Phosphorylation of the transferrin receptor in isolated sheep reticulocyte plasma membranes

1984 ◽  
Vol 62 (9) ◽  
pp. 927-934 ◽  
Author(s):  
Rose M. Johnstone ◽  
Mohammed Adam ◽  
Claire Turbide ◽  
James Larrick
Keyword(s):  
Monoclonal Antibody ◽  
Cyclic Nucleotides ◽  
Transferrin Receptor ◽  
Plasma Membranes ◽  
Detectable Effect ◽  
Half Time ◽  
Serine Residue ◽  
Apparent Effect ◽  
Intact Cells ◽  
Receptor Phosphorylation

The transferrin receptor of sheep reticulocyte plasma membranes undergoes phosphorylation with [γ-32P]ATP in isolated plasma membranes. The phosphorylation is stimulated by Mn2+, Co2+, and Mg2+, but not by Ca2+, Ba2+, Zn2+, Fe2+, or Cu2+. There is no detectable effect of cyclic nucleotides on the phosphorylation of the receptor. Transferrin and a monoclonal antibody against the transferrin receptor have no apparent effect on receptor phosphorylation in intact cells or isolated membranes. Immunoprecipitates of the receptor retain ability to phosphorylate the receptor. The phosphorylation appears to be at a serine residue which turns over with a half time of 20–30 min. ATP appears to be the best, but not the only substrate for receptor phosphorylation.

The fate of the transferrin receptor during maturation of sheep reticulocytes in vitro

1984 ◽  
Vol 62 (11) ◽  
pp. 1246-1254 ◽  
Author(s):  
R. M. Johnstone ◽  
M. Adam ◽  
B. T. Pan
Keyword(s):  
Transferrin Receptor ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Molecular Size ◽  
Binding Activity ◽  
Intact Cells ◽  
Receptor Phosphorylation ◽  
Receptor Antibody ◽  
Peptide Map

The transferrin receptor of sheep reticulocytes is excised from the cell during in vitro maturation to erythrocytes. The excised receptor may be recovered from the medium by centrifugation at 100 000 × g. Loss of transferrin-binding activity parallels the loss of binding of anti-receptor antibody, as well as RNA content. The released receptor retains the molecular size of the receptor isolated from the plasma membranes (93 000 monomer, 186 000 dimer), has an identical iodotyrosyl peptide map, and is still capable of binding transferrin, as well as an antibody directed against the receptor. The receptor is released in a vesicular form. The major peptides of the vesicles are the receptor and an unidentified peptide of 70 000 whose iodotyrosyl peptide map is distinct from that of the receptor. Although the transferrin receptor has been shown to undergo posttranslational modification (phosphorylation, acylation, and glycosylation) in cultured cells, it has not been established whether any of the transformations are retained in nongrowing cells. In the present communication, it is shown that isolated reticulocyte plasma membranes are capable of receptor phosphorylation, a process previously shown only with intact, cultured cells. The phosphorylating activity is retained in immunoprecipitates of the receptor, but is absent in the vesicles released during maturation. No evidence has been obtained for an effect of either transferrin or an anti-receptor antibody on receptor phosphorylation in intact cells or isolated membranes.

scholarly journals Protein kinase C does not phosphorylate the externalized form of the transferrin receptor

1987 ◽  
Vol 242 (1) ◽  
pp. 151-161 ◽  
Author(s):  
M A Adam ◽  
R M Johnstone
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Transferrin Receptor ◽  
Plasma Membranes ◽  
Kinase Activity ◽  
Intact Cells ◽  
Kinase C ◽  
Exogenous Protein ◽  
Heat Treated ◽  
Receptor Pool

We have investigated the phosphorylation of transferrin receptors both in intact sheep reticulocytes and in isolated plasma membranes. Phosphorylation of the receptor in intact cells or isolated plasma membranes is stimulated by phorbol diesters, suggesting that protein kinase C may be involved. Identical [32P] phosphopeptide tryptic maps are formed in the presence and absence of phorbol diesters. Using heat-treated membranes (which are devoid of endogenous kinase activity) exogenous protein kinase C phosphorylates the same peptides as the endogenous kinase(s). During maturation of reticulocytes to erythrocytes, the transferrin receptor is released to the medium in vesicular form. In cells labelled with [32P]Pi, the released receptor is not labelled with 32P and the exocytosed vesicles do not phosphorylate receptor with [gamma-32P]ATP. The absence of 32P in the released receptor appears to be due to a change in the receptor, since, even in the presence of exogenous protein kinase C, the exocytosed receptor is phosphorylated to approximately 8% of the level obtained with receptors from the plasma membrane. These data suggest that during maturation and externalization the receptor is altered so that it loses its capacity to act as a substrate for exogenous protein kinase C as well as the endogenous kinase(s). This change may be a signal which segregates the receptor for externalization from the receptor pool remaining for transferrin recycling during the final stages of red cell maturation.

scholarly journals Quantification of cAMP and cGMP analogs in intact cells: pitfals in enzyme immunoassays for cyclic nucleotides

2011 ◽  
Vol 11 (S1) ◽  
Author(s):  
Katharina Werner ◽  
Frank Schwede ◽  
Hans-Gottfried Genieser ◽  
Jörg Geiger ◽  
Elke Butt
Keyword(s):  
Cyclic Nucleotides ◽  
Intact Cells ◽  
Enzyme Immunoassays ◽  
Camp And Cgmp

Ultrastructure of the hepatopancreas of the Pacific white shrimp, Penaeus vannamei (Crustacea: Decapoda)

1988 ◽  
Vol 68 (2) ◽  
pp. 323-337 ◽  
Author(s):  
Thomas Caceci ◽  
Kay F. Neck ◽  
Donal D H. Lewis ◽  
Raymond F. Sis
Keyword(s):  
B Cells ◽  
Plasma Membranes ◽  
Ultrastructural Level ◽  
Pacific White Shrimp ◽  
White Shrimp ◽  
Penaeus Vannamei ◽  
Intact Cells ◽  
The Pacific ◽  
Total Complement ◽  
Electron Microscopes

Fourteen specimens of the hepatopancreas of the Pacific white shrimp, Penaeus vannamei, were prepared for examination with the transmission and scanning electron microscopes and with the light microscope. The histology and ultrastructure of this organ is similar to that seen in other Decapoda. At the ultrastructural level, it was observed that B-cells rupture at approximately the level of gap junctions located on the lateral plasma membranes of the cells, and discharge the contents of their large vacuoles into the intercellular space. This efflux of enzymatic material may be the mechanism by which cells are released from the wall of the tubule at the proximal end: the rupture and collapse of a B-cell may be analagous to the removal of the keystone which supports an arch. Deprived of support, and lacking structural adaptations for cohesion (there are no desmosomes or interdigitations in the epithelium) and with the intercellular material digested, the remaining intact cells collapse into the lumen of the tubule. The lysis of individual cells of all types - R-, F-, and B-cells - may contribute to the tubules’ total complement of digestive enzymes.

Effect of tumour angiogenesis factors on the membrane of capillary and aortic endothelial cells: an e.s.r. spin-label study

1984 ◽  
Vol 68 (1) ◽  
pp. 153-162
Author(s):  
N.J. Dodd ◽  
S. Kumar
Keyword(s):  
Endothelial Cells ◽  
Spin Label ◽  
Plasma Membranes ◽  
Detectable Effect ◽  
Aortic Endothelial Cells ◽  
Whole Cells ◽  
Membrane Order ◽  
Migration Factor ◽  
Aortic Endothelial

Two distinct factors have been separated from an angiogenic extract of a rat Walker 256 carcinoma, one inducing proliferation and the other migration of capillary endothelial cells in vitro, but having no detectable effect on aortic endothelial cells. The influence of these factors on the order of plasma membranes of these cells was examined by electron spin resonance, using the lipophilic spin label 5-doxyl stearic acid. No detectable effect was observed on treating whole cells or isolated membranes with proliferation factor. In contrast, exposure of capillary endothelial cell membranes to migration factor caused a reduction of membrane order, particularly at temperatures above 30 degrees C. The migration factor had no detectable effect on membrane order of aortic endothelial cells.

Spreading of a sperm surface antigen within the plasma membrane of the egg after fertilization in the rat

Development ◽  
1983 ◽  
Vol 75 (1) ◽  
pp. 259-270
Author(s):  
Stephen J. Gaunt
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Guinea Pig ◽  
Surface Antigen ◽  
Plasma Membranes ◽  
Entire Surface ◽  
Sperm Tail ◽  
Sperm Surface ◽  
Sperm Antigens

The rat sperm surface antigen 2D6, located over the entire surface of the spermatozoon, is shown by use of a monoclonal antibody in indirect immunofluorescence experiments to spread laterally over the surface of the egg after fusion of sperm and egg plasma membranes at fertilization. Freshly fertilized eggs, obtained from superovulated rats 14h after hCG injection, showed the 2D6 antigen to have spread in a gradient over a discrete fan-shaped area of the egg surface anterior to the protruding sperm tail. Eggs at a later stage of sperm incorporation, obtained 20 h after hCG injection, snowed that the spread of antigen had extended to cover most or all of their surfaces. By 40 h after hCG injection, the approximate time that fertilized eggs cleaved to form 2-cell embryos, most of the 2D6 antigen had been lost from the cell surface. Fertilized eggs, but not unfertilized eggs or 2-cell embryos, were lysed by 2D6 monoclonal antibody in the presence of guinea pig complement. A model for sperm-egg fusion is presented to account for the observed pattern of spreading shown by the 2D6 antigen. The possible role of sperm antigens on the egg surface is discussed.

scholarly journals Desensitization of tumour Leydig cells by lutropin: evidence for uncoupling of the lutropin receptor from the guanine nucleotide-binding protein

1982 ◽  
Vol 202 (3) ◽  
pp. 739-745 ◽  
Author(s):  
Clive J. Dix ◽  
Matthias Schumacher ◽  
Brian A. Cooke
Keyword(s):  
Cyclic Amp ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Linear Increase ◽  
Guanine Nucleotide ◽  
Production Rates ◽  
Guanine Nucleotide Binding Protein ◽  
Intact Cells ◽  
Guanine Nucleotide Binding ◽  
Guanine Nucleotide Regulatory Protein

Purified rat Leydig tumour cells were pretreated with lutropin and the effect on the subsequent response to lutropin was determined. Maximal cyclic AMP production was achieved with the same concentration of lutropin in control and lutropin-pretreated cells; however, the maximum stimulated level in pretreated cells was only 30% of controls. The sensitivity to lutropin was decreased in lutropin-pretreated cells [ED50 (dose that produces a response that is 50% of the maximum response) 60±5.7ng/ml and 8±1.8ng/ml (mean±s.d., n=3) for controls], as was the rate of maximal cyclic AMP production (0.58, compared with 1.89pmol/106 cells per min for controls). However, cholera-toxin-stimulated cyclic AMP production was not decreased by lutropin pretreatment, and a potentiation was seen at all time points studied (up to 6h). Pre-incubation with lutropin caused a decrease in specific 125I-labelled human choriogonadotropin binding; however, this decrease was abolished if the cells were washed under acidic conditions (pH3.0 for 2min at 4°C), indicating that occupation but not loss of the lutropin receptors had taken place. The effect of pretreating the cells with lutropin on adenylate cyclase activity in purified plasma membranes was also investigated. In plasma membranes from control cells both guanosine 5′-[β,γ-imido]triphosphate [p(NH)ppG] plus lutropin and NaF plus lutropin caused a 50–60-fold linear increase in cyclic AMP production over 40min compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone. In plasma membranes isolated from lutropin-treated cells the NaF-plus-lutropin- and the p(NH)ppG-stimulated cyclic AMP production rates were unchanged but no effect of lutropin could be demonstrated with or without added p(NH)ppG. In contrast the plasma membranes from dibutyryl cyclic AMP-treated cells had similar cyclic AMP production rates to control cells with all stimulants studied. The present evidence obtained from studies both with intact cells and with isolated plasma membranes indicates that the initial lutropin-induced desensitization of the rat Leydig tumour cell is due to a lesion in the hormone-receptor coupling to the guanine nucleotide regulatory protein. This process is apparently not mediated by cyclic AMP.

scholarly journals Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes.

1987 ◽  
Vol 104 (5) ◽  
pp. 1239-1248 ◽  
Author(s):  
E S Sztul ◽  
D Biemesderfer ◽  
M J Caplan ◽  
M Kashgarian ◽  
J L Boyer
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Alpha Subunit ◽  
Gamma Glutamyl Transferase ◽  
Surface Distribution ◽  
Western Blots ◽  
Glutamyl Transferase

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.

scholarly journals Size heterogeneity, phosphorylation and transmembrane organisation of desmosomal glycoproteins 2 and 3 (desmocollins) in MDCK cells

1990 ◽  
Vol 96 (2) ◽  
pp. 239-248
Author(s):  
E.P. Parrish ◽  
J.E. Marston ◽  
D.L. Mattey ◽  
H.R. Measures ◽  
R. Venning ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Adhesion ◽  
Adhesion Molecules ◽  
Cell Adhesion Molecules ◽  
Mdck Cells ◽  
Intact Cells ◽  
Specific Antibodies ◽  
Metabolic Labelling ◽  
Membrane Spanning ◽  
Size Heterogeneity

Metabolic labelling with [35S]methionine and immunoprecipitation with specific antibodies to bovine desmosomal glycoproteins 2 and 3 (dg2 and dg3: desmocollins) reveals a triplet of polypeptides of Mr 115,000, 107,000 and 104,000 in MDCK cells. Tunicamycin treatment shows that this heterogeneity does not arise through differential N-linked glycosylation. Under conditions in which cells are actively forming desmosomes, the largest polypeptide, dg2, becomes phosphorylated on serine, but the two smaller polypeptides, dg3a and 3b, do not. Controlled trypsinisation of intact cells yields three membrane-protected fragments (Mr 28,000, 24,000 and 23,000) derived from these glycoproteins. The largest of these fragments is phosphorylated but the two smaller fragments are not. A monoclonal antibody to bovine dg2 and dg3 stains MDCK cells cytoplasmically. In immunoblotting of MDCK cells the monoclonal antibody recognises dg2 strongly and shows a weaker reaction with a band of lower Mr corresponding to dg3a. It also recognises the immunoprecipitated 28,000 Mr fragment from trypsinised cells and a smaller fragment of 24,000 Mr. The simplest interpretation of these data is that all three glycoproteins have a transmembrane configuration with a single membrane-spanning domain, and show heterogeneity of size and phosphorylation in their cytoplasmic domains. The data are discussed in relation to the known structures of some cell adhesion molecules. Questions about the relative roles and distributions of the different polypeptides in desmosomal organisation are raised.

scholarly journals Fluorescent, short-chain C6-NBD-sphingomyelin, but not C6-NBD-glucosylceramide, is subject to extensive degradation in the plasma membrane: implications for signal transduction related to cell differentiation

1995 ◽  
Vol 309 (3) ◽  
pp. 905-912 ◽  
Author(s):  
J W Kok ◽  
T Babia ◽  
K Klappe ◽  
D Hoekstra
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Cell Differentiation ◽  
Plasma Membranes ◽  
Cell Types ◽  
Short Chain ◽  
Synthetic Activity ◽  
Intact Cells ◽  
Ht29 Cells ◽  
Extensive Degradation

The involvement of the plasma membrane in the metabolism of the sphingolipids sphingomyelin (SM) and glucosylceramide (GlcCer) was studied, employing fluorescent short-chain analogues of these lipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoylsphingosylphosphorylcholine (C6-NBD-SM), C6-NBD-GlcCer and their common biosynthetic precursor C6-NBD-ceramide (C6-NBD-Cer). Although these fluorescent short-chain analogues are metabolically active, some caution is to be taken in view of potential changes in biophysical/biochemical properties of the lipid compared with its natural counterpart. However, these short-chain analogues offer the advantage of studying the lipid metabolic enzymes in their natural environment, since detergent solubilization is not necessary for measuring their activity. These studies were carried out with several cell types, including two phenotypes (differing in state of differentiation) of HT29 cells. Degradation and biosynthesis of C6-NBD-SM and C6-NBD-GlcCer were determined in intact cells, in their isolated plasma membranes, and in plasma membranes isolated from rat liver tissue. C6-NBD-SM was found to be subject to extensive degradation in the plasma membrane, due to neutral sphingomyelinase (N-SMase) activity. The extent of C6-NBD-SM hydrolysis showed a general cell-type dependence and turned out to be dependent on the state of cell differentiation, as revealed for HT29 cells. In undifferentiated HT29 cells N-SMase activity was at least threefold higher than in its differentiated counterpart. In contrast, in all cell types studied, very little if any biosynthesis of C6-NBD-SM from the precursor C6-NBD-Cer occurred. Moreover, in the case of C6-NBD-GlcCer, neither hydrolytic nor synthetic activity was found to be associated with the plasma membrane. These results are discussed in the context of the involvement of the sphingolipids SM and GlcCer in signal transduction pathways in the plasma membrane.

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