scholarly journals Fate of Internalized Thyrotropin-Releasing Hormone Receptors Monitored with a Timer Fusion Protein

Endocrinology ◽  
2004 ◽  
Vol 145 (7) ◽  
pp. 3095-3100 ◽  
Author(s):  
Laurie B. Cook ◽  
Patricia M. Hinkle
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Hormone Receptors ◽  
Inositol Phosphate ◽  
Fold Increase ◽  
Receptor Internalization ◽  
Free Calcium ◽  
Surface Binding ◽  
Cytoplasmic Vesicles ◽  
Trh Receptors

Abstract Trafficking of TRH receptors was studied in a stable HEK293 cell line expressing receptor fused to a Timer protein (TRHR-Timer) that spontaneously changes from green to red over 10 h. Cells expressing TRHR-Timer responded to TRH with an 11-fold increase in inositol phosphate formation, increased intracellular free calcium, and internalization of 75% of bound [3H][N3-methyl-His2]TRH within 10 min. After a 20-min exposure to TRH at 37 C, 75–80% of surface binding sites disappeared as receptors internalized. When TRH was removed and cells incubated in hormone-free medium, approximately 75% of [3H][N3-methyl-His2]TRH binding sites reappeared at the surface over the next 2 h with or without cycloheximide. Trafficking of TRHR-Timer was monitored microscopically after addition and withdrawal of TRH. In untreated cells, both new (green) and old (red) receptors were seen at the plasma membrane, and TRH caused rapid movement of young and old receptors into cytoplasmic vesicles. When TRH was withdrawn, some TRHR-Timer reappeared at the plasma membrane after several hours, but much of the internalized receptor remained intracellular in vesicles that condensed to larger structures in perinuclear regions deeper within the cell. Strikingly, receptors that moved to the plasma membrane were generally younger (more green) than those that underwent endocytosis. There was no change in the red to green ratio over the course of the experiment in cells exposed to vehicle. The results indicate that, after agonist-driven receptor internalization, the plasma membrane is replenished with younger receptors, arising either from an intracellular pool or preferential recycling of younger receptors.

Evidence for direct non-genomic effects of triiodothyronine on bone rudiments in rats: Stimulation of the inositol phosphate second messenger system

1991 ◽  
Vol 125 (6) ◽  
pp. 603-608 ◽  
Author(s):  
Peter Lakatos ◽  
Paula H. Stern
Keyword(s):  
Plasma Membrane ◽  
Thyroid Hormones ◽  
Time Course ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messenger ◽  
Cytosolic Calcium ◽  
Membrane Receptors ◽  
Free Calcium ◽  
Stimulation Of

Abstract. Thyroid hormones increase cytosolic free calcium by binding to plasma membrane receptors in several tissues. This calcium increase appears to initiate extranuclear effects in these tissues. Increases in cytosolic calcium are often a consequence of stimulation of inositol phosphate second messenger pathway. Several calcemic hormones act via this signal transduction route. Therefore we investigated the effects of the metabolically active T3 and the inactive analogues 3,5-diiodotyrosine and rT3 on the inositol phosphate pathway in fetal rat limb bone cultures prelabeled with [3H]myoinositol. Labelled inositol and inositol phosphates were separated by HPLC. There was a significant increase in the radioactivity in inositol bis- and trisphosphates after 1 min of exposure to 10−7 mol/l T3. Stimulation was also observed at 10−6 mol/l T3, but not at 10−5 mol/l. Time course studies demonstrated a rapid effect of T3 on inositol phosphates within 30 seconds that lasted through 5 min. After 20 min incubation with T3, no increase was observed in inositol mono- and bisphosphates, and a decrease was seen in inositol trisphosphate. Pretreatment with indomethacin prevented these effects of T3. 3,5-diiodothyrosine and rT3 did not affect inositol phosphate metabolism. These results suggest the existence of plasma membrane-associated receptors for T3 in bone, in addition to the nuclear receptors demonstrated previously. The role of these receptors in the effects of thyroid hormones on bone remains to be established.

scholarly journals Complement-induced vesiculation and exposure of membrane prothrombinase sites in platelets of paroxysmal nocturnal hemoglobinuria

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1192-1196 ◽  
Author(s):  
T Wiedmer ◽  
SE Hall ◽  
TL Ortel ◽  
WH Kane ◽  
WF Rosse ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Plasma Membranes ◽  
Membrane Binding ◽  
Coagulation Factor ◽  
Fold Increase ◽  
Factor Va ◽  
Membrane Binding Sites ◽  
Normal Controls

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder in which the glycolipid-anchored membrane proteins, including the cell-surface complement inhibitors, CD55 and CD59, are partially or completely deleted from the plasma membranes of mature blood cells. To gain insight into the pathogenesis of thrombosis that is frequently observed in this disorder, the procoagulant responses of PNH platelets exposed to the human terminal complement proteins C5b-9 were investigated. C5b-9 complexes were assembled on gel-filtered platelets by incubation with purified C5b6, C7, C9, and limiting amounts of C8. Platelet microparticle formation and exposure of plasma membrane- binding sites for coagulation factor Va were then analyzed by flow cytometry. PNH platelets exhibiting undetectable levels of surface CD59 antigen showed an approximately 10-fold increase in sensitivity to C5b- 9-stimulated expression of membrane-binding sites for factor Va when compared with platelets from normal controls. Expression of catalytic surface for the prothrombinase complex (VaXa) paralleled the exposure of factor Va-binding sites; the rate of prothrombin conversion by C5b-9- treated PNH platelets exceeded that of C5b-9-treated normal controls by approximately 10-fold at the maximal input of C8 tested (500 ng/mL). These data indicate that PNH platelets deficient in plasma membrane CD59 antigen are exquisitely sensitive to C5b-9-induced expression of prothrombinase activity, and suggest that the tendency toward thrombosis in these patients may be due, at least in part, to the deletion of this complement inhibitor from the platelet plasma membrane.

scholarly journals Complement-induced vesiculation and exposure of membrane prothrombinase sites in platelets of paroxysmal nocturnal hemoglobinuria

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1192-1196 ◽  
Author(s):  
T Wiedmer ◽  
SE Hall ◽  
TL Ortel ◽  
WH Kane ◽  
WF Rosse ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Plasma Membranes ◽  
Membrane Binding ◽  
Coagulation Factor ◽  
Fold Increase ◽  
Factor Va ◽  
Membrane Binding Sites ◽  
Normal Controls

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder in which the glycolipid-anchored membrane proteins, including the cell-surface complement inhibitors, CD55 and CD59, are partially or completely deleted from the plasma membranes of mature blood cells. To gain insight into the pathogenesis of thrombosis that is frequently observed in this disorder, the procoagulant responses of PNH platelets exposed to the human terminal complement proteins C5b-9 were investigated. C5b-9 complexes were assembled on gel-filtered platelets by incubation with purified C5b6, C7, C9, and limiting amounts of C8. Platelet microparticle formation and exposure of plasma membrane- binding sites for coagulation factor Va were then analyzed by flow cytometry. PNH platelets exhibiting undetectable levels of surface CD59 antigen showed an approximately 10-fold increase in sensitivity to C5b- 9-stimulated expression of membrane-binding sites for factor Va when compared with platelets from normal controls. Expression of catalytic surface for the prothrombinase complex (VaXa) paralleled the exposure of factor Va-binding sites; the rate of prothrombin conversion by C5b-9- treated PNH platelets exceeded that of C5b-9-treated normal controls by approximately 10-fold at the maximal input of C8 tested (500 ng/mL). These data indicate that PNH platelets deficient in plasma membrane CD59 antigen are exquisitely sensitive to C5b-9-induced expression of prothrombinase activity, and suggest that the tendency toward thrombosis in these patients may be due, at least in part, to the deletion of this complement inhibitor from the platelet plasma membrane.

HORMONAL REGULATION OF FOLLICLE-STIMULATING HORMONE RECEPTORS IN THE TESTES OF JAPANESE QUAIL

1980 ◽  
Vol 85 (3) ◽  
pp. 511-518 ◽  
Author(s):  
KAZUYOSHI TSUTSUI ◽  
SUSUMU ISHII
Keyword(s):  
Sertoli Cell ◽  
Japanese Quail ◽  
Binding Sites ◽  
Hormonal Regulation ◽  
Hormone Receptors ◽  
Fold Increase ◽  
Hormone Treatment ◽  
Testicular Development ◽  
Plot Analysis ◽  
Testicular Weight

The effects of gonadotrophins and testosterone on the binding of 125I-labelled rat FSH to a testicular homogenate of hypophysectomized Japanese quail have been studied. Hypophysectomy resulted in rapid decrease in the binding of FSH per testis but did not change the binding per unit testicular weight, suggesting a decrease in FSH binding per Sertoli cell. Injections of NIH-FSH-S12 to hypophysectomized birds (100 pg/day) for 5 days induced hypertrophy of Sertoli cells and 2·2-fold increase in the binding of FSH per Sertoli cell. Injections of testosterone to hypophysectomized birds (5·0 mg/day) for 5 days also increased the binding of FSH per Sertoli cell 2·3-fold, but did not change the cell size. Combined administration of FSH and testosterone induced 4·4-fold increase in the binding of FSH per Sertoli cell and also marked hypertrophy. The Scatchard plot analysis of the binding of FSH to the testicular preparation of hypophysectomized birds treated simultaneously with FSH and testosterone showed that the number of the binding sites, but not the affinity of the binding, was increased by the hormone treatment. Synergistic action of FSH and testosterone was observed in the increase of not only the number of the FSH binding sites but also in testicular weight and the number of spermatogonia and spermatocytes. These results indicated that FSH receptors in the testes of Japanese quail are regulated by FSH and testosterone. It is suggested that FSH acting synergistically with testosterone increases the sensitivity of the testis to FSH itself by increasing FSH receptors. This self-potentiation mechanism may enable rapid testicular development in photostimulated birds and mammals at puberty.

μ1A deficiency induces a profound increase in MPR300/IGF-II receptor internalization rate

2001 ◽  
Vol 114 (24) ◽  
pp. 4469-4476 ◽  
Author(s):  
Christoph Meyer ◽  
Eeva-Liisa Eskelinen ◽  
Medigeshi Ramarao Guruprasad ◽  
Kurt von Figura ◽  
Peter Schu
Keyword(s):  
Plasma Membrane ◽  
Fold Increase ◽  
Retrograde Transport ◽  
Receptor Internalization ◽  
Recycling Rate ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
Internalization Rate ◽  
Clathrin Adaptor ◽  
Golgi Network

The mannose-6-phosphate/IGF-II receptor MPR300 mediates sorting of lysosomal enzymes from the trans-Golgi network to endosomes and endocytosis of hormones, for example, of IGF-II. We analyzed transport of MPR300 in μ1A-adaptin-deficient fibroblasts, which lack a functional AP-1 clathrin adaptor complex. In μ1A-adaptin-deficient fibroblasts, the homologous MPR46 accumulates in endosomes due to a block in retrograde transport to the trans-Golgi network. The MPR300-mediated endocytosis is markedly enhanced. We demonstrate that the seven-fold increase in endocytosis is not associated with an increased steady-state concentration of receptors at the plasma membrane, but with an increased internalization rate of MPR300. Internalization of other receptors that are also endocytosed by AP-2 is not affected. More MPR300 receptors are found in clathrin-coated pits of the plasma membrane, whereas outside coated-areas, more MPR300 are concentrated in clusters and all intracellular receptors reside in endosomes, which are in equilibrium with the plasma membrane. Thus AP-1-mediated transport of MPR300 from endosomes to the TGN controls indirectly the recycling rate of the receptor between the plasma membrane and endosomes.

scholarly journals Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

1982 ◽  
Vol 92 (3) ◽  
pp. 634-647 ◽  
Author(s):  
PL Zeitlin ◽  
AL Hubbard
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Binding Sites ◽  
Rat Hepatocytes ◽  
Surface Binding ◽  
Isolated Rat Hepatocytes ◽  
Electron Microscopic Autoradiography ◽  
Surface Receptors ◽  
Receptor Mediated Endocytosis ◽  
Isolated Rat

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.

scholarly journals Inhibition of MLKL-dependent necroptosis via downregulating interleukin-1R1 contributes to neuroprotection of hypoxic preconditioning in transient global cerebral ischemic rats

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Lixuan Zhan ◽  
Xiaomei Lu ◽  
Wensheng Xu ◽  
Weiwen Sun ◽  
En Xu
Keyword(s):  
Plasma Membrane ◽  
Cerebral Ischemia ◽  
Neuronal Death ◽  
Interleukin 1 ◽  
Hypoxic Preconditioning ◽  
Global Cerebral Ischemia ◽  
Free Calcium ◽  
Membrane Translocation ◽  
Vessel Occlusion ◽  
Transient Global Cerebral Ischemia

Abstract Background Our previous study indicated that hypoxic preconditioning reduced receptor interacting protein (RIP) 3-mediated necroptotic neuronal death in hippocampal CA1 of adult rats after transient global cerebral ischemia (tGCI). Although mixed lineage kinase domain-like (MLKL) has emerged as a crucial molecule for necroptosis induction downstream of RIP3, how MLKL executes necroptosis is not yet well understood. In this study, we aim to elucidate the molecular mechanism underlying hypoxic preconditioning that inactivates MLKL-dependent neuronal necroptosis after tGCI. Methods Transient global cerebral ischemia was induced by the four-vessel occlusion method. Twenty-four hours before ischemia, rats were exposed to systemic hypoxia with 8% O2 for 30 min. Western blotting was used to detect the expression of MLKL and interleukin-1 type 1 receptor (IL-1R1) in CA1. Immunoprecipitation was used to assess the interactions among IL-1R1, RIP3, and phosphorylated MLKL (p-MLKL). The concentration of intracellular free calcium ion (Ca2+) was measured using Fluo-4 AM. Silencing and overexpression studies were used to study the role of p-MLKL in tGCI-induced neuronal death. Results Hypoxic preconditioning decreased the phosphorylation of MLKL both in neurons and microglia of CA1 after tGCI. The knockdown of MLKL with siRNA decreased the expression of p-MLKL and exerted neuroprotective effects after tGCI, whereas treatment with lentiviral delivery of MLKL showed opposite results. Mechanistically, hypoxic preconditioning or MLKL siRNA attenuated the RIP3-p-MLKL interaction, reduced the plasma membrane translocation of p-MLKL, and blocked Ca2+ influx after tGCI. Furthermore, hypoxic preconditioning downregulated the expression of IL-1R1 in CA1 after tGCI. Additionally, neutralizing IL-1R1 with its antagonist disrupted the interaction between IL-1R1 and the necrosome, attenuated the expression and the plasma membrane translocation of p-MLKL, thus alleviating neuronal death after tGCI. Conclusions These data support that the inhibition of MLKL-dependent neuronal necroptosis through downregulating IL-1R1 contributes to neuroprotection of hypoxic preconditioning against tGCI.

scholarly journals Cell surface-binding sites for progesterone mediate calcium uptake in human sperm.

1991 ◽  
Vol 266 (28) ◽  
pp. 18655-18659 ◽  
Author(s):  
P.F. Blackmore ◽  
J. Neulen ◽  
F. Lattanzio ◽  
S.J. Beebe
Keyword(s):  
Cell Surface ◽  
Binding Sites ◽  
Calcium Uptake ◽  
Human Sperm ◽  
Surface Binding ◽  
Cell Surface Binding
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