scholarly journals Biosynthesis and assembly of platelet GPIIb-IIIa in human megakaryocytes: evidence that assembly between pro-GPIIb and GPIIIa is a prerequisite for expression of the complex on the cell surface

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1603-1611 ◽  
Author(s):  
A Duperray ◽  
A Troesch ◽  
R Berthier ◽  
E Chagnon ◽  
P Frachet ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Liquid Cultures ◽  
Calcium Dependent ◽  
Large Pool ◽  
Adhesive Proteins ◽  
Kinetics Of ◽  
Megakaryocytic Cell

Abstract The platelet membrane glycoproteins GPIIb and GPIIIa form a calcium- dependent heterodimer that functions as a receptor for adhesive proteins on stimulated platelets. In this study, we have investigated the kinetics of the assembly reaction that result in GPIIb-IIIa dimerization. Pulse-chase experiments analysis performed on human megakaryocytes obtained from liquid cultures of chronic myelogenous leukemic patients with antibodies specific for GPIIIa or GPIIb demonstrated the existence of a pro-GPIIb-GPIIIa complex and of a large pool (60%) of unassociated GPIIIa; nearly all the GPIIb and the pro- GPIIb molecules were found associated with GPIIIa. This free GPIIIa was not exposed on the cell surface. Pulse-chase experiments on a subclone of the human megakaryocytic cell line LAMA-84 revealed that the cells from this subclone produced only the pro-GPIIb, which was neither processed into mature GPIIb nor expressed on the cell surface. The expression of GPIIIa in PMA treated cells resulted in the production of the mature GPIIb form and the expression of the GPIIb-IIIa complex on the cell surface. These results indicate that assembly between the early forms of pro-GPIIb and GPIIIa is an obligatory step for the maturation of the heterodimer and its expression on the cell surface.

scholarly journals Biosynthesis and assembly of platelet GPIIb-IIIa in human megakaryocytes: evidence that assembly between pro-GPIIb and GPIIIa is a prerequisite for expression of the complex on the cell surface

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1603-1611 ◽  
Author(s):  
A Duperray ◽  
A Troesch ◽  
R Berthier ◽  
E Chagnon ◽  
P Frachet ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Liquid Cultures ◽  
Calcium Dependent ◽  
Large Pool ◽  
Adhesive Proteins ◽  
Kinetics Of ◽  
Megakaryocytic Cell

The platelet membrane glycoproteins GPIIb and GPIIIa form a calcium- dependent heterodimer that functions as a receptor for adhesive proteins on stimulated platelets. In this study, we have investigated the kinetics of the assembly reaction that result in GPIIb-IIIa dimerization. Pulse-chase experiments analysis performed on human megakaryocytes obtained from liquid cultures of chronic myelogenous leukemic patients with antibodies specific for GPIIIa or GPIIb demonstrated the existence of a pro-GPIIb-GPIIIa complex and of a large pool (60%) of unassociated GPIIIa; nearly all the GPIIb and the pro- GPIIb molecules were found associated with GPIIIa. This free GPIIIa was not exposed on the cell surface. Pulse-chase experiments on a subclone of the human megakaryocytic cell line LAMA-84 revealed that the cells from this subclone produced only the pro-GPIIb, which was neither processed into mature GPIIb nor expressed on the cell surface. The expression of GPIIIa in PMA treated cells resulted in the production of the mature GPIIb form and the expression of the GPIIb-IIIa complex on the cell surface. These results indicate that assembly between the early forms of pro-GPIIb and GPIIIa is an obligatory step for the maturation of the heterodimer and its expression on the cell surface.

scholarly journals The in vitro synthesis of polypeptides for the platelet membrane glycoproteins IIb and IIIa

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1031-1037 ◽  
Author(s):  
SM Silver ◽  
MM McDonough ◽  
G Vilaire ◽  
JS Bennett
Keyword(s):  
Leukemia Cell ◽  
K562 Cells ◽  
Platelet Membrane ◽  
Human Leukemia ◽  
Membrane Glycoproteins ◽  
In Vitro Synthesis ◽  
Calcium Dependent ◽  
Hel Cells ◽  
Von Willebrand

Abstract The platelet membrane glycoproteins IIb (GpIIb) and GpIIIa form calcium- dependent heterodimers containing binding sites for fibrinogen, von Willebrand factor, and fibronectin. Although GpIIb and GpIIIa are distinct proteins, both GpIIb and GpIIIa are deficient in platelets from individuals with the recessive disorder Glanzmann's thrombasthenia. To gain a better understanding of the genetic basis for GpIIb and GpIIIa synthesis, we studied their synthesis by two human leukemia cell lines, HEL and K562. HEL cells contained complexes of GpIIb and GpIIIa, and K562 cells expressed GpIIIa, but not GpIIb, when stimulated with phorbol-12-myristate-13-acetate (PMA). RNA from HEL cells directed the in vitro synthesis of a 110,000-Mr precursor for GpIIb and a 92,000-Mr precursor for GpIIIa, which indicates that the synthesis of GpIIb and GpIIIa by HEL cells is directed by separate mRNAs. In contrast, RNA from PMA-stimulated K562 cells only directed the synthesis of a 92,000-Mr precursor for GpIIIa. The dissociation of GpIIb and GpIIIa synthesis in K562 cells suggests that GpIIb and GpIIIa may be the products of separate genes.

scholarly journals Membrane-bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 641-649 ◽  
Author(s):  
K Miyazawa ◽  
DA Williams ◽  
A Gotoh ◽  
J Nishimaki ◽  
HE Broxmeyer ◽  
...  
Keyword(s):  
Cell Surface ◽  
Tyrosine Kinase ◽  
Life Span ◽  
Protein Degradation ◽  
Cell Line ◽  
Soluble Form ◽  
Steel Factor ◽  
Degradation Rates ◽  
Membrane Bound ◽  
Kinetics Of

Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (SI/SI4) established from SI/SI homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. Interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, SI4- h220 (membrane-bound form) induced more persistent activation of c-kit kinase than SI4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that SI4-h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas SI4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to SI4- h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a “turn-off switch” for activated c-kit kinase.

Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL).

1984 ◽  
Vol 3 (2) ◽  
pp. 453-459 ◽  
Author(s):  
A. Tabilio ◽  
J.P. Rosa ◽  
U. Testa ◽  
N. Kieffer ◽  
A.T. Nurden ◽  
...  
Keyword(s):  
Cell Line ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Erythroleukemia Cell ◽  
Granule Proteins

scholarly journals Differences in internalization and intracellular processing of interleukin-1 associated with the two forms of interleukin-1 receptor found in B-cells and T-cells

1991 ◽  
Vol 273 (1) ◽  
pp. 79-83 ◽  
Author(s):  
R Horuk
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cell Surface ◽  
Cell Line ◽  
Interleukin 1 ◽  
Cell Types ◽  
Single Chain ◽  
Surface Binding ◽  
Raji Cells ◽  
Kinetics Of

There are at least two classes of interleukin-1 (IL-1) receptor, namely p80, and 80 kDa single-chain protein found in T-cells, fibroblasts and many other cell types, and p68, a 68 kDa protein expressed in B-cells and macrophages. The to classes of IL-1 receptor show distinct differences in their substrate-binding site and molecular properties. In this study we show that the kinetics of IL-1 internalization and the consequences of ligand processing in the two subtypes of IL-1 receptor are also very different. The Raji cell line was used as a source of the p68 form of the IL-1 receptor, whereas the YT cell line was used as a source of p80 receptor. Under conditions of steady-state binding the IL-1 was equally distributed between cell-surface and intracellular sites in YT cells, compared with predominantly cell-surface binding (85%) in Raji cells. The mechanism of IL-1 processing was also different in the two cell types. In Raji cells 60% of internalized IL-1 was released from the cells in an intact form, whereas the remainder was degraded. All of the IL-1 extruded from YT cells was intact. The kinetics of IL-1 release was faster in Raji cells, with a half-time of 4.5 h compared with over 15 h in YT cells. SDS/PAGE analysis of internalized IL-1 in Raji cells revealed that the ligand was sequentially processed to trichloroacetic acid-soluble products. The YT receptor-ligand complex was resistant to dissociation at pH 5, whereas that in Raji cells rapidly dissociated at this pH. Treatment of Raji cells with the lysosomotropic agent chloroquine inhibited the degradation of IL-1 without having any effect on the amount of intact IL-1 in the intracellular compartments. From these data we conclude that the pathways of internalization, intracellular trafficking and overall processing of IL-1 are different for p68 IL-1 receptors compared with p80. This could have direct consequences for IL-1 action and IL-1 receptor regulation in the cell.

scholarly journals The in vitro synthesis of polypeptides for the platelet membrane glycoproteins IIb and IIIa

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1031-1037 ◽  
Author(s):  
SM Silver ◽  
MM McDonough ◽  
G Vilaire ◽  
JS Bennett
Keyword(s):  
Leukemia Cell ◽  
K562 Cells ◽  
Platelet Membrane ◽  
Human Leukemia ◽  
Membrane Glycoproteins ◽  
In Vitro Synthesis ◽  
Calcium Dependent ◽  
Hel Cells ◽  
Von Willebrand

The platelet membrane glycoproteins IIb (GpIIb) and GpIIIa form calcium- dependent heterodimers containing binding sites for fibrinogen, von Willebrand factor, and fibronectin. Although GpIIb and GpIIIa are distinct proteins, both GpIIb and GpIIIa are deficient in platelets from individuals with the recessive disorder Glanzmann's thrombasthenia. To gain a better understanding of the genetic basis for GpIIb and GpIIIa synthesis, we studied their synthesis by two human leukemia cell lines, HEL and K562. HEL cells contained complexes of GpIIb and GpIIIa, and K562 cells expressed GpIIIa, but not GpIIb, when stimulated with phorbol-12-myristate-13-acetate (PMA). RNA from HEL cells directed the in vitro synthesis of a 110,000-Mr precursor for GpIIb and a 92,000-Mr precursor for GpIIIa, which indicates that the synthesis of GpIIb and GpIIIa by HEL cells is directed by separate mRNAs. In contrast, RNA from PMA-stimulated K562 cells only directed the synthesis of a 92,000-Mr precursor for GpIIIa. The dissociation of GpIIb and GpIIIa synthesis in K562 cells suggests that GpIIb and GpIIIa may be the products of separate genes.

scholarly journals Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes.

1986 ◽  
Vol 103 (4) ◽  
pp. 1249-1256 ◽  
Author(s):  
T Haylett ◽  
L Thilo
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
State Level ◽  
Background Level ◽  
Fluid Phase ◽  
Membrane Glycoproteins ◽  
First Order Kinetics ◽  
Secondary Lysosomes ◽  
Kinetics Of ◽  
Lysosome Membrane

Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D1, was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane on self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label in secondary lysosomes increased by first order kinetics (k = [56 min]-1) from less than 0.1% (background level) to a steady-state level of approximately 2.5% of the total label. As analyzed by NaDodSO4 PAGE, labeled molecules of Mr 160-190 kD were depleted and of Mr 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all Mr classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constituents to secondary lysosomes is a limited and selective process, and that only approximately 1% of internalized membrane is recycled via a membrane pool of secondary lysosomes.

scholarly journals Activation of the Mitogen-Activated Protein Kinase Pathway Is Involved in and Sufficient for Megakaryocytic Differentiation of CMK Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3462-3470 ◽  
Author(s):  
Allen S. Melemed ◽  
John W. Ryder ◽  
Terry A. Vik
Keyword(s):  
Cell Surface ◽  
Map Kinase ◽  
Cell Line ◽  
Specific Inhibitor ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Kinase Pathway ◽  
Megakaryocytic Cell

Abstract Activation of the mitogen-activated protein (MAP) kinase pathway has been associated with both cell proliferation and differentiation. Constitutively activated forms of Mek (MAP kinase/Erk kinase) and Erk (MAP kinase) have been previously shown capable of inducing differentiation or proliferation in nonhematopoietic cells. To specifically examine the role of Erk activation in megakaryocytic growth and development, we activated the MAP kinase pathway by the transfection of constitutively activated Mek or Erk cDNA into a human megakaryoblastic cell line, CMK, by electroporation. The CMK transfectant clones that expressed constitutively activated Mek or Erk showed morphologic changes of differentiation. Transfected cells also showed expression of mature megakaryocytic cell surface markers. The MAP kinase pathway was also activated by treatment of the hematopoietic cells with a cytokine that activates Erk. The treatment of CMK cells with stem cell factor (SCF ) caused MAP kinase activation and induced differentiation by the expression of mature megakaryocytic cell surface markers. The effects of the SCF treatment were inhibited by pretreatment with a specific inhibitor of the MAP kinase pathway, PD98059. In this report, we conclude that activation of the MAP kinase pathway was both necessary and sufficient to induce differentiation in this megakaryoblastic cell line.

Sign in / Sign up

Export Citation Format

Share Document