scholarly journals Evidence that a pertussis-toxin-sensitive substrate is involved in the stimulation by epidermal growth factor and vasopressin of plasma-membrane Ca2+ inflow in hepatocytes

1987 ◽  
Vol 248 (3) ◽  
pp. 911-918 ◽  
Author(s):  
B P Hughes ◽  
J N Crofts ◽  
A M Auld ◽  
L C Read ◽  
G J Barritt
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Pertussis Toxin ◽  
Glycogen Phosphorylase ◽  
Regulatory Protein ◽  
Phosphorylase Activity ◽  
Rate Of Increase ◽  
Epidermal Growth ◽  
Glycogen Phosphorylase Activity

1. In hepatocytes, epidermal growth factor (EFG) (a) increased the rate of 45Ca2+ exchange in cells incubated at 1.3 mM extracellular Ca2+, (b) increased the activity of glycogen phosphorylase a and the intracellular free Ca2+ concentration (measured with quin2) in a process dependent on the concentration of extracellular Ca2+, and (c) enhanced the increase in glycogen phosphorylase activity which follows the addition of Ca2+ to cells previously incubated in the absence of Ca2+. It is concluded that EGF stimulates plasma-membrane Ca2+ inflow. 2. The effects of the combination of EGF and vasopressin on the rate of 45Ca2+ exchange and on the rate of increase in glycogen phosphorylase activity were the same as those of vasopressin alone. 3. The amount of 45Ca2+ released by EGF from internal stores was about 30% of that released by vasopressin. No detectable increase in [3H]inositol mono-, bis- or tris-phosphate was observed after the addition of EGF to cells labelled with myo-[3H]inositol. 4. In hepatocytes isolated from rats treated with pertussis toxin, the effects of EGF and vasopressin on phosphorylase activity (measured at 1.3 mM-Ca2+) and on the rate of Ca2+ inflow (measured with quin2) were markedly decreased compared with those in normal cells. 5. Treatment with pertussis toxin did not impair the ability of vasopressin to release Ca2+ from internal stores, but decreased vasopressin-stimulated [3H]inositol polyphosphate formation by 50%. 6. It is concluded that the mechanism(s) by which vasopressin and EGF stimulate plasma-membrane Ca2+-inflow transporters in hepatocytes involves a GTP-binding regulatory protein sensitive to pertussis toxin, and does not require an increase in the concentration of inositol trisphosphate comparable with that which induces the release of Ca2+ from the endoplasmic reticulum.

scholarly journals Epidermal Growth Factor Receptor Distribution during Chemotactic Responses

2000 ◽  
Vol 11 (11) ◽  
pp. 3873-3883 ◽  
Author(s):  
Maryse Bailly ◽  
Jeffrey Wyckoff ◽  
Boumediene Bouzahzah ◽  
Ross Hammerman ◽  
Vonetta Sylvestre ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Spatial Gradient ◽  
Spatial Gradients ◽  
Epidermal Growth ◽  
Green Fluorescent

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

Orientation of the v-erb-B gene product in the plasma membrane

1986 ◽  
Vol 6 (4) ◽  
pp. 1329-1333
Author(s):  
R C Schatzman ◽  
G I Evan ◽  
M L Privalsky ◽  
J M Bishop
Keyword(s):  
Endoplasmic Reticulum ◽  
Epidermal Growth Factor Receptor ◽  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Kinase Domain ◽  
Amino Terminus ◽  
Protein Kinase Domain ◽  
Epidermal Growth

The retroviral oncogene v-erb-B encodes a truncated version of the receptor for epidermal growth factor. To define the disposition of the v-erb-B protein within cells and across the plasma membrane, we raised antibodies against defined epitopes in the protein and used these in immunofluorescence to analyze cells transformed by v-erb-B. A small fraction of the v-erb-B protein was found on the plasma membrane in a clustered configuration. The bulk of the protein was located in the endoplasmic reticulum and Golgi apparatus. Epitopes near the amino terminus of the v-erb-B protein were displayed on the surface of the cell, whereas epitopes in the protein kinase domain were located exclusively within cells. We conclude that the v-erb-B protein spans the plasma membrane in a manner similar or identical to that of the epidermal growth factor receptor, even though the viral transforming protein does not possess the signal peptide that is thought to direct insertion of the receptor into the membrane.

scholarly journals Evidence that guanosine 5′-[γ-thio]triphosphate stimulates plasma membrane Ca2+ inflow when introduced into hepatocytes

1989 ◽  
Vol 257 (2) ◽  
pp. 591-598 ◽  
Author(s):  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Plasma Membrane ◽  
Liver Cell ◽  
Glycogen Phosphorylase ◽  
Initial Rate ◽  
Effective Concentration ◽  
Phosphorylase Activity ◽  
Glycogen Phosphorylase Activity ◽  
Rate Of Activation ◽  
In Cells

1. Slowly hydrolysable analogues of GTP were introduced into hepatocytes by incubating the cells in the absence of Mg2+ and in the presence of ATP4-. Experiments using guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S])indicated that about 50% of the GTP[S] loaded into the cells was subsequently hydrolysed. 2. In cells loaded with GTP[S] and incubated in the absence of added extracellular Ca2+ (Ca2+o), the rate of activation of glycogen phosphorylase observed after addition of 1.3 mM-Ca2+o was 250% greater than the rate observed in unloaded cells. Smaller effects (130%) were observed in cells loaded with either guanyl-5′-yl imidodiphosphate or guanosine 5-[beta-thio]diphosphate (GDP[S]). Cells loaded with adenosine 5′-[gamma-thio]triphosphate showed no increase in glycogen phosphorylase activity on addition of Ca2+o. 3. The effect of a submaximal concentration of GTP[S] on the Ca2+-induced activation of glycogen phosphorylase was additive with that of a half-maximally effective concentration of vasopressin. GTP[S] did not increase the effect of a maximally effective concentration of the hormone. 4. Cells loaded with GTP[S] exhibited an increased initial rate of 45Ca2+ exchange measured at 1.3 mM-Ca2+o. 5. GTP[S] did not affect the amount of 45Ca2+ exchanged by cells incubated at 0.1 mM-Ca2+o or the ability of vasopressin to release 45Ca2+ from these cells. 6. It is concluded that the introduction of slowly hydrolysable analogues of GTP to the liver cell cytoplasmic space stimulates the inflow of Ca2+ across the plasma membrane through a channel similar to that activated by vasopressin.

scholarly journals Epidermal Growth Factor Facilitates Epinephrine Inhibition of P2X7-Receptor-Mediated Pore Formation and Apoptosis: A Novel Signaling Network

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 164-174 ◽  
Author(s):  
Liqin Wang ◽  
Ying-Hong Feng ◽  
George I. Gorodeski
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Epithelial Cells ◽  
P2x7 Receptor ◽  
Pore Formation ◽  
Cell Number ◽  
Epidermal Growth ◽  
Phosphoinositide 3 Kinase ◽  
Dependent Mechanism

Epidermal growth factor (EGF), epinephrine, and the P2X7 receptor system regulate growth of human uterine cervical epithelial cells, but little is known about how these systems intercommunicate in exerting their actions. The objective of this study was to understand the mechanisms of EGF and epinephrine regulation of growth of cervical cells. Treatment of cultured CaSki cells with 0.2 nm EGF increased cell number via a PD98059-sensitive pathway. Treatment with 2 nm epinephrine increased cell number, and the effect was facilitated by cotreatment with EGF. Whereas the effect of EGF alone involved up-regulation of [3H]-thymidine incorporation and an increase in cell proliferation, the effect of epinephrine was mediated by inhibition of apoptosis. Epinephrine inhibited apoptosis induced by the P2X7 receptor ligand 2′,3′-0-(4-benzoylbenzoyl)-ATP, by attenuation of P2X7 receptor plasma membrane pore formation. Cotreatment with EGF facilitated epinephrine effect via a phosphoinositide 3-kinase-dependent mechanism. CaSki cells express the β2-adrenoceptor, and the epinephrine antiapoptotic effect could be mimicked by β2-adrenoceptor agonists and by activators of adenylyl cyclase. Likewise, the effect could be blocked by β2-adrenoceptor blockers and by the inhibitor of protein kinase-A H-89. Western immunoblot analysis revealed that epinephrine decreased the levels of the glycosylated 85-kDa form of the P2X7 receptor and increased receptor degradation, and that EGF potentiated these effects of epinephrine. EGF did not affect cellular levels of the β2-adrenoceptor. In contrast, EGF, acting via the EGF receptor, augmented β2-adrenoceptor recycling, and it inhibited β2-adrenoceptor internalization via a phosphoinositide 3-kinase-dependent mechanism. We conclude that, in cervical epithelial cells, EGF has a dual role: as mitogen, acting via the MAPK/MAPK kinase pathway, and as an antiapoptotic factor by facilitating epinephrine effect and resulting in greater expression of β2-adrenoceptors in the plasma membrane. These findings underscore a novel signaling network of communication between the receptor tyrosine kinases, the G protein-coupled receptors, and the purinergic P2X7 receptor.

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