scholarly journals PI3P signaling regulates receptor sorting but not transport in the endosomal pathway

2003 ◽  
Vol 162 (6) ◽  
pp. 971-979 ◽  
Author(s):  
A. Petiot ◽  
J. Fauré ◽  
H. Stenmark ◽  
J. Gruenberg
Keyword(s):  
Growth Factor ◽  
Signaling Pathway ◽  
Egf Receptor ◽  
Fluid Phase ◽  
Kinase Substrate ◽  
Early Endosomes ◽  
Endosomal Pathway ◽  
Bulk Transport

While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

scholarly journals An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor

2008 ◽  
Vol 180 (6) ◽  
pp. 1205-1218 ◽  
Author(s):  
Ingrid Roxrud ◽  
Camilla Raiborg ◽  
Nina Marie Pedersen ◽  
Espen Stang ◽  
Harald Stenmark
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Coated Pits ◽  
Kinase Substrate ◽  
Epidermal Growth

Down-regulation of activated and ubiquitinated growth factor (GF) receptors by endocytosis and subsequent lysosomal degradation ensures attenuation of GF signaling. The ubiquitin-binding adaptor protein Eps15 (epidermal growth factor receptor [EGFR] pathway substrate 15) functions in endocytosis of such receptors. Here, we identify an Eps15 isoform, Eps15b, and demonstrate its expression in human cells and conservation across vertebrate species. Although both Eps15 and Eps15b interact with the endosomal sorting protein Hrs (hepatocyte growth factor–regulated tyrosine kinase substrate) in vitro, we find that Hrs specifically binds Eps15b in vivo (whereas adaptor protein 2 preferentially interacts with Eps15). Although Eps15 mainly localizes to clathrin-coated pits at the plasma membrane, Eps15b localizes to Hrs-positive microdomains on endosomes. Eps15b overexpression, similarly to Hrs overexpression, inhibits ligand-mediated degradation of EGFR, whereas Eps15 is without effect. Similarly, depletion of Eps15b but not Eps15 delays degradation and promotes recycling of EGFR. These results indicate that Eps15b is an endosomally localized isoform of Eps15 that is present in the Hrs complex via direct Hrs interaction and important for the sorting function of this complex.

scholarly journals The Neuroprotective Effects of Insulin-Like Growth Factor 1 via the Hippo/YAP Signaling Pathway is Mediated by the PI3K/AKT Pathway Following Cerebral Ischemia-Reperfusion Injury

2021 ◽  
Author(s):  
Pian Gong ◽  
Yichun Zou ◽  
Wei Zhang ◽  
Qi Tian ◽  
Shoumeng Han ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Growth Factor ◽  
Signaling Pathway ◽  
Neuronal Death ◽  
Infarct Volume ◽  
Akt Signaling ◽  
Akt Signaling Pathway ◽  
Neuroprotective Effects

Abstract Insulin-like growth factor 1 (IGF-1) exhibits neuroprotective properties, such as vasodilatory and anti-inflammatory effects following ischemic stroke. However, the specific molecular mechanisms of action of IGF-1 following ischemic stroke remain elusive. We wanted to explore whether IGF-1 regulates Hippo/YAP signaling pathway, potentially via activation of the PI3K/AKT signaling pathway to exert its neuroprotective effects following ischemic stroke. In the in vitro study, we used oxygen–glucose deprivation to injure cultured PC12 and SH-5YSY cells, and cortical primary neurons. Cell viability was measured using CCK-8 assay. For the in vivo analyses, Sprague–Dawley rats were subjected to middle cerebral artery occlusion; neurological function was assessed using the neurological deficit score; infarct volume was measured using triphenyltetrazolium chloride staining, and neuronal death and apoptosis was evaluated by TUNEL staining, H&E staining and Nissl staining. Western blot was used to measure the levels of YAP/TAZ, PI3K and phosphorylated AKT (p-AKT) both in vitro and in vivo. We found that IGF-1 induced activation of YAP/TAZ, which resulted in improved cell viability in vitro, and decreased neurological deficits, neuronal death and apoptosis, and cerebral infarct volume in vivo. Notably, the neuroprotective effects of IGF-1 were reversed by an inhibitor of the PI3K/AKT signaling pathway, LY294002, which not only reduced expressions of PI3K and p-AKT, but also down-regulated expression of YAP/TAZ, leading to aggravation of neurological dysfunction. These findings indicate that neuroprotective effect of IGF-1 is partly realized by up-regulation of YAP/TAZ, which is mediated by activation of the PI3K/AKT signaling pathway following cerebral ischemic stroke.

RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis

2000 ◽  
Vol 113 (16) ◽  
pp. 2837-2844 ◽  
Author(s):  
V. Jullien-Flores ◽  
Y. Mahe ◽  
G. Mirey ◽  
C. Leprince ◽  
B. Meunier-Bisceuil ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Growth Factor ◽  
Egf Receptor ◽  
The Other ◽  
Ras Signaling ◽  
Receptor Endocytosis ◽  
Epidermal Growth ◽  
Two Hybrid

RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral-RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. Mu2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates mu2 from mu1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.

scholarly journals Differential Downregulation of E-Cadherin and Desmoglein by Epidermal Growth Factor

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Miquella G. Chavez ◽  
Christian A. Buhr ◽  
Whitney K. Petrie ◽  
Angela Wandinger-Ness ◽  
Donna F. Kusewitt ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Wound Repair ◽  
Egf Receptor ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Epidermal Growth ◽  
E Cadherin

Modulation of cell : cell junctions is a key event in cutaneous wound repair. In this study we report that activation of the epidermal growth factor (EGF) receptor disrupts cel : cell adhesion, but with different kinetics and fates for the desmosomal cadherin desmoglein and for E-cadherin. Downregulation of desmoglein preceded that of E-cadherinin vivoand in an EGF-stimulatedin vitrowound reepithelialization model. Dual immunofluorescence staining revealed that neither E-cadherin nor desmoglein-2 internalized with the EGF receptor, or with one another. In response to EGF, desmoglein-2 entered a recycling compartment based on predominant colocalization with the recycling marker Rab11. In contrast, E-cadherin downregulation was accompanied by cleavage of the extracellular domain. A broad-spectrum matrix metalloproteinase inhibitor protected E-cadherin but not the desmosomal cadherin, desmoglein-2, from EGF-stimulated disruption. These findings demonstrate that although activation of the EGF receptor regulates adherens junction and desmosomal components, this stimulus downregulates associated cadherins through different mechanisms.

scholarly journals Targeted Antiepidermal Growth Factor Receptor (Cetuximab) Immunoliposomes Enhance Cellular Uptake In Vitro and Exhibit Increased Accumulation in an Intracranial Model of Glioblastoma Multiforme

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Joachim Høg Mortensen ◽  
Maria Jeppesen ◽  
Linda Pilgaard ◽  
Ralf Agger ◽  
Meg Duroux ◽  
...  
Keyword(s):  
Growth Factor ◽  
Glioblastoma Multiforme ◽  
Egf Receptor ◽  
Xenograft Model ◽  
Growth Factor Receptor ◽  
Liposomal Formulations ◽  
Cancer Types ◽  
Epidermal Growth

Therapeutic advances do not circumvent the devastating fact that the survival rate in glioblastoma multiforme (GBM) is less than 5%. Nanoparticles consisting of liposome-based therapeutics are provided against a variety of cancer types including GBM, but available liposomal formulations are provided without targeting moieties, which increases the dosing demands to reach therapeutic concentrations with risks of side effects. We prepared PEGylated immunoliposomes (ILs) conjugated with anti-human epidermal growth factor receptor (EGFR) antibodies Cetuximab (α-hEGFR-ILs). The affinity of the α-hEGFR-ILs for the EGF receptor was evaluated in vitro using U87 mg and U251 mg cells and in vivo using an intracranial U87 mg xenograft model. The xenograft model was additionally analyzed with respect to permeability to endogenous albumin, tumor size, and vascularization. The in vitro studies revealed significantly higher binding of α-hEGFR-ILs when compared with liposomes conjugated with isotypic nonimmune immunoglobulin. The uptake and internalization of the α-hEGFR-ILs by U87 mg cells were further confirmed by 3D deconvolution analyses. In vivo, the α-hEGFR-ILs accumulated to a higher extent inside the tumor when compared to nonimmune liposomes. The data show that α-hEGFR-ILs significantly enhance the uptake and accumulation of liposomes in this experimental model of GBM suggestive of improved specific nanoparticle-based delivery.

scholarly journals Plasticity of early endosomes

1992 ◽  
Vol 103 (2) ◽  
pp. 335-348 ◽  
Author(s):  
R.G. Parton ◽  
P. Schrotz ◽  
C. Bucci ◽  
J. Gruenberg
Keyword(s):  
Fluid Phase ◽  
Early Endosome ◽  
Cross Linking ◽  
High Plasticity ◽  
Late Endosomes ◽  
Low Concentrations ◽  
Cell Surface Biotinylation ◽  
Early Endosomes

We observed that the structural organization of early endosomes was significantly modified after cell surface biotinylation followed by incubation in the presence of low concentrations of avidin. Under these conditions early endosomes increased in size to form structures which extended over several micrometers and which had an intra-luminal content with a characteristic electron-dense appearance. The modified early endosomes were not formed when either avidin or biotinylation was omitted, suggesting that they resulted from the cross-linking of internalized biotinylated proteins by avidin. Accumulation of a fluid-phase tracer was increased after the avidin-biotin treatment (145% after 45 min). Both recycling and transport to the late endosomes still occurred, albeit to a somewhat lower extent than in control cells. Quantitative electron microscopy showed that the volume of the endosomal compartment was increased approximately 1.5-fold but that the surface area of the compartment decreased relative to its volume after avidin-biotin treatment. Finally, overexpression of a rab5 mutant, which is known to inhibit early endosome fusion in vitro, prevented the formation of these structures in vivo and caused early endosome fragmentation. Altogether, our data suggest that early endosomes exhibit a high plasticity in vivo. Cross-linking appears to interfere with this dynamic process but does not arrest membrane traffic to/from early endosomes.

scholarly journals Coronavirus Genetically Redirected to the Epidermal Growth Factor Receptor Exhibits Effective Antitumor Activity against a Malignant Glioblastoma

2009 ◽  
Vol 83 (15) ◽  
pp. 7507-7516 ◽  
Author(s):  
Monique H. Verheije ◽  
Martine L. M. Lamfers ◽  
Thomas Würdinger ◽  
Guy C. M. Grinwis ◽  
Winald R. Gerritsen ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Human Tumor ◽  
Adaptor Protein ◽  
Progeny Virus ◽  
Epidermal Growth ◽  
Positive Strand Rna

ABSTRACT Coronaviruses are positive-strand RNA viruses with features attractive for oncolytic therapy. To investigate this potential, we redirected the coronavirus murine hepatitis virus (MHV), which is normally unable to infect human cells, to human tumor cells by using a soluble receptor (soR)-based expression construct fused to an epidermal growth factor (EGF) receptor targeting moiety. Addition of this adapter protein to MHV allowed infection of otherwise nonsusceptible, EGF receptor (EGFR)-expressing cell cultures. We introduced the sequence encoding the adaptor protein soR-EGF into the MHV genome to generate a self-targeted virus capable of multiround infection. The resulting recombinant MHV was viable and had indeed acquired the ability to infect all glioblastoma cell lines tested in vitro. Infection of malignant human glioblastoma U87ΔEGFR cells gave rise to release of progeny virus and efficient cell killing in vitro. To investigate the oncolytic capacity of the virus in vivo, we used an orthotopic U87ΔEGFR xenograft mouse model. Treatment of mice bearing a lethal intracranial U87ΔEGFR tumor by injection with MHVsoR-EGF significantly prolonged survival compared to phosphate-buffered saline-treated (P = 0.001) and control virus-treated (P = 0.004) animals, and no recurrent tumor load was observed. However, some adverse effects were seen in normal mouse brain tissues that were likely caused by the natural murine tropism of MHV. This is the first demonstration of oncolytic activity of a coronavirus in vivo. It suggests that nonhuman coronaviruses may be attractive new therapeutic agents against human tumors.

scholarly journals miR-142-5p regulates the progression of diabetic retinopathy by targeting IGF1

2020 ◽  
Vol 34 ◽  
pp. 205873842090904 ◽  
Author(s):  
Xiuming Liu ◽  
Jianchang Li ◽  
Xiaofeng Li
Keyword(s):  
Cell Proliferation ◽  
Diabetic Retinopathy ◽  
Growth Factor ◽  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Igf1r Signaling

As one of leading causes of blindness, diabetic retinopathy (DR) is a progressive microvascular complication of diabetes mellitus (DM). Despite significant efforts have been devoted to investigate DR over the years, the molecular mechanisms still remained unclear. Emerging evidences demonstrated that microRNAs (miRNAs) were tightly associated with pathophysiological development of DR. Hence, this study was aimed to illustrate the role and molecular mechanisms of miR-412-5p in progression of DR. Streptozotocin (STZ) treatment in rats and human retinal endothelial cell (HREC) models were used to simulate DR conditions in vivo and in vitro. Hematoxylin-eosin (HE) staining was used to demonstrate the morphology of retinal tissues of rats. Qualitative real-time polymerase chain reaction (qRT-PCR) detected miR-142-5p and vascular endothelial growth factor (VEGF) expression levels. Cell counting kit-8 (CCK8) assay and immunofluorescence (IF) measured the cell proliferation rates. Western blot tested the expression status of IGF1/IGF1R-mediated signaling pathway. Dual-luciferase reporter assays demonstrated the molecular mechanism of miR-142-5p. miR-142-5p level was down-regulated in retinal tissues of DR rats and high glucose (HG)-treated HRECs. Insulin-like growth factor 1 (IGF1) was identified as a direct target of miR-142-5p. The reduced miR-142-5p level enhanced HRECs proliferation via activating IGF/IGF1R-mediated signaling pathway including p-PI3K, p-ERK, p-AKT, and VEGF activation, ultimately giving rise to cell proliferation. Either miR-142-5p overexpression or IGF1 knockdown alleviated the pathological effects on retinal tissues in DR rats. Collectively, miR-142-5p participated in DR development by targeting IGF1/p-IGF1R signaling pathway and VEGF generation. This miR-142-5p/IGF1/VEGF axis provided a novel therapeutic target for DR clinical treatment.

scholarly journals ADAM17 is the Principal Ectodomain Sheddase of the EGF-Receptor Ligand Amphiregulin

2017 ◽  
Author(s):  
Vishnu Hosur ◽  
Michelle L. Farley ◽  
Lisa M. Burzenski ◽  
Leonard D. Shultz ◽  
Michael V. Wiles
Keyword(s):  
Wound Healing ◽  
Growth Factor ◽  
Egf Receptor ◽  
Skin Diseases ◽  
Ectodomain Shedding ◽  
Receptor Ligand ◽  
Epithelial Cancers ◽  
Protein Levels

AbstractThe epidermal growth factor (EGF)-receptor ligand amphiregulin (AREG) is a potent growth factor implicated in proliferative skin diseases and in primary and metastatic epithelial cancers. AREG in vitro, synthesized as a pro-peptide, requires conversion to an active peptide by metalloproteases by a process known as ectodomain shedding. Although, (Adam17) a disintegrin and metalloprotease 17 is implicated in ectodomain shedding of AREG, it remains to be established in vivo whether ADAM17 contributes to AREG shedding. In the present study, using a curly bare (Rhbdf2cub) mouse model that shows loss-of-hair, enlarged sebaceous glands, and rapid cutaneous wound-healing phenotypes mediated by enhanced Areg mRNA and protein levels, we sought to identify the principal ectodomain sheddase of AREG. To this end, we generated Rhbdf2cub mice lacking ADAM17 specifically in the skin and examined the above phenotypes of Rhbdf2cub mice. We find that ADAM17 deficiency in the skin of Rhbdf2cub mice restores a full hair coat, prevents sebaceous-gland enlargement, and impairs the rapid wound-healing phenotype observed in Rhbdf2cub mice. Furthermore, in vitro, stimulated shedding of AREG is abolished in Rhbdf2cub mouse embryonic keratinocytes lacking ADAM17. Thus, our data demonstrate that ADAM17 is the major ectodomain sheddase of AREG.

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