Protein Kinase Reactions Associated with Parotid Gland Secretory Granule Membranes

Journal of Dental Research ◽

10.1177/00220345870660022901 ◽

1987 ◽

Vol 66 (2) ◽

pp. 557-563 ◽

Cited By ~ 8

Author(s):

F. Dowd ◽

E.L. Watson ◽

Y.-S. Lau ◽

J. Justin ◽

J. Pasieniuk ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Secretory Granule ◽

Dependent Protein Kinase ◽

Calcium Dependent ◽

Kinase C ◽

Protein Kinase C Activity ◽

Granule Membrane ◽

Dependent Protein ◽

Calcium Dependent Protein Kinase

Rat parotid secretory granule membranes were examined for the presence of calcium-dependent protein kinase activities and kinase substrates. Protein kinase C (C-kinase), which is stimulated by certain phospholipids, was present in the membranes, as indicated by its ability to catalyze the phosphorylation of histone. Two substrates for protein kinase C were seen in the granule membranes. The cytosolic fraction from the cell contained kinase activity, which was stimulated by phosphatidylserine and which caused the phosphorylation of two granule membrane polypeptides. In addition, when both granule membranes and cytosol were incubated together, phosphorylation of the cytosolic substrates was inhibited, indicating that the granule membrane substrates were phosphorylated preferentially. The results indicate that the granule membranes may react with cytosolic protein kinase C activity in a way which would direct an intracellular calcium and diacylglycerol signal toward the granule membrane. Since these signals occur during stimulation by various agonists, the mechanism may contribute to secretion.

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