scholarly journals Secretagogue-dependent phosphorylation of phogrin, an insulin granule membrane protein tyrosine phosphatase homologue

1999 ◽  
Vol 341 (3) ◽  
pp. 563-569 ◽  
Author(s):  
Christina WASMEIER ◽  
John C. HUTTON
Keyword(s):  
Protein Kinase ◽  
Membrane Protein ◽  
Time Course ◽  
Secretory Granules ◽  
Tyrosine Phosphatase ◽  
Signal Transduction Pathway ◽  
Integral Membrane Protein ◽  
Neuroendocrine Cells ◽  
Calmodulin Antagonist ◽  
Min6 Cells

Phogrin, a 60/64 kDa integral membrane protein localized to dense-core secretory granules of neuroendocrine cells, was found to be reversibly phosphorylated in intact pancreatic β-cells. Phosphorylation occurred in response to a variety of secretory stimuli, including glucose and depolarizing concentrations of K+. In MIN6 cells, the glucose dose-response and time course of phogrin phosphorylation paralleled that of insulin secretion. Like secretion, glucose- or K+-stimulated phosphorylation required the presence of Ca2+. The calmodulin antagonist W-7 and the Ca2+/calmodulin-dependent kinase II inhibitor KN-93 dose-dependently inhibited both phosphorylation and secretion, while the ‘inactive’ analogue KN-92 was effective only at significantly higher concentrations. Phosphorylation of phogrin was also stimulated in cells exposed to forskolin, an effect presumably mediated by protein kinase A (cAMP-dependent protein kinase). Under these conditions, phogrin phosphorylation could be dissociated from the secretory response. In MIN6 cells, as in pancreatic islets, cAMP potentiates rather than initiates insulin release. Thus our observations are consistent with a role for phogrin phosphorylation in the signal-transduction pathway at a site proximal to the exocytic event itself, possibly regulating secretory-granule mobilization and recruitment to the exocytic site.

scholarly journals Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells.

1986 ◽  
Vol 103 (6) ◽  
pp. 2511-2527 ◽  
Author(s):  
F Navone ◽  
R Jahn ◽  
G Di Gioia ◽  
H Stukenbrok ◽  
P Greengard ◽  
...  
Keyword(s):  
Nervous System ◽  
Membrane Protein ◽  
Synaptic Vesicles ◽  
Secretory Granules ◽  
Light And Electron Microscopy ◽  
Immunoblot Analysis ◽  
Integral Membrane Protein ◽  
Neuroendocrine Cells ◽  
Synapsin I ◽  
Large Dense Core Vesicles

An intrinsic membrane protein of brain synaptic vesicles with Mr 38,000 (p38, synaptophysin) has recently been partially characterized (Jahn, R., W. Schiebler, C. Ouimet, and P. Greengard, 1985, Proc. Natl. Acad. Sci. USA, 83:4137-4141; Wiedenmann, B., and W. W. Franke, 1985, Cell, 41:1017-1028). We have now studied the presence of p38 in a variety of tissues by light and electron microscopy immunocytochemistry and by immunochemistry. Our results indicate that, within the nervous system, p38, like the neuron-specific phosphoprotein synapsin I, is present in virtually all nerve terminals and is selectively associated with small synaptic vesicles (SSVs). No p38 was detectable on large dense-core vesicles (LDCVs). p38 and synapsin I were found to be present in similar concentrations throughout the brain. Outside the nervous system, p38 was found in a variety of neuroendocrine cells, but not in any other cell type. In neuroendocrine cells p38 was localized on a pleiomorphic population of small, smooth-surfaced vesicles, which were interspersed among secretory granules and concentrated in the Golgi area, but not on the secretory granules themselves. Immunoblot analysis of endocrine tissues and cell lines revealed a band with a mobility slightly different from that of neuronal p38. This difference was attributable to a difference in glycosylation. The finding that p38, like synapsin I, is a component of SSVs of virtually all neurons, but not of LDCVs, supports the idea that SSVs and LDCVs are organelles of two distinct pathways for regulated neuronal secretion. In addition, our results indicate the presence in a variety of neuroendocrine cells of an endomembrane system, which is related to SSVs of neurons but is distinct from secretory granules.

scholarly journals Study of the transit of an integral membrane protein from secretory granules through the plasma membrane of secreting rat basophilic leukemia cells using a specific monoclonal antibody.

1986 ◽  
Vol 102 (2) ◽  
pp. 516-522 ◽  
Author(s):  
J S Bonifacino ◽  
P Perez ◽  
R D Klausner ◽  
I V Sandoval
Keyword(s):  
Monoclonal Antibody ◽  
Bovine Serum Albumin ◽  
Membrane Protein ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Secretory Granules ◽  
Integral Membrane Protein ◽  
Rat Basophilic Leukemia Cells ◽  
Rbl Cells ◽  
Basophilic Leukemia

The monoclonal antibody 5G10 reacted specifically with an 80-kD integral membrane protein in rat basophilic leukemia (RBL) cells. Immunofluorescence microscopy studies of RBL cells, fixed and permeabilized, revealed that the 80-kD protein was located in the membrane of cytoplasmic vesicles. The vesicles were identified as secretory granules by their content in immunoreactive serotonin. Expression of the 5G10 antigen on the surface of unstimulated RBL cells was low. However, RBL cells stimulated to secrete with anti-dinitrophenyl IgE followed by dinitrophenyl-bovine serum albumin or with the Ca2+ ionophore A-23187 displayed an increased expression of the antigen on their surface. Surface exposure of the 5G10 antigen was maximal at 5 min after stimulation of secretion. Removal of dinitrophenyl-bovine serum albumin from the incubation medium resulted in internalization of 50% of the antigen within 10 min.

scholarly journals Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis.

1995 ◽  
Vol 131 (5) ◽  
pp. 1231-1242 ◽  
Author(s):  
M Berryman ◽  
R Gary ◽  
A Bretscher
Keyword(s):  
Time Course ◽  
Tyrosine Phosphatase ◽  
Gel Filtration ◽  
Signal Transduction Pathway ◽  
Major Protein ◽  
Phosphatase Inhibitors ◽  
Protein Mass ◽  
Cell Surface Structures ◽  
Total Protein Mass

Ezrin is a component of the microvillus cytoskeleton of a variety of polarized epithelial cells and is believed to function as a membrane-cytoskeletal linker. In this study, we isolated microvilli from human placental syncytiotrophoblast as a model system for biochemical analysis of ezrin function. In contrast to intestinal microvilli, ezrin is a major protein component of placental microvilli, comprising approximately 5% of the total protein mass and present at about one quarter of the molar abundance of actin. Gel filtration and chemical cross-linking studies demonstrated that ezrin exists mainly in the form of noncovalent dimers and higher order oligomers in extracts of placental microvilli. A novel form of ezrin, apparently representing covalently cross-linked adducts, was present as a relatively minor constituent of placental microvilli. Both oligomers and adducts remained associated with the detergent-insoluble cytoskeleton, indicating a tight interaction with actin filaments. Moreover, stimulation of human A431 carcinoma cells with EGF induces the rapid formation of ezrin oligomers in vivo, thus identifying a signal transduction pathway involving ezrin oligomerization coincident with microvillus assembly. In addition to time course studies, experiments with tyrosine kinase and tyrosine phosphatase inhibitors revealed a correlation between the phosphorylation of ezrin on tyrosine and the onset of oligomer formation, consistent with the possibility that phosphorylation of ezrin might be required for the generation of stable oligomers. Based on these observations, a model for the assembly of cell surface structures is proposed.

scholarly journals Occludin-deficient Embryonic Stem Cells Can Differentiate into Polarized Epithelial Cells Bearing Tight Junctions

1998 ◽  
Vol 141 (2) ◽  
pp. 397-408 ◽  
Author(s):  
Mitinori Saitou ◽  
Kazushi Fujimoto ◽  
Yoshinori Doi ◽  
Masahiko Itoh ◽  
Toyoshi Fujimoto ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Membrane Protein ◽  
Tight Junctions ◽  
Time Course ◽  
Es Cells ◽  
Embryonic Stem ◽  
Freeze Fracture ◽  
Integral Membrane Protein ◽  
Embryoid Bodies ◽  
Wild Type

Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.

scholarly journals The Lumenal Domain of the Integral Membrane Protein Phogrin Mediates Targeting to Secretory Granules

Traffic ◽  
2002 ◽  
Vol 3 (9) ◽  
pp. 654-665 ◽  
Author(s):  
Christina Wasmeier ◽  
Nicholas A. Bright ◽  
John C. Hutton
Keyword(s):  
Membrane Protein ◽  
Secretory Granules ◽  
Integral Membrane Protein ◽  
Lumenal Domain

Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic β-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy

2002 ◽  
Vol 363 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Mica OHARA-IMAIZUMI ◽  
Yoko NAKAMICHI ◽  
Toshiaki TANAKA ◽  
Hidenori KATSUTA ◽  
Hitoshi ISHIDA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Time Course ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Ph Sensitive ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Min6 Cells ◽  
Green Fluorescent

The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic β-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH7.4, but not at approximately pH5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin—pHluorin, time-lapse confocal laser scanning microscopy (5 or 10s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50mM KCl and 22mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin—pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.

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