scholarly journals Exophilin4/Slp2-a Targets Glucagon Granules to the Plasma Membrane through Unique Ca2+-inhibitory Phospholipid-binding Activity of the C2A Domain

2007 ◽  
Vol 18 (2) ◽  
pp. 688-696 ◽  
Author(s):  
Miao Yu ◽  
Kazuo Kasai ◽  
Kazuaki Nagashima ◽  
Seiji Torii ◽  
Hiromi Yokota-Hashimoto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Cell Types ◽  
Binding Activity ◽  
Effector Proteins ◽  
Membrane Phospholipids ◽  
Β Cells ◽  
Phospholipid Binding ◽  
Lysosome Related Organelles ◽  
C2a Domain

Rab27a and Rab27b have recently been recognized to play versatile roles in regulating the exocytosis of secretory granules and lysosome-related organelles by using multiple effector proteins. However, the precise roles of these effector proteins in particular cell types largely remain uncharacterized, except for those in pancreatic β cells and in melanocytes. Here, we showed that one of the Rab27a/b effectors, exophilin4/Slp2-a, is specifically expressed in pancreatic α cells, in contrast to another effector, granuphilin, in β cells. Like granuphilin toward insulin granules, exophilin4 promotes the targeting of glucagon granules to the plasma membrane. Although the interaction of granuphilin with syntaxin-1a is critical for the targeting activity, exophilin4 does this primarily through the affinity of its C2A domain toward the plasma membrane phospholipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Notably, the binding activity to phosphatidylserine is inhibited by a physiological range of the Ca2+ concentration attained after secretagogue stimulation, which presents a striking contrast to the Ca2+-stimulatory activity of the C2A domain of synaptotagmin I. Analyses of the mutant suggested that this novel Ca2+-inhibitory phospholipid-binding activity not only mediates docking but also modulates the subsequent fusion of the secretory granules.

scholarly journals The Rab27a effector exophilin7 promotes fusion of secretory granules that have not been docked to the plasma membrane

2013 ◽  
Vol 24 (3) ◽  
pp. 319-330 ◽  
Author(s):  
Hao Wang ◽  
Ray Ishizaki ◽  
Jun Xu ◽  
Kazuo Kasai ◽  
Eri Kobayashi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Knockout Mice ◽  
Secretory Granules ◽  
Small Gtpase ◽  
Membrane Phospholipids ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Insulin Granules ◽  
Insulin Exocytosis

Granuphilin, an effector of the small GTPase Rab27a, mediates the stable attachment (docking) of insulin granules to the plasma membrane and inhibits subsequent fusion of docked granules, possibly through interaction with a fusion-inhibitory Munc18-1/syntaxin complex. However, phenotypes of insulin exocytosis differ considerably between Rab27a- and granuphilin-deficient pancreatic β cells, suggesting that other Rab27a effectors function in those cells. We found that one of the putative Rab27a effector family proteins, exophilin7/JFC1/Slp1, is expressed in β cells; however, unlike granuphilin, exophilin7 overexpressed in the β-cell line MIN6 failed to show granule-docking or fusion-inhibitory activity. Furthermore, exophilin7 has no affinities to either Munc18-1 or Munc18-1–interacting syntaxin-1a, in contrast to granuphilin. Although β cells of exophilin7-knockout mice show no apparent abnormalities in intracellular distribution or in ordinary glucose-induced exocytosis of insulin granules, they do show impaired fusion in response to some stronger stimuli, specifically from granules that have not been docked to the plasma membrane. Exophilin7 appears to mediate the fusion of undocked granules through the affinity of its C2A domain toward the plasma membrane phospholipids. These findings indicate that the two Rab27a effectors, granuphilin and exophilin7, differentially regulate the exocytosis of either stably or minimally docked granules, respectively.

Transferrin receptor hyperexpression in primary erythroblasts is lost on transformation by avian erythroblastosis virus

Blood ◽  
2002 ◽  
Vol 100 (1) ◽  
pp. 289-298 ◽  
Author(s):  
Lioba Lobmayr ◽  
Thomas Sauer ◽  
Iris Killisch ◽  
Matthias Schranzhofer ◽  
Robert B. Wilson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Messenger Rna ◽  
Large Fraction ◽  
Regulatory Protein ◽  
Regulatory System ◽  
Cell Types ◽  
Binding Activity ◽  
Chicken Cell ◽  
Avian Erythroblastosis Virus

Abstract In primary chicken erythroblasts (stem cell factor [SCF] erythroblasts), transferrin receptor (TfR) messenger RNA (mRNA) and protein were hyperexpressed as compared to nonerythroid chicken cell types. This erythroid-specific hyperexpression was abolished in transformed erythroblasts (HD3E22 cells) expressing the v-ErbA and v-ErbB oncogenes of avian erythroblastosis virus. TfR expression in HD3E22 cells could be modulated by changes in exogenous iron supply, whereas expression in SCF erythroblasts was not subject to iron regulation. Measurements of TfR mRNA half-life indicated that hyperexpression in SCF erythroblasts was due to a massive stabilization of transcripts even in the presence of high iron levels. Changes in mRNA binding activity of iron regulatory protein 1 (IRP1), the primary regulator of TfR mRNA stability in these cells, correlated well with TfR mRNA expression; IRP1 activity in HD3E22 cells and other nonerythroid cell types tested was iron dependent, whereas IRP1 activity in primary SCF erythroblasts could not be modulated by iron administration. Analysis of avian erythroblasts expressing v-ErbA alone indicated that v-ErbA was responsible for these transformation-specific alterations in the regulation of iron metabolism. In SCF erythroblasts high amounts of TfR were detected on the plasma membrane, but a large fraction was also located in early and late endosomal compartments, potentially concealing temporary iron stores from the IRP regulatory system. In contrast, TfR was almost exclusively located to the plasma membrane in HD3E22 cells. In summary, stabilization of TfR mRNA and redistribution of Fe-Tf/TfR complexes to late endosomal compartments may contribute to TfR hyperexpression in primary erythroblasts, effects that are lost on leukemic transformation.

scholarly journals Plasma membrane electron transport in pancreatic β-cells is mediated in part by NQO1

2011 ◽  
Vol 301 (1) ◽  
pp. E113-E121 ◽  
Author(s):  
Joshua P. Gray ◽  
Timothy Eisen ◽  
Gary W. Cline ◽  
Peter J. S. Smith ◽  
Emma Heart
Keyword(s):  
Plasma Membrane ◽  
Electron Transport ◽  
Potassium Cyanide ◽  
Cell Types ◽  
Glycolytic Flux ◽  
Dependent Manner ◽  
Β Cells ◽  
Quinone Oxidoreductase ◽  
Plasma Membrane Electron Transport ◽  
Diphenylene Iodonium

Plasma membrane electron transport (PMET), a cytosolic/plasma membrane analog of mitochondrial electron transport, is a ubiquitous system of cytosolic and plasma membrane oxidoreductases that oxidizes cytosolic NADH and NADPH and passes electrons to extracellular targets. While PMET has been shown to play an important role in a variety of cell types, no studies exist to evaluate its function in insulin-secreting cells. Here we demonstrate the presence of robust PMET activity in primary islets and clonal β-cells, as assessed by the reduction of the plasma membrane-impermeable dyes WST-1 and ferricyanide. Because the degree of metabolic function of β-cells (reflected by the level of insulin output) increases in a glucose-dependent manner between 4 and 10 mM glucose, PMET was evaluated under these conditions. PMET activity was present at 4 mM glucose and was further stimulated at 10 mM glucose. PMET activity at 10 mM glucose was inhibited by the application of the flavoprotein inhibitor diphenylene iodonium and various antioxidants. Overexpression of cytosolic NAD(P)H-quinone oxidoreductase (NQO1) increased PMET activity in the presence of 10 mM glucose while inhibition of NQO1 by its inhibitor dicoumarol abolished this activity. Mitochondrial inhibitors rotenone, antimycin A, and potassium cyanide elevated PMET activity. Regardless of glucose levels, PMET activity was greatly enhanced by the application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle. We propose a model for the role of PMET as a regulator of glycolytic flux and an important component of the metabolic machinery in β-cells.

scholarly journals Secretory-granule dynamics visualized in vivo with a phogrin–green fluorescent protein chimaera

1998 ◽  
Vol 333 (1) ◽  
pp. 193-199 ◽  
Author(s):  
Aristea E. POULI ◽  
Evaggelia EMMANOUILIDOU ◽  
Chao ZHAO ◽  
Christina WASMEIER ◽  
John C. HUTTON ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Pc12 Cells ◽  
Secretory Granule ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Dense Core ◽  
Β Cells ◽  
Green Fluorescent ◽  
Insulinoma Cells

To image the behaviour in real time of single secretory granules in neuroendocrine cells we have expressed cDNA encoding a fusion construct between the dense-core secretory-granule-membrane glycoprotein, phogrin (phosphatase on the granule of insulinoma cells), and enhanced green fluorescent protein (EGFP). Expressed in INS-1 β-cells and pheochromocytoma PC12 cells, the chimaera was localized efficiently (up to 95%) to dense-core secretory granules (diameter 200–1000 nm), identified by co-immunolocalization with anti-(pro-)insulin antibodies in INS-1 cells and dopamine β-hydroxylase in PC12 cells. Using laser-scanning confocal microscopy and digital image analysis, we have used this chimaera to monitor the effects of secretagogues on the dynamics of secretory granules in single living cells. In unstimulated INS-1 β-cells, granule movement was confined to oscillatory movement (dithering) with period of oscillation 5–10 s and mean displacement < 1 µm. Both elevated glucose concentrations (30 mM), and depolarization of the plasma membrane with K+, provoked large (5–10 µm) saltatory excursions of granules across the cell, which were never observed in cells maintained at low glucose concentration. By contrast, long excursions of granules occurred in PC12 cells without stimulation, and occurred predominantly from the cell body towards the cell periphery and neurite extensions. Purinergic-receptor activation with ATP provoked granule movement towards the membrane of PC12 cells, resulting in the transfer of fluorescence to the plasma membrane consistent with fusion of the granule and diffusion of the chimaera in the plasma membrane. These results illustrate the potential use of phogrin–EGFP chimeras in the study of secretory-granule dynamics, the regulation of granule–cytoskeletal interactions and the trafficking of a granule-specific transmembrane protein during the cycle of exocytosis and endocytosis.

scholarly journals Regulation by bivalent cations of phospholipid binding to the C2A domain of synaptotagmin III

1997 ◽  
Vol 323 (2) ◽  
pp. 421-425 ◽  
Author(s):  
Mitsunori FUKUDA ◽  
Toshio KOJIMA ◽  
Katsuhiko MIKOSHIBA
Keyword(s):  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Binding Proteins ◽  
Binding Activity ◽  
Binding Property ◽  
Dependent Manner ◽  
Phospholipid Binding ◽  
Bivalent Cations ◽  
Dependent Interaction ◽  
C2a Domain

Synaptotagmins are Ca2+-and phospholipid-binding proteins of synaptic vesicles that might function as Ca2+ receptors for neurotransmitter release via their first C2 (C2A) domain. Here we describe the effect of Mg2+ on phospholipid binding to the C2A domains of multiple synaptotagmins (II–VI), and demonstrate that only synaptotagmin III can bind negatively charged phospholipids [phosphatidylserine (PS) and phosphatidylinositol] in a Mg2+-dependent manner. The Mg2+-dependent interaction with PS was found to have an EC50 of approx. 30 μM Mg2+, which is comparable to that of Sr2+ and Ba2+ (EC50 values of approx. 10 μM). This binding property of the C2A domain is specific to synaptotagmin III, because none of the C2A domains of other proteins, such as rabphilin 3A, Doc2α, Doc2β or Gap1m, showed phospholipid binding activity in the presence of 1 mM Mg2+. Our results suggest that synaptotagmin III is involved in presynaptic functions different from those of synaptotagmins I and II.

scholarly journals Mutation-deletion analysis of a Ca2+-dependent phospholipid binding (CaLB) domain within p120 GAP, a GTPase-activating protein for p21 ras

1995 ◽  
Vol 307 (2) ◽  
pp. 487-491 ◽  
Author(s):  
D J Gawler ◽  
L J W Zhang ◽  
M F Moran
Keyword(s):  
Amino Acids ◽  
Sequence Similarity ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Binding Activity ◽  
Membrane Phospholipids ◽  
Gtpase Activating Protein ◽  
Phospholipid Binding ◽  
P21 Ras

p120 GAP is a GTPase activating protein for p21 ras. It is a multidomain protein which exhibits sequence similarity with other GTPase-activating proteins, src, pleckstrin and a central portion of the protein kinase C conserved region 2 domain known as CaLB (Ca(2+)-dependent phospholipid-binding). The presence of this CaLB motif has led to the speculation that p120 GAP may be a member of a family of structurally related proteins containing a Ca(2+)-dependent membrane/lipid-binding domain. Here we have studied the in vitro Ca(2+)-dependent phospholipid-binding properties of the isolated proposed CaLB sequence in human GAP and deduce that a phospholipid-binding sequence is indeed located between amino acids 606 and 648. Binding of phosphatidylserine and phosphatidylinositol, but not phosphatidylcholine, within this sequence is Ca(2+)-dependent, with an estimated EC50 for Ca2+ of approx. 1 microM. Using deletion-mutation analysis we have further defined the minimal boundaries for this in vitro phospholipid-binding activity. p120 GAP amino acids 612-643 exhibit full phospholipid-binding activity, but further deletion of either amino acids 612-617 or amino acids 633-648 significantly decreased or abolished phospholipid binding. These studies establish that amino acids 612-643 of p120 GAP indeed constitute a functional CaLB domain and thereby imply a role for Ca2+ in the regulation of p120 GAP association with cellular (membrane) phospholipids.

scholarly journals SNAP-25 is expressed in islets of Langerhans and is involved in insulin release.

1995 ◽  
Vol 128 (6) ◽  
pp. 1019-1028 ◽  
Author(s):  
K Sadoul ◽  
J Lang ◽  
C Montecucco ◽  
U Weller ◽  
R Regazzi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
B Cell ◽  
Insulin Release ◽  
Islet Cells ◽  
Secretory Granules ◽  
Cell Types ◽  
Pancreatic B Cell ◽  
Pancreatic Endocrine Cells ◽  
Presynaptic Plasma Membrane

SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP-25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.

scholarly journals A unique combination of plasma membrane Ca2+-ATPase isoforms is expressed in islets of Langerhans and pancreatic β-cell lines

1996 ◽  
Vol 314 (2) ◽  
pp. 663-669 ◽  
Author(s):  
Anikó VÁRADI ◽  
Elek MOLNÁR ◽  
Stephen J. H. ASHCROFT
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Islets Of Langerhans ◽  
Cell Lines ◽  
Cell Types ◽  
Human Islets ◽  
Unique Combination ◽  
Β Cells ◽  
Β Cell ◽  
Calmodulin Binding

Changes in free intracellular Ca2+ concentration regulate insulin secretion from pancreatic β-cells. The existence of steep Ca2+ gradients within the β-cell requires the presence of specialized Ca2+ exclusion systems. In this study we have characterized the plasma membrane Ca2+-ATPases (PMCAs) which extrude Ca2+ from the cytoplasm. PMCA isoform- and subtype-specific mRNA expression was investigated in rodent pancreatic α- and β-cell lines, and in human and rat islets of Langerhans using reverse-transcription PCR with primers flanking the calmodulin-binding region of rat PMCA. The expression pattern of PMCA 1 and 2 was conserved in different species and islet-cell types since both rat and human islets of Langerhans and all cell lines tested contained the 1b and 2b forms. PMCA 4 isoform subtypes, however, were expressed in a cell-type-specific manner since β-cells expressed PMCA 4b only, whereas in islets of Langerhans, which contain α, β, δ and polypeptide-secreting cells, PMCA 4a and 4b were simultaneously present. No evidence was obtained for the expression of PMCA 3. Characterization of the β-cell Ca2+-pump protein showed that it shared several similarities with the erythrocyte PMCA. It is a P-type ATPase; its phosphorylated intermediate was stabilized by La3+; it reacted with a PMCA-specific antibody; and it was not N-glycosylated. However, the β-cell PMCA had a higher molecular mass than that of the erythrocyte; this difference could be explained by either predominant translation of the PMCA 2 form, which has a molecular mass 3–8 kDa higher than the erythrocyte PMCA 1 and 4 proteins, or by a possible sequence insertion. Thus a unique combination of functionally distinct PMCA isoforms (1b, 2b, 4b) participates in Ca2+ homoeostasis in the β-cell.

scholarly journals TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic β-cells: different behaviour of granule motion between normal and Goto–Kakizaki diabetic rat β-cells

2004 ◽  
Vol 381 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Mica OHARA-IMAIZUMI ◽  
Chiyono NISHIWAKI ◽  
Toshiteru KIKUTA ◽  
Shintaro NAGAI ◽  
Yoko NAKAMICHI ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Release ◽  
Secretory Granules ◽  
Dynamic Change ◽  
Second Phase ◽  
Β Cells ◽  
Gk Rat ◽  
Pancreatic Β Cells ◽  
Insulin Granules ◽  
Granule Motion

We imaged and analysed the motion of single insulin secretory granules near the plasma membrane in live pancreatic β-cells, from normal and diabetic Goto–Kakizaki (GK) rats, using total internal reflection fluorescence microscopy (TIRFM). In normal rat primary β-cells, the granules that were fusing during the first phase originate from previously docked granules, and those during the second phase originate from ‘newcomers’. In diabetic GK rat β-cells, the number of fusion events from previously docked granules were markedly reduced, and, in contrast, the fusion from newcomers was still preserved. The dynamic change in the number of docked insulin granules showed that, in GK rat β-cells, the total number of docked insulin granules was markedly decreased to 35% of the initial number after glucose stimulation. Immunohistochemistry with anti-insulin antibody observed by TIRFM showed that GK rat β-cells had a marked decline of endogenous insulin granules docked to the plasma membrane. Thus our results indicate that the decreased number of docked insulin granules accounts for the impaired insulin release during the first phase of insulin release in diabetic GK rat β-cells.

Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic β Cells

2013 ◽  
Vol 6 (273) ◽  
pp. ra29-ra29 ◽  
Author(s):  
Olof Idevall-Hagren ◽  
Ida Jakobsson ◽  
Yunjian Xu ◽  
Anders Tengholm
Keyword(s):  
Plasma Membrane ◽  
Cell Types ◽  
Dominant Negative ◽  
Nucleotide Exchange ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Spatiotemporal Regulation ◽  
Nucleotide Exchange Factor ◽  
Multiple Cell ◽  
Guanosine Triphosphatase

The cAMP (adenosine 3′,5′-monophosphate)–activated guanine nucleotide exchange factor (GEF) Epac2 is an important mediator of cAMP-dependent processes in multiple cell types. We used real-time confocal and total internal reflection fluorescence microscopy to examine the spatiotemporal regulation of Epac2, which is a GEF for the guanosine triphosphatase (GTPase) Rap. We demonstrated that increases in the concentration of cAMP triggered the translocation of Epac2 from the cytoplasm to the plasma membrane in insulin-secreting β cells. Glucose-induced oscillations of the submembrane concentration of cAMP were associated with cyclic translocation of Epac2, and this translocation could be amplified by increases in the cytoplasmic Ca2+ concentration. Analyses of Epac2 mutants identified the high-affinity cAMP-binding and the Ras association domains as crucial for the translocation. Expression of a dominant-negative Ras mutant reduced Epac2 translocation, and Ca2+-dependent oscillations in Ras activity synchronized with Epac2 translocation in single β cells. The cyclic translocation of Epac2 was accompanied by oscillations of Rap GTPase activity at the plasma membrane, and expression of an inactive Rap1B mutant decreased insulin secretion. Thus, Epac2 localization is dynamically controlled by cAMP as well as by Ca2+-mediated activation of Ras. These results help to explain how oscillating signals can produce pulses of insulin release from pancreatic β cells.

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