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1
DiCello, Jesse J., Pradeep Rajasekhar, Emily M. Eriksson, Ayame Saito, Arisbel B. Gondin, Nicholas A. Veldhuis, Meritxell Canals, Simona E. Carbone, and Daniel P. Poole. "Clathrin and GRK2/3 inhibitors block δ-opioid receptor internalization in myenteric neurons and inhibit neuromuscular transmission in the mouse colon." American Journal of Physiology-Gastrointestinal and Liver Physiology 317, no. 2 (August 1, 2019): G79—G89. http://dx.doi.org/10.1152/ajpgi.00085.2019.
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Endocytosis is a major mechanism through which cellular signaling by G protein-coupled receptors (GPCRs) is terminated. However, recent studies demonstrate that GPCRs are internalized in an active state and continue to signal from within endosomes, resulting in effects on cellular function that are distinct to those arising at the cell surface. Endocytosis inhibitors are commonly used to define the importance of GPCR internalization for physiological and pathophysiological processes. Here, we provide the first detailed examination of the effects of these inhibitors on neurogenic contractions of gastrointestinal smooth muscle, a key preliminary step to evaluate the importance of GPCR endocytosis for gut function. Inhibitors of clathrin-mediated endocytosis (Pitstop2, PS2) or G protein-coupled receptor kinase-2/3-dependent phosphorylation (Takeda compound 101, Cmpd101), significantly reduced GPCR internalization. However, they also attenuated cholinergic contractions through different mechanisms. PS2 abolished contractile responses by colonic muscle to SNC80 and morphine, which strongly and weakly internalize δ-opioid and μ-opioid receptors, respectively. PS2 did not affect the increased myogenic contractile activity following removal of an inhibitory neural influence (tetrodotoxin) but suppressed electrically evoked neurogenic contractions. Ca2+ signaling by myenteric neurons in response to exogenous ATP was unaffected by PS2, suggesting inhibitory actions on neurotransmitter release rather than neurotransmission. In contrast, Cmpd101 attenuated contractions to the cholinergic agonist carbachol, indicating direct effects on smooth muscle. We conclude that, although PS2 and Cmpd101 are effective blockers of GPCR endocytosis in enteric neurons, these inhibitors are unsuitable for the study of neurally mediated gut function due to their inhibitory effects on neuromuscular transmission and smooth muscle contractility. NEW & NOTEWORTHY Internalization of activated G protein-coupled receptors is a major determinant of the type and duration of subsequent downstream signaling events. Inhibitors of endocytosis effectively block opioid receptor internalization in enteric neurons. The clathrin-dependent endocytosis inhibitor Pitstop2 blocks effects of opioids on neurogenic contractions of the colon in an internalization-independent manner. These inhibitors also significantly impact cholinergic neuromuscular transmission. We conclude that these tools are unsuitable for examination of the contribution of neuronal G protein-coupled receptor endocytosis to gastrointestinal motility.
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Bigby, M., P. Wang, J. F. Fierro, and M. S. Sy. "Phorbol myristate acetate-induced down-modulation of CD4 is dependent on calmodulin and intracellular calcium." Journal of Immunology 144, no. 8 (April 15, 1990): 3111–16. http://dx.doi.org/10.4049/jimmunol.144.8.3111.
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Abstract PMA causes rapid down-modulation of CD4 molecules on murine immature thymocytes, human PBL, and CD4-positive human tumor cell lines, but not on murine peripheral lymphocytes. The mechanisms of phorbol ester-induced down modulation of CD4 molecules, however, have not been elucidated. To determine how PMA down-modulates CD4 expression by T lymphocytes, we studied the ability of inhibitors of protein kinase C, calmodulin, actin, and tubulin to block PMA-induced modulation of CD4 in several murine and human cell types. We also tested the ability of intracellular and extracellular calcium chelators to block CD4 internalization. There was marked variability in the degree of PMA-induced down-modulation of CD4 among various cell types. The effects of PMA on CD4 expression were greater for murine thymocytes, for human PBL, and for the human lymphoblastic leukemia cell line, MOLT-3, than for any of the other cell types studied. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, blocked phosphorylation but not internalization of CD4 molecules induced by PMA. Therefore, phosphorylation of CD4 molecules by protein kinase C is not required for the internalization of the molecules. Internalization was blocked by both inhibitors of calmodulin, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, and trifluoperazine. PMA-induced internalization of CD4 was blocked by Quin-2 AM, which chelates intracellular calcium. EGTA, which chelates extracellular calcium, did not block internalization. Inhibitors of actin or tubulin did not block internalization. These results suggest that PMA-induced modulation of CD4 can occur in the absence of phosphorylation of the CD4 molecules and is calmodulin and intracellular calcium dependent.
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van Kerkhof, P., and G. J. Strous. "The ubiquitin-proteasome pathway regulates lysosomal degradation of the growth hormone receptor and its ligand." Biochemical Society Transactions 29, no. 4 (August 1, 2001): 488–93. http://dx.doi.org/10.1042/bst0290488.
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The growth hormone (GH) receptor (GHR) is a mammalian plasma membrane protein whose internalization is mediated by the ubiquitin-proteasome pathway. GH internalization and degradation are inhibited when cells are treated with proteasome inhibitors. Here we show that a GHR truncated at residue 369 can enter the cells in the presence of a proteasome inhibitor, but that the subsequent lysosomal degradation of GH is blocked. Lysosomal inhibitors prolong the half-life of both receptor and ligand. Experiments with antibodies against different receptor tail sections show that degradation of the GHR cytosolic domain precedes degradation of the extracellular GH-binding domain. A possible role for the ubiquitin-proteasome pathway in the degradation of the receptor and ligand is discussed.
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Luton, F., M. Buferne, J. Davoust, A. M. Schmitt-Verhulst, and C. Boyer. "Evidence for protein tyrosine kinase involvement in ligand-induced TCR/CD3 internalization and surface redistribution." Journal of Immunology 153, no. 1 (July 1, 1994): 63–72. http://dx.doi.org/10.4049/jimmunol.153.1.63.
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Abstract Triggering of the TCR/CD3 complex can lead to its internalization and modulation from the cell surface. In the present study, we address the question of the dependence of internalization on protein tyrosine kinase (PTK) activation. With use of an activating anti-clonotypic (anti-Ti) mAb on a CTL clone, we have shown that the PTK inhibitors genistein and tyrphostin 25 delayed anti-Ti-induced internalization, but did not affect fluid phase protein uptake or transferrin receptor cycling. Confocal microscopy with use of fluorescent anti-Ti mAb revealed that the inhibition of TCR internalization corresponded to the induction of large patches that were localized in cell membrane areas depleted of polymerized actin, the formation of which was dependent on the combined action of the anti-Ti mAb and the PTK inhibitors. In contrast to the effect of these PTK inhibitors, depletion of Src-like PTKs by T cell pretreatment with herbimycin A led to an increased rate of anti-Ti-induced internalization. Internalization induced by the monovalent Fab fraction of anti-Ti mAb was similarly affected by the PTK inhibitors, although the extent of induced internalization was less by approximately one-half. An analysis of substrates phosphorylated in kinase assays on TCR/CD3 immunoprecipitates of the CTL, which were activated by anti-Ti mAb in both the absence and presence of genistein, identified protein bands in which phosphorylation or association with CD3 was inhibited in the presence of genistein. Thus, a genistein-sensitive PTK activity seems to control ligand-induced TCR/CD3 complex redistribution and internalization.
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Chen, Yang, Shan Wang, Xinan Lu, Haoran Zhang, Yan Fu, and Yongzhang Luo. "Cholesterol sequestration by nystatin enhances the uptake and activity of endostatin in endothelium via regulating distinct endocytic pathways." Blood 117, no. 23 (June 9, 2011): 6392–403. http://dx.doi.org/10.1182/blood-2010-12-322867.
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Abstract Specific internalization of endostatin into endothelial cells has been proved to be important for its biologic functions. However, the mechanism of endostatin internalization still remains elusive. In this study, we report for the first time that both caveolae/lipid rafts and clathrin-coated pits are involved in endostatin internalization. Inhibition of either the caveolae pathway or the clathrin pathway with the use of chemical inhibitors, small interfering RNAs, or dominant-negative mutants alters endostatin internalization in vitro. Intriguingly, cholesterol sequestration by nystatin, a polyene antifungal drug, significantly enhances endostatin uptake by endothelial cells through switching endostatin internalization predominantly to the clathrin-mediated pathway. Nystatin-enhanced internalization of endostatin also increases its inhibitory effects on endothelial cell tube formation and migration. More importantly, combined treatment with nystatin and endostatin selectively enhances endostatin uptake and biodistribution in tumor blood vessels and tumor tissues but not in normal tissues of tumor-bearing mice, ultimately resulting in elevated antiangiogenic and antitumor efficacies of endostatin in vivo. Taken together, our data show a novel mechanism of endostatin internalization and support the potential application of enhancing the uptake and therapeutic efficacy of endostatin via regulating distinct endocytic pathways with cholesterol-sequestering agents.
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Lazari, Maria de Fatima M., Xuebo Liu, Kazuto Nakamura, Jeffrey L. Benovic, and Mario Ascoli. "Role of G Protein-Coupled Receptor Kinases on the Agonist-Induced Phosphorylation and Internalization of the Follitropin Receptor." Molecular Endocrinology 13, no. 6 (June 1, 1999): 866–78. http://dx.doi.org/10.1210/mend.13.6.0289.
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Abstract The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4α, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or Gαt, a scavenger of Gβγ. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and Gαt inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.
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Almeida, Raul A., John R. Dunlap, and Stephen P. Oliver. "Binding of Host Factors Influences Internalization and Intracellular Trafficking ofStreptococcus uberisin Bovine Mammary Epithelial Cells." Veterinary Medicine International 2010 (2010): 1–8. http://dx.doi.org/10.4061/2010/319192.
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We showed that internalization ofStreptococcus uberisinto bovine mammary epithelial cells occurred through receptor- (RME) and caveolae-mediated endocytosis (CME). We reported also that treatment ofS. uberiswith host proteins including lactoferrin (LF) enhanced its internalization into host cells. Since the underlying mechanism(s) involved in such enhancement was unknown we investigated if preincubation ofS. uberiswith host proteins drives internalization of this pathogen into host cells through CME. Thus, experiments involving coculture of collagen-, fibronectin-, and LF-pretreatedS. uberiswith bovine mammary epithelial cells treated with RME and CME inhibitors were conducted. Results showed that internalization of host proteins-pretreatedS. uberisinto mammary epithelial cells treated with RME inhibitors was higher than that of untreated controls. These results suggest that pretreatment with selected host proteins commitsS. uberisto CME, thus avoiding intracellular bactericidal mechanisms and allowing its persistence into bovine mammary epithelial cells.
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Van Hamme, Evelien, Hannah L. Dewerchin, Els Cornelissen, Bruno Verhasselt, and Hans J. Nauwynck. "Clathrin- and caveolae-independent entry of feline infectious peritonitis virus in monocytes depends on dynamin." Journal of General Virology 89, no. 9 (September 1, 2008): 2147–56. http://dx.doi.org/10.1099/vir.0.2008/001602-0.
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Feline infectious peritonitis virus (FIPV), a coronavirus that causes a lethal chronic disease in cats, enters feline monocytes via endocytosis. In this study, the pathway of internalization is characterized by evaluating the effect of chemical inhibitors and/or expression of dominant-negative (DN) proteins on the percentage of internalized virions per cell and infection. Further, co-localization studies were performed to determine the involvement of certain cellular internalization proteins. FIPV is not internalized through a clathrin-mediated pathway, as chlorpromazine, amantadine and DN eps15 did not influence virus uptake and FIPV did not co-localize with clathrin. The caveolae-mediated pathway could be excluded based on the inability of genistein and DN caveolin-1 to inhibit virus uptake and lack of co-localization between FIPV and caveolin-1. Dynamin inhibitory peptide and DN dynamin effectively inhibited virus internalization. The inhibitor strongly reduced uptake to 20.3±1.1 % of uptake in untreated cells. In the presence of DN dynamin, uptake was 58.7±3.9 % relative to uptake in untransduced cells. Internalization of FIPV was slightly reduced to 85.0±1.4 and 87.4±6.1 % of internalization in control cells by the sterol-binding drugs nystatin and methyl-β-cyclodextrin, respectively. Rho GTPases were inhibited by Clostridium difficile toxin B, but no effect was observed. These results were confirmed with infection studies showing that infection was not influenced by chlorpromazine, amantadine and genistein, but was significantly reduced by dynamin inhibition and nystatin. In conclusion, these results indicate that FIPV enters monocytes through a clathrin- and caveolae-independent pathway that strongly depends on dynamin and is slightly sensitive to cholesterol depletion.
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Goretzki, L., and B. M. Mueller. "Receptor-mediated endocytosis of urokinase-type plasminogen activator is regulated by cAMP-dependent protein kinase." Journal of Cell Science 110, no. 12 (June 15, 1997): 1395–402. http://dx.doi.org/10.1242/jcs.110.12.1395.
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Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.
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Huang, Dianshuai, Qingjie Fan, Zhiyi Liu, Shuqin Zhang, Wei Huang, Hongrui Li, Chongyang Liang, and Fei Sun. "An Epitope on EGFR Loading Catastrophic Internalization Serve as a Novel Oncotarget for Hepatocellular Carcinoma Therapy." Cancers 12, no. 2 (February 16, 2020): 456. http://dx.doi.org/10.3390/cancers12020456.
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The precise role of Epidermal Growth Factor Receptor (EGFR) in Hepatocellular carcinoma (HCC) cells is unknown and EGFR inhibitors have not achieved positive clinical results. The rapid and drastic internalization of EGFR has been proved to successfully treat EGFR inhibitor-resistant patients in recent clinical trials. Here, the anti-tumor efficacy of a protein (rLZ-8) from Ganoderma lucidum was evaluated, it was demonstrated that rLZ-8 could bind to EGFR specifically, drastically enter into Hepatoma cells, abrogate endosomal recycling and induce HCC cell death. Surprisingly, we screened a monoclonal antibody which possesses competitive binding site with rLZ-8, it also trigger catastrophic EGFR internalization. This result suggests that it is necessary to investigate the interface of EGFR and rLZ-8 complex. An internalization related epitope (S222/K269) was identified on the dimerization arm of EGFR extracellular domain (ECD). These results suggest vulnerability of HCC cells to catastrophic EGFR internalization that can be targeted by a novel epitope and point to the possible exploitation in the design of anti-EGFR therapeutic biologics for HCC therapy.
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Narazaki, Masashi, Marta Segarra, Xu Hou, Toshio Tanaka, Xuri Li, and Giovanna Tosato. "Oligo-guanosine nucleotide induces neuropilin-1 internalization in endothelial cells and inhibits angiogenesis." Blood 116, no. 16 (October 21, 2010): 3099–107. http://dx.doi.org/10.1182/blood-2010-01-265801.
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Abstract Ligand interaction with cognate cell-surface receptor often promotes receptor internalization, protecting cells from prolonged or excessive signaling from extracellular ligands. Compounds that induce internalization of surface receptors prevent ligand binding to cognate cell-surface receptors serving as inhibitors. Here, we show that synthetic polyriboguanosine (poly G) and oligo-deoxyriboguanosine (oligo G) reduce endothelial levels of surface neuropilin-1 (NRP1), a receptor shared by semaphorin 3A and vascular endothelial growth factor (VEGF), which plays critical roles in angiogenesis. Oligo G also reduces levels of cell-surface scavenger receptor expressed by endothelial cells I (SREC-I), but not levels of NRP2, gp130, CD31, VEGFR-1, or VEGFR-2. Poly or oligo A, T, and C do not promote NRP1 or SREC-I internalization. We find that oligo G binds to NRP1 with high affinity (Kd:1.3 ± 0.16nM), bridges the extracellular domain of NRP1 to that of SREC-I, and induces coordinate internalization of NRP1 and SREC-I. In vitro, oligo G blocks the binding and function of VEGF165 in endothelial cells. In vivo, intravitreal administration of oligo G reduces choroidal neovascularization in mice. These results demonstrate that synthetic oligo G is an inhibitor of pathologic angiogenenesis that reduces cell-surface levels and function of NRP1 acting as an internalization inducer.
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Xi, Chunhua, Xuan Liang, Chunhua Chen, Hasan Babazada, Tianzuo Li, and Renyu Liu. "Hypoxia Induces Internalization of κ-Opioid Receptor." Anesthesiology 126, no. 5 (May 1, 2017): 842–54. http://dx.doi.org/10.1097/aln.0000000000001571.
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Abstract Background It has been demonstrated that κ-opioid receptor agonists can reduce hypoxia–ischemia brain injury in animal models. However, it is unclear how the κ-opioid receptor responds to hypoxia–ischemia. In the current study, the authors used an in vitro model of oxygen–glucose deprivation and reoxygenation to explore how κ-opioid receptors respond to hypoxia and reoxygenation. Methods Mouse neuroblastoma Neuro2A cells were stably transfected with mouse κ-opioid receptor–tdTomato fusion protein or Flag-tagged mouse κ-opioid receptor, divided into several groups (n = 6 to 12), and used to investigate the κ-opioid receptor movement. Observations were performed under normal oxygen, at 30 min to 1 h after oxygen–glucose deprivation and at 1 h after reoxygenation using high-resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of κ-opioid receptor antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor. Results Hypoxic conditions caused the κ-opioid receptor to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific κ-opioid receptor antagonist norbinaltorphimine blocked internalization, suggesting the involvement of activation of a specific κ-opioid receptor. κ-Opioid receptor internalization appears to be reversed by reoxygenation. Quantities of intracellular κ-opioid receptor-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 to 85% (P < 0.01) after oxygen–glucose deprivation. Potassium channel blockers and dopamine receptor inhibitor failed to block hypoxia-induced κ-opioid receptor internalization. Conclusions Hypoxia induces reversible κ-opioid receptor internalization, which was inhibited by selective κ-opioid receptor antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between κ-opioid receptor and hypoxia via κ-opioid receptor activation and the dynamin-dependent mechanism.
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Orloff, G. M., S. L. Orloff, M. S. Kennedy, P. J. Maddon, and J. S. McDougal. "Penetration of CD4 T cells by HIV-1. The CD4 receptor does not internalize with HIV, and CD4-related signal transduction events are not required for entry." Journal of Immunology 146, no. 8 (April 15, 1991): 2578–87. http://dx.doi.org/10.4049/jimmunol.146.8.2578.
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Abstract Receptor binding of HIV to the CD4 molecule is required for efficient infection of T cells, but the post-binding steps that result in penetration of HIV are not well understood. CD4 is induced to internalize upon T cell activation, and mAb to CD4 modify signal transduction and T cell activation as does HIV in some systems. It is not known whether HIV binding triggers CD4 endocytosis or whether signal transduction events are required for penetration. Selected inhibitors of signal transduction were evaluated for their effects on penetration using two assays that are dependent on penetration. After short term exposure to inhibitor and HIV, cells were analyzed for reverse-transcribed HIV DNA (DNA amplification assay), or productive infection is monitored (infectivity assay). Viral penetration was tested in the presence of H7 (protein kinase C inhibition), EGTA (extracellular Ca2+ chelation), cyclosporine A (inhibition of Ca2+/calmodulin-dependent activation), or pertussis toxin (inhibition of G protein function). All agents were used at concentrations that were inhibitory for their respective signal transduction pathways. None of the inhibitors affected viral penetration. We tracked the CD4 molecule with fluorescent probes that do not interfere with HIV binding in a system where CD4 T cells were saturated with HIV and the penetration event was relatively synchronized. Under conditions where detection of CD4 was more sensitive than the detection of HIV, HIV internalization was readily detected but CD4 internalization was not.
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Mueller, Anja, Eamonn Kelly, and Philip G. Strange. "Pathways for internalization and recycling of the chemokine receptor CCR5." Blood 99, no. 3 (February 1, 2002): 785–91. http://dx.doi.org/10.1182/blood.v99.3.785.
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Abstract M-tropic human immunodeficiency virus (HIV-1) strains enter the cell after interaction with their receptors, CD4 and the G-protein–coupled chemokine receptor CCR5. The number of cell surface CCR5 molecules is thought to be important in determining the infection rate for HIV. Cell surface CCR5 is dependent on the rate of receptor internalization and recycling. Internalization of G-protein–coupled receptors after agonist activation is thought to occur either through clathrin-coated pits or through caveolae. In this study, the role of these different pathways was investigated in Chinese hamster ovary cells expressing CCR5 using specific inhibitors. Internalization of CCR5 after chemokine treatment was inhibited by sucrose, indicating a role for the clathrin-coated pit pathway. Activation of CCR5 leads to arrestin-2 movement in the cells, providing further evidence for the involvement of clathrin-coated pits. Nystatin and filipin also affected the rate of internalization of CCR5, indicating a role for caveolae. Using inhibitors of vesicle transport in the cell, it was found that the CCR5 recycling pathway is independent of the Golgi apparatus and late endosomes. Protein synthesis is not involved in receptor recovery. It seems likely that after internalization, CCR5 is directed to early endosomes and subsequently recycled to the cell surface.
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Tompkins, John D., Todd A. Clason, Thomas R. Buttolph, Beatrice M. Girard, Anne K. Linden, Jean C. Hardwick, Laura A. Merriam, Victor May, and Rodney L. Parsons. "Src family kinase inhibitors blunt PACAP-induced PAC1 receptor endocytosis, phosphorylation of ERK, and the increase in cardiac neuron excitability." American Journal of Physiology-Cell Physiology 314, no. 2 (February 1, 2018): C233—C241. http://dx.doi.org/10.1152/ajpcell.00223.2017.
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Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.
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Zhang, Yaguang, Hongmei Shu, Jing Hu, Min Zhang, Junweng Wu, Kehai Liu, and Qing Zhu. "Binding Affinity, Cellular Uptake, and Subsequent Intracellular Trafficking of the Nano-Gene Vector P123-PEI-R13." Journal of Nanomaterials 2016 (2016): 1–8. http://dx.doi.org/10.1155/2016/7064246.
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A nano-gene vector PEI-P123-R13 was synthesized by cross-linking low molecular weight PEI with P123 and further coupling bifunctional peptide R13 to the polymer for targeting tumor and increasing cellular uptake. The binding assessment of R13 toαvβ3 positive cells was performed by HRP labeling. The internalization pathways of P123-PEI-R13/DNA complexes were investigated based on the effect of specific endocytic inhibitors on transfection efficiency. The mechanism of intracellular trafficking was investigated based on the effect of endosome-lysosome acidification inhibitors, cytoskeleton, and dynein inhibitors on transfection efficiency. The results indicated that the bifunctional peptide R13 had the ability of binding toαvβ3 positive cellsin vitro. The modification of P123-PEI-R13 with R13 made it display new property of internalization. P123-PEI-R13/DNA complexes were conducted simultaneously via clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis, and possible energy-independent route. After internalization, P123-PEI-R13/DNA complexes could escape from the endosome-lysosome system because of its acidification and further took microtubule as the track and dynein as the dynamic source to be transported toward the microtubule (+) end, to wit nucleus, under the action of microfilament, and with the aid of intermediate filament.
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Chou, Hsien-Yeh, Namasivayam Elangovan, Ying-Chu Lee, Hsiou Hsia Lin, and Sin-Tak Chu. "Internalization and trafficking of mouse 24p3 protein in L929 cells." Journal of Endocrinology 191, no. 1 (October 2006): 239–47. http://dx.doi.org/10.1677/joe.1.06909.
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It has been shown that mouse 24p3 protein is a dexamethasone-inducible secretory protein which suggests the involvement of an autocrine mechanism in the L929 cell line. The aim of this study was to investigate the possibilityof this mechanism in L929 cells and to also demonstrate further processing of this protein after association with L929 cells. We have characterized the internalization of 24p3 protein in L929 cells with fluorescein isothiocyanate- and Alexa555-labeled protein supplement instead of secreted 24p3 protein. As endocytotic inhibitors could reveal the inhibition of protein internalization, we confirmed the existence of receptor-mediated 24p3 protein internalization in L929 cells by carrying out an inhibition test. Plus-chase experiment of L929 cells with biotinylated 24p3 protein demonstrated a release of internalized 24p3 protein into the extracellular region. The protein recycling inhibitor, bafilomycin A1, arrests the transport of 24p3 protein by accumulating in early endosome, but no effect could be observed in protein release from early to late endosome by nocodazole. Theseresults providethe first evidence on recycling of apo-24p3 protein in L929 cells.
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Matsuoka, Hidetada, and Masumi Inoue. "Src mediates endocytosis of TWIK-related acid-sensitive K+ 1 channels in PC12 cells in response to nerve growth factor." American Journal of Physiology-Cell Physiology 309, no. 4 (August 15, 2015): C251—C263. http://dx.doi.org/10.1152/ajpcell.00354.2014.
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TWIK-related acid-sensitive K+ (TASK) channels produce background K+ currents. We elucidated that TASK1 channels in rat adrenal medullary cells and PC12 cells are internalized in a clathrin-dependent manner in response to nerve growth factor (NGF). Here, the molecular mechanism for this internalization in PC12 cells was explored. The combination of enzyme inhibitors with tropomyosin receptor kinase A mutants revealed that the internalization was mediated by both phospholipase C and phosphatidylinositol 3-kinase pathways that converge on protein kinase C with the consequent activation of Src, a nonreceptor tyrosine kinase. The NGF-induced endocytosis of TASK1 channels did not occur in the presence of the Src inhibitor or with the expression of a kinase-dead Src mutant. Additionally, NGF induced a transient colocalization of Src with the TASK1 channel, but not the TASK1 mutant, in which tyrosine at 370 was replaced with phenylalanine. This TASK1 mutant showed no increase in tyrosine phosphorylation and markedly diminished internalization in response to NGF. We concluded that NGF induces endocytosis of TASK1 channels via tyrosine phosphorylation in its carboxyl terminus.
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Jochen, A. L., and P. Berhanu. "Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes." Journal of Cell Biology 103, no. 5 (November 1, 1986): 1807–16. http://dx.doi.org/10.1083/jcb.103.5.1807.
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To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.
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Janes, Mandy E., K. M. Emily Chu, Adrian J. L. Clark, and Peter J. King. "Mechanisms of Adrenocorticotropin-Induced Activation of Extracellularly Regulated Kinase 1/2 Mitogen-Activated Protein Kinase in the Human H295R Adrenal Cell Line." Endocrinology 149, no. 4 (January 3, 2008): 1898–905. http://dx.doi.org/10.1210/en.2007-0949.
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The role of ACTH in stimulating or inhibiting growth of adrenal cells has been a subject of some controversy. Reports that ACTH may stimulate ERK/MAPK in Y1 cells have suggested a role for cAMP in this process. In attempting to extend this work, the ACTH responses in the human H295R cell line have been studied. This cell line makes only a very modest cAMP response to ACTH, yet the ERK1/2 response is highly reproducible and immediate but not prolonged. It is minimally reduced by the protein kinase A inhibitor, H89, but unaffected by protein kinase C and calcium inhibitors. Inhibition of epidermal growth factor receptor or other tyrosine kinase receptor transactivation was without effect, as was inhibition of c-Src activity or c-Src phosphorylation. The most effective inhibitor of this pathway was dansylcadaverine, an inhibitor of receptor internalization. These findings imply that ACTH-induced ERK1/2 activation in H295R cells is dependent on a mechanism distinct from that by which most G protein-coupled receptors activate ERK1/2 but that nevertheless seems to depend on receptor internalization.
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Nedellec, Rebecca, Mia Coetzer, Michael M. Lederman, Robin E. Offord, Oliver Hartley, and Donald E. Mosier. "“Resistance” to PSC-RANTES Revisited: Two Mutations in Human Immunodeficiency Virus Type 1 HIV-1SF162 or Simian-Human Immunodeficiency Virus SHIVSF162-p3 Do Not Confer Resistance." Journal of Virology 84, no. 11 (March 24, 2010): 5842–45. http://dx.doi.org/10.1128/jvi.01907-09.
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ABSTRACT Resistance of human immunodeficiency virus type 1 (HIV-1) to small-molecule CCR5 inhibitors is well demonstrated, but resistance to macromolecular CCR5 inhibitors (e.g., PSC-RANTES) that act by both CCR5 internalization and receptor blockade had not been reported until recently (3). The report of a single simian-human immunodeficiency virus SHIVSF162-p3 variant with one V3 and one gp41 sequence change in gp160 that conferred both altered replicative fitness and resistance to PSC-RANTES was therefore surprising. We introduced the same two mutations into both the parental HIV-1SF162 and the macaque-adapted SHIVSF162-p3 and found minor differences in entry fitness but no changes in sensitivity to inhibition by either PSC-RANTES or the small-molecule allosteric inhibitor TAK-779. We attribute the earlier finding to confounding fitness effects with inhibitor sensitivity.
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Kierbel, A., A. Gassama-Diagne, K. Mostov, and J. N. Engel. "The Phosphoinositol-3-Kinase–Protein Kinase B/Akt Pathway Is Critical forPseudomonas aeruginosaStrain PAK Internalization." Molecular Biology of the Cell 16, no. 5 (May 2005): 2577–85. http://dx.doi.org/10.1091/mbc.e04-08-0717.
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Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.
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Sneddon, John M., and Wanda M. Wenman. "The effect of ions on the adhesion and internalization of Chlamydia trachomatis by HeLa cells." Canadian Journal of Microbiology 31, no. 4 (April 1, 1985): 371–74. http://dx.doi.org/10.1139/m85-071.
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The adhesion and internalization of Chlamydia trachomatis by HeLa cells was unaffected by removal of K+, Mg2+, or glucose from the incubation medium, slightly reduced by removal of Na+, and significantly reduced by omission of Ca2+. Sr2+, Mg2+, and Mn2+ could replace Ca2+ in the adhesion but only Sr2+ supported internalization, and La3+, Co2+, Fe3+, Ba2+, and Zn2+ all reduced internalization more than adhesion. During initial infection there was no measurable difference in the uptake or release of 45Ca2+ or 86Rb+ between infected and noninfected HeLa monolayers. Infection was not prevented by pretreatment of the monolayers with the calcium channel blockers, verapamil, D600, and nitrendipine, or the calmodulin inhibitors, TMB-8 or trifluperazine. The results suggest that divalent cations are not essential for chlamydial infection but that the process of internalization is facilitated by the presence of cations, particularly Na+ and Ca2+.
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Rabehi, Lila, Théano Irinopoulou, Béatrice Cholley, Nicole Haeffner-Cavaillon, and Marie-Paule Carreno. "Gram-Positive and Gram-Negative Bacteria Do Not Trigger Monocytic Cytokine Production through Similar Intracellular Pathways." Infection and Immunity 69, no. 7 (July 1, 2001): 4590–99. http://dx.doi.org/10.1128/iai.69.7.4590-4599.2001.
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ABSTRACT Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) and Staphylococcus aureus Cowan (SAC), suggesting that gram-positive and gram-negative bacteria may trigger similar intracellular events. Treatment with specific kinase inhibitors prior to cell stimulation dramatically decreased LPS-induced cytokine production. Blocking of the p38 pathway prior to LPS stimulation decreased interleukin-1α (IL-1α), IL-1ra, and tumor necrosis factor alpha (TNF-α) production, whereas blocking of the ERK1/2 pathways inhibited IL-1α, IL-1β, and IL-1ra but not TNF-α production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1α production was decreased in the presence of ERK kinase inhibitor. We also demonstrated that although LPS and SAC have been shown to bind to CD14 before transmitting signals to TLR4 and TLR2, respectively, internalization of CD14 occurred only in monocytes triggered by LPS. Pretreatment of the cells with SB203580, U0126, or a mixture of both inhibitors did not affect internalization of CD14. Altogether, these results suggest that TLR2 signaling does not involve p38 mitogen-activated protein kinase signaling pathways, indicating that divergent pathways are triggered by gram-positive and gram-negative bacteria, thereby inducing cytokine production.
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Khandaker, Masud H., Gordon Mitchell, Luoling Xu, Joseph D. Andrews, Rajkumari Singh, Harry Leung, Joaquı́n Madrenas, Stephen S. G. Ferguson, Ross D. Feldman, and David J. Kelvin. "Metalloproteinases Are Involved in Lipopolysaccharide– and Tumor Necrosis Factor-–Mediated Regulation of CXCR1 and CXCR2 Chemokine Receptor Expression." Blood 93, no. 7 (April 1, 1999): 2173–85. http://dx.doi.org/10.1182/blood.v93.7.2173.
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Abstract The neutrophil-specific G-protein–coupled chemokine receptors, CXCR1 and CXCR2, bind with high affinity to the potent chemoattractant interleukin-8 (IL-8). The mechanisms of IL-8 receptor regulation are not well defined, although previous studies have suggested a process of ligand-promoted internalization as a putative regulatory pathway. Herein, we provide evidence for two distinct processes of CXCR1 and CXCR2 regulation. Confocal microscopy data showed a redistribution of CXCR1 expression from the cell surface of neutrophils to internal compartments after stimulation with IL-8, whereas stimulation with bacterial lipopolysaccharide (LPS) or tumor necrosis factor- (TNF-) did not induce CXCR1 internalization but instead mediated a significant loss of membrane-proximal CXCR1 staining intensity. To investigate whether proteolytic cleavage was the mechanism responsible for LPS- and TNF-–induced downmodulation of IL-8 receptors, we tested a panel of proteinase inhibitors. The downmodulation of CXCR1 and CXCR2 by LPS and TNF- was most dramatically inhibited by metalloproteinase inhibitors; 1,10-phenanthroline and EDTA significantly attenuated LPS- and TNF-–induced loss of CXCR1 and CXCR2 cell surface expression. Metalloproteinase inhibitors also blocked the release of CXCR1 cleavage fragments into the cell supernatants of LPS- and TNF-–stimulated neutrophils. In addition, while treatment of neutrophils with LPS and TNF- inhibited IL-8 receptor–mediated calcium mobilization and IL-8–directed neutrophil chemotaxis, both 1,10-phenanthroline and EDTA blocked these inhibitory processes. In contrast, metalloproteinase inhibitors did not affect IL-8–mediated downmodulation of CXCR1 and CXCR2 cell surface expression or receptor signaling. Thus, these findings may provide further insight into the mechanisms of leukocyte regulation during immunologic and inflammatory responses.
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Khandaker, Masud H., Gordon Mitchell, Luoling Xu, Joseph D. Andrews, Rajkumari Singh, Harry Leung, Joaquı́n Madrenas, Stephen S. G. Ferguson, Ross D. Feldman, and David J. Kelvin. "Metalloproteinases Are Involved in Lipopolysaccharide– and Tumor Necrosis Factor-–Mediated Regulation of CXCR1 and CXCR2 Chemokine Receptor Expression." Blood 93, no. 7 (April 1, 1999): 2173–85. http://dx.doi.org/10.1182/blood.v93.7.2173.407a06_2173_2185.
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The neutrophil-specific G-protein–coupled chemokine receptors, CXCR1 and CXCR2, bind with high affinity to the potent chemoattractant interleukin-8 (IL-8). The mechanisms of IL-8 receptor regulation are not well defined, although previous studies have suggested a process of ligand-promoted internalization as a putative regulatory pathway. Herein, we provide evidence for two distinct processes of CXCR1 and CXCR2 regulation. Confocal microscopy data showed a redistribution of CXCR1 expression from the cell surface of neutrophils to internal compartments after stimulation with IL-8, whereas stimulation with bacterial lipopolysaccharide (LPS) or tumor necrosis factor- (TNF-) did not induce CXCR1 internalization but instead mediated a significant loss of membrane-proximal CXCR1 staining intensity. To investigate whether proteolytic cleavage was the mechanism responsible for LPS- and TNF-–induced downmodulation of IL-8 receptors, we tested a panel of proteinase inhibitors. The downmodulation of CXCR1 and CXCR2 by LPS and TNF- was most dramatically inhibited by metalloproteinase inhibitors; 1,10-phenanthroline and EDTA significantly attenuated LPS- and TNF-–induced loss of CXCR1 and CXCR2 cell surface expression. Metalloproteinase inhibitors also blocked the release of CXCR1 cleavage fragments into the cell supernatants of LPS- and TNF-–stimulated neutrophils. In addition, while treatment of neutrophils with LPS and TNF- inhibited IL-8 receptor–mediated calcium mobilization and IL-8–directed neutrophil chemotaxis, both 1,10-phenanthroline and EDTA blocked these inhibitory processes. In contrast, metalloproteinase inhibitors did not affect IL-8–mediated downmodulation of CXCR1 and CXCR2 cell surface expression or receptor signaling. Thus, these findings may provide further insight into the mechanisms of leukocyte regulation during immunologic and inflammatory responses.
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Kwon, Yong-Jun, Weontae Lee, Auguste Genovesio, and Neil Emans. "A High-Content Subtractive Screen for Selecting Small Molecules Affecting Internalization of GPCRs." Journal of Biomolecular Screening 17, no. 3 (November 15, 2011): 379–85. http://dx.doi.org/10.1177/1087057111427347.
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G-protein–coupled receptors (GPCRs) are pivotal in cellular responses to the environment and are common drug targets. Identification of selective small molecules acting on single GPCRs is complicated by the shared machinery coupling signal transduction to physiology. Here, we demonstrate a high-content screen using a panel of GPCR assays to identify receptor selective molecules acting within the kinase/phosphatase inhibitor family. A collection of 88 kinase and phosphatase inhibitors was screened against seven agonist-induced GPCR internalization cell models as well as transferrin uptake in human embryonic kidney cells. Molecules acting on a single receptor were identified through excluding pan-specific compounds affecting housekeeping endocytosis or disrupting internalization of multiple receptors. We identified compounds acting on a sole GPCR from activities in a broad range of chemical structures that could not be easily sorted by conventional means. Selective analysis can therefore rapidly select compounds selectively affecting GPCR activity with specificity to one receptor class through high-content screening.
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Mortensen, Ole Valente, Mads Breum Larsen, Balakrishna M. Prasad, and Susan G. Amara. "Genetic Complementation Screen Identifies a Mitogen-activated Protein Kinase Phosphatase, MKP3, as a Regulator of Dopamine Transporter Trafficking." Molecular Biology of the Cell 19, no. 7 (July 2008): 2818–29. http://dx.doi.org/10.1091/mbc.e07-09-0980.
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The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization, we used a genetic complementation screen in Xenopus oocytes to identify a mitogen-activated protein (MAP) kinase phosphatase, MKP3/Pyst1/DUSP6, as a molecule that inhibits protein kinase C–induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several well-studied MAP kinases (ERK1/2, p38, and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin-dependent endocytosis.
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Gemei, Marica, Carmine Talarico, Laura Brandolini, Candida Manelfi, Lorena Za, Silvia Bovolenta, Chiara Liberati, et al. "Binding Mode Exploration of B1 Receptor Antagonists’ by the Use of Molecular Dynamics and Docking Simulation—How Different Target Engagement Can Determine Different Biological Effects." International Journal of Molecular Sciences 21, no. 20 (October 16, 2020): 7677. http://dx.doi.org/10.3390/ijms21207677.
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The kinin B1 receptor plays a critical role in the chronic phase of pain and inflammation. The development of B1 antagonists peaked in recent years but almost all promising molecules failed in clinical trials. Little is known about these molecules’ mechanisms of action and additional information will be necessary to exploit the potential of the B1 receptor. With the aim of contributing to the available knowledge of the pharmacology of B1 receptors, we designed and characterized a novel class of allosteric non-peptidic inhibitors with peculiar binding characteristics. Here, we report the binding mode analysis and pharmacological characterization of a new allosteric B1 antagonist, DFL20656. We analyzed the binding of DFL20656 by single point mutagenesis and radioligand binding assays and we further characterized its pharmacology in terms of IC50, B1 receptor internalization and in vivo activity in comparison with different known B1 antagonists. We highlighted how different binding modes of DFL20656 and a Merck compound (compound 14) within the same molecular pocket can affect the biological and pharmacological properties of B1 inhibitors. DFL20656, by its peculiar binding mode, involving tight interactions with N114, efficiently induced B1 receptor internalization and evoked a long-lasting effect in an in vivo model of neuropathic pain. The pharmacological characterization of different B1 antagonists highlighted the effects of their binding modes on activity, receptor occupancy and internalization. Our results suggest that part of the failure of most B1 inhibitors could be ascribed to a lack of knowledge about target function and engagement.
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Baidya, Gaurav, Rameshvar Tiwary, Madeeha Mudassir, Neha Singh, Suman Saha, Kunzang Chosdol, Subrata Sinha, and Parthaprasad Chattopadhyay. "Passive internalization and active extrusion determines PLGA-nanoparticle concentration in cancer cell lines." Nanomedicine 15, no. 23 (August 2020): 2229–39. http://dx.doi.org/10.2217/nnm-2020-0229.
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Background: Poly(lactic-co-glycolic) acid nanoparticle (PLGA-NP) trafficking across cell membranes was investigated to confirm preliminary results that contradicted existing studies. Materials & methods: Uptake and retention of PLGA-NPs at 37 and 4°C in the presence and absence of metabolic inhibitors in various cell lines was estimated. Results: Pulse experiments with metabolic inhibitors and culturing at 4°C demonstrated the predominantly passive nature of PLGA-NP uptake. Chase experiments with metabolic inhibitors indicated the role of active exocytosis in the extrusion of these NPs. PLGA-NPs with ionic or nonionic hydrophilic coats with highly positive or negative ζ-potential also showed similar results. Conclusion: Our study opens up the possibility of modulation of active exocytosis to increase intracellular retention of NPs for an extended period of drug delivery.
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Morinelli, Thomas A., Mi-Hye Lee, Ryan T. Kendall, Louis M. Luttrell, Linda P. Walker, and Michael E. Ullian. "Angiotensin II activates NF-κB through AT1A receptor recruitment of β-arrestin in cultured rat vascular smooth muscle cells." American Journal of Physiology-Cell Physiology 304, no. 12 (June 15, 2013): C1176—C1186. http://dx.doi.org/10.1152/ajpcell.00235.2012.
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Activation of the angiotensin type 1A receptor (AT1AR) in rat aorta vascular smooth muscle cells (RASMC) results in increased synthesis of the proinflammatory enzyme cyclooxygenase-2 (COX-2). We previously showed that nuclear localization of internalized AT1AR results in activation of transcription of the gene for COX-2, i.e., prostaglandin-endoperoxide synthase-2. Others have suggested that ANG II stimulation of COX-2 protein synthesis is mediated by NF-κB. The purpose of the present study was to examine the interrelationship between AT1AR activation, β-arrestin recruitment, and NF-κB activation in the ability of ANG II to increase COX-2 protein synthesis in RASMC. In the present study we utilized RASMC, inhibitors of the NF-κB pathway, β-arrestin knockdown, radioligand binding, immunoblotting, and immunofluorescence to characterize the roles of AT1AR internalization, NF-κB activation, and β-arrestin in ANG II-induced COX-2 synthesis. Ro-106-9920 or parthenolide, agents that inhibit the initial steps of NF-κB activation, blocked ANG II-induced p65 NF-κB nuclear localization, COX-2 protein expression, β-arrestin recruitment, and AT1AR internalization without inhibiting ANG II-induced p42/44 ERK activation. Curcumin, an inhibitor of NF-κB-induced transcription, blocked ANG II-induced COX-2 protein expression without altering AT1AR internalization, ANG II-induced p65 NF-κB nuclear localization, or p42/44 ERK activation. Small interfering RNA-induced knockdown of β-arrestin-1 and -2 inhibited ANG II-induced p65 NF-κB nuclear localization. In vascular smooth muscle cells, internalization of the activated AT1AR mediated by β-arrestins activates the NF-κB pathway, producing nuclear localization of the transcription factor and initiation of COX-2 protein synthesis, thereby linking internalization of the receptor with the NF-κB pathway.
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Apirakaramwong, A., Perayot Pamonsinlapatham, S. Techaarpornkul, Praneet Opanasopit, Suwannee Panomsuk, and S. Soksawatmaekhin. "Mechanisms of Cellular Uptake with Chitosan/DNA Complex in Hepatoma Cell Line." Advanced Materials Research 506 (April 2012): 485–88. http://dx.doi.org/10.4028/www.scientific.net/amr.506.485.
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Chitosan (CS) has a high potential for gene delivery into mammalian cells. However, its uptake mechanism is not well clarified. We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine), caveolae-mediated endocytosis (genistein), macropinocytosis (LY 29004 and wortmannin), microtubuli polymerization (nocodazole) and of membrane cholesterol recycle (methyl-β-cyclodextrin) on the transfection efficiency with CS/pEGFP complexes and on the internalization of CS/rhodamine-labeled pEGFP complexes by hepatoma cell line (Huh 7 cells). The transfection was blocked by nocodazole, genistein, and methyl-β-cyclodextrin, respectively. CS/DNA complexes internalization was clearly inhibited by genistein. We conclude that the complexes uptake predominantly by caveolin-mediated pathways. In addition, fluorescence colocalization studies with acidotropic probes, LysoSensor dye, illustrated that CS/DNA complexes are targeted to lysosomes for the degradation after internalization.
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Casartelli, M., G. Cermenati, S. Rodighiero, F. Pennacchio, and B. Giordana. "A megalin-like receptor is involved in protein endocytosis in the midgut of an insect (Bombyx mori, Lepidoptera)." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 295, no. 4 (October 2008): R1290—R1300. http://dx.doi.org/10.1152/ajpregu.00036.2008.
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The mechanism responsible for fluorescein isothiocyanate (FITC)-albumin internalization by columnar cells in culture obtained from the midgut of Bombyx mori larvae was examined by confocal laser scanning microscopy. Protein uptake changed over time, and it appeared to be energy dependent, since it was strongly reduced by both low temperatures and metabolic inhibitors. Labeled albumin uptake as a function of increasing protein concentration showed a saturation kinetics with a Michaelis constant value of 2.0 ± 0.6 μM. These data are compatible with the occurrence of receptor-mediated endocytosis. RT-PCR analysis and colocalization experiments with an anti-megalin primary antibody indicated that the receptor involved was a putative homolog of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein receptor family, responsible for the uptake of various molecules, albumin included, in many epithelial cells of mammals. This insect receptor, like the mammalian counterpart, required Ca2+ for albumin internalization and was inhibited by gentamicin. FITC-albumin internalization was clathrin mediated, since two inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalized in the intermicrovillar areas of the apical plasma membrane. The integrity of actin and microtubule organization was essential for the correct functioning of the endocytic machinery.
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Kansra, Sanjay, Stefan W. Stoll, Jessica L. Johnson, and James T. Elder. "Autocrine Extracellular Signal-regulated Kinase (ERK) Activation in Normal Human Keratinocytes: Metalloproteinase-mediated Release of Amphiregulin Triggers Signaling from ErbB1 to ERK." Molecular Biology of the Cell 15, no. 9 (September 2004): 4299–309. http://dx.doi.org/10.1091/mbc.e04-03-0233.
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ErbB signaling through extracellular signal-regulated kinase (ERK) has been implicated in regulating the expression of ErbB ligands in hyperproliferative skin disorders and wound healing. Here, we characterize the process of autocrine ERK activation in cultured normal human keratinocytes (NHKs) subjected to growth factor (GF) deprivation. Basal ERK phosphorylation was lower after 48 h than after 24 h of GF deprivation, and lowest at 30–60 min after an additional medium change. ERK phosphorylation was markedly increased by low concentrations of epidermal growth factor (EGF) (0.2–1 ng/ml) that provoked only a limited increase in ErbB1 tyrosine phosphorylation and internalization. Basal ErbB tyrosine phosphorylation and ERK phosphorylation were inhibited by two different ErbB receptor tyrosine kinase inhibitors, by the ErbB1-specific neutralizing monoclonal antibody 225 IgG, by two different metalloproteinase inhibitors, and by neutralizing antibodies against amphiregulin (AR). In contrast, these responses were unaffected by neutralizing antibodies against other ErbB1 ligands or the ErbB2 inhibitors geldanamycin and AG825. The time course of autocrine ERK phosphorylation correlated with the appearance of soluble AR, and two different metalloproteinase inhibitors blocked AR release. These results define an amphiregulin- and ErbB1-dependent mechanism by which autocrine ERK activation is maintained in NHKs, even when ErbB1 autophosphorylation and internalization are limited.
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Estall, Jennifer L., Bernardo Yusta, and Daniel J. Drucker. "Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization." Molecular Biology of the Cell 15, no. 8 (August 2004): 3673–87. http://dx.doi.org/10.1091/mbc.e03-11-0825.
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The intestinotrophic and cytoprotective actions of glucagon-like peptide-2 (GLP-2) are mediated by the GLP-2 receptor (GLP-2R), a member of the class II glucagon-secretin G protein-coupled receptor superfamily. Although native GLP-2 exhibits a short circulating half-life, long-acting degradation-resistant GLP-2 analogues are being evaluated for therapeutic use in human subjects. Accordingly, we examined the mechanisms regulating signaling, internalization, and trafficking of the GLP-2R to identify determinants of receptor activation and desensitization. Heterologous cells expressing the transfected rat or human GLP-2R exhibited a rapid, dose-dependent, and prolonged desensitization of the GLP-2–stimulated cAMP response and a sustained GLP-2–induced decrease in levels of cell surface receptor. Surprisingly, inhibitors of clathrin-dependent endocytosis failed to significantly decrease GLP-2R internalization, whereas cholesterol sequestration inhibited ligand-induced receptor internalization and potentiated homologous desensitization. The hGLP-2R localized to both Triton X-100–soluble and –insoluble (lipid raft) cellular fractions and colocalized transiently with the lipid raft marker caveolin-1. Although GLP-2R endocytosis was dependent on lipid raft integrity, the receptor transiently associated with green fluorescent protein tagged-early endosome antigen 1–positive vesicles and inhibitors of endosomal acidification attenuated the reappearance of the GLP-2R on the cell surface. Our data demonstrate that GLP-2R desensitization and raft-dependent trafficking represent distinct and independent cellular mechanisms and provide new evidence implicating the importance of a clathrin- and dynamin-independent, lipid raft-dependent pathway for homologous G protein-coupled receptor internalization.
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Pratt, Jonathan, Khadidja Haidara, and Borhane Annabi. "MT1-MMP Expression Levels and Catalytic Functions Dictate LDL Receptor-Related Protein-1 Ligand Internalization Capacity in U87 Glioblastoma Cells." International Journal of Molecular Sciences 23, no. 22 (November 17, 2022): 14214. http://dx.doi.org/10.3390/ijms232214214.
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Modulations in cell surface receptor ectodomain proteolytic shedding impact on receptor function and cancer biomarker expression. As such, heavily pursued therapeutic avenues have exploited LDL receptor-related protein-1 (LRP-1)-mediated capacity in internalizing Angiopep-2 (An2), a brain-penetrating peptide that allows An2–drug conjugates to cross the blood–brain tumor barrier (BBTB). Given that LRP-1 is proteolytically shed from the cell surface through matrix metalloproteinase (MMP) activity, the balance between MMP expression/function and LRP-1-mediated An2 internalization is unknown. In this study, we found that membrane type-1 (MT1)-MMP expression increased from grade 1 to 4 brain tumors, while that of LRP-1 decreased inversely. MMP pharmacological inhibitors such as Ilomastat, Doxycycline and Actinonin increased in vitro An2 internalization by up to 2.5 fold within a human grade IV-derived U87 glioblastoma cell model. Transient siRNA-mediated MT1-MMP gene silencing resulted in increased basal An2 cell surface binding and intracellular uptake, while recombinant MT1-MMP overexpression reduced both cell surface LRP-1 expression as well as An2 internalization. The addition of Ilomastat to cells overexpressing recombinant MT1-MMP restored LRP-1 expression at the cell surface and An2 uptake to levels comparable to those observed in control cells. Collectively, our data suggest that MT1-MMP expression status dictates An2-mediated internalization processes in part by regulating cell surface LRP-1 functions. Such evidence prompts preclinical evaluations of combined MMP inhibitors/An2–drug conjugate administration to potentially increase the treatment of high-MT1-MMP-expressing brain tumors.
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Soltész, Dóra, Ildikó Szabó, and Zoltán Bánóczi. "The Balance between Hydrophobicity/Aromaticity and Positively Charged Residues May Influence the Cell Penetration Ability." Pharmaceutics 15, no. 4 (April 18, 2023): 1267. http://dx.doi.org/10.3390/pharmaceutics15041267.
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Cell-penetrating peptides (CPPs) are commonly modified to increase their cellular uptake, alter the mechanism of penetration or enhance their endosomal release. Earlier, we described the internalization enhancement ability of the 4-((4-(dimethylamino)phenyl)azo)benzoyl (Dabcyl) group. We proved that this modification on the N-terminus of tetra- and hexaarginine enhanced their cellular uptake. The introduction of an aromatic ring 4-(aminomethyl) benzoic acid, AMBA) into the peptide backbone has a synergistic effect with Dabcyl, and the tetraarginine derivatives had outstanding cellular uptake. Based on these results, the effect of Dabcyl or Dabcyl-AMBA modification on the internalization of oligoarginines was studied. Oligoarginines were modified with these groups and their internalization was measured using flow cytometry. The concentration dependence of the cellular uptake of selected constructs was compared too. Their internalization mechanism was also examined by using different endocytosis inhibitors. While the effect of the Dabcyl group was optimal for hexaarginine, the Dabcyl-AMBA group increased the cellular uptake in the case of all oligoarginines. All derivatives, with the exception of only tetraarginine, were more effective than the octaarginine control. The internalization mechanism was dependent on the size of the oligoarginine and was independent of the modification. Our findings suggest that these modifications enhanced the internalization of oligoarginines and resulted in novel, very effective CPPs.
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Xu, Yi, Betty Revon Liu, Han-Jung Lee, Katie B. Shannon, Jeffrey G. Winiarz, Tien-Chun Wang, Huey-Jenn Chiang, and Yue-wern Huang. "Nona-Arginine Facilitates Delivery of Quantum Dots into Cells via Multiple Pathways." Journal of Biomedicine and Biotechnology 2010 (2010): 1–11. http://dx.doi.org/10.1155/2010/948543.
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Semiconductor quantum dots (QDs) have recently been used to deliver and monitor biomolecules, such as drugs and proteins. However, QDs alone have a low efficiency of transport across the plasma membrane. In order to increase the efficiency, we used synthetic nona-arginine (SR9), a cell-penetrating peptide, to facilitate uptake. We found that SR9 increased the cellular uptake of QDs in a noncovalent binding manner between QDs and SR9. Further, we investigated mechanisms of QD/SR9 cellular internalization. Low temperature and metabolic inhibitors markedly inhibited the uptake of QD/SR9, indicating that internalization is an energy-dependent process. Results from both the pathway inhibitors and the RNA interference (RNAi) technique suggest that cellular uptake of QD/SR9 is predominantly a lipid raft-dependent process mediated by macropinocytosis. However, involvement of clathrin and caveolin-1 proteins in transducing QD/SR9 across the membrane cannot be completely ruled out.
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Klimovich, Maria, Latifa Zekri, Gundram Jung, and Helmut R. Salih. "Abstract 2886: Antigen internalization and its prevention during treatment with bispecific antibodies." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2886. http://dx.doi.org/10.1158/1538-7445.am2022-2886.
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Abstract Antigen-mediated antibody internalization is an important factor influencing kinetics and efficacy of antibody-based therapeutics. Antibodies directed against HER2, CD20, FLT3, EGFR, CD10 and CD22 have been shown to undergo internalization upon binding to their target antigens. Although antibody-induced internalization may be desirable under some circumstances, for example in case of antibody-drug conjugates, it can subvert the therapeutic efficacy of ADCC-inducing antibodies and bispecific antibodies (bsAbs) aiming to mobilize T cells against cancer. Here we examined the turnover of PSMA, a target antigen widely used for diagnostic and therapeutic interventions in prostate cancer. As a model system, we used prostate cancer cell lines expressing varying levels of PSMA in functional analyses with our PSMAxCD3 bsAb CC-1 which is currently being evaluated in two clinical trials (NCT04104607 and NCT04496674). Antigen internalization kinetics was analyzed by flow cytometric-internalization assays and live-cell imaging of antibody internalization. These analyses revealed that surface PSMA expression is rapidly downregulated after binding of PSMAxCD3 bsAb. Internalization occured in a dose-dependent manner, with complete negativity observed at concentrations ≥0.1 µg/ml. After withdrawal of the bsAb, antigen surface levels were fully restored within 24 hours. PSMA internalization was found to be partially prevented by inhibiting clathrin-dependent endocytosis. Currently, we are screening various clinically approved clathrin-dependent endocytosis inhibitors (e.g. statins and chloroquine) to identify compounds for combinatorial approaches to prevent undesired PSMA internalization in patients receiving PSMAxCD3 bsAbs. Altogether, our results document the involvement of clathrin-dependent endocytosis in bsAbs-driven PSMA internalization and provide a basis for combinatorial approaches to increase the efficacy of PSMA-directed therapeutics. Citation Format: Maria Klimovich, Latifa Zekri, Gundram Jung, Helmut R. Salih. Antigen internalization and its prevention during treatment with bispecific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2886.
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Alam, Syed Benazir, and Marianna Kulka. "Internalization of benzylisoquinoline alkaloids by resting and activated bone marrow-derived mast cells utilizes energy-dependent mechanisms." Inflammation Research 71, no. 3 (January 25, 2022): 343–56. http://dx.doi.org/10.1007/s00011-021-01526-2.
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Abstract Objective and design Drug delivery to inflammatory cells is dependent upon poorly understood, complex endocytic processes. Berberine (BBR), a benzylisoquinoline alkaloid, binds to heparin and targets glycosaminoglycan-rich granules in mast cells (MC), but the mechanism of BBR internalization is unknown. Methods BMMC were treated with various concentrations of BBR for different amounts of time and BBR internalization was assessed by flow cytometry and fluorescence microscopy. BMMC were pretreated with endocytic inhibitors or a growth factor (IL-3) prior to BBR exposure to access mechanisms of its internalization. Results After 24 h, 48 ± 0.8% of BMMC internalized BBR and this process was dependent upon temperature and the presence of glucose in the medium. Methanol fixation reduced BBR internalization, suggesting the involvement of an energy-dependent active transport mechanism. To determine mode of internalization, BBR was encapsulated into Lipofectamine TM lipoplexes since these are known to circumvent classical endocytic pathways. Incorporating BBR into lipoplexes decreased BBR internalization by 26% and 10% (10 μg/ml and 100 μg/ml Lipo-BBR respectively) by BMMC. BBR endocytosis was significantly reduced by Latrunculin B (88%), Cytochalasin B (87%), Chloroquine (86.5%) and 3-methyladenine (91%), indicating that actin polymerization, lysosomal pH and lysosomal self-degradation via the autophagy pathway was involved. In contrast, IL-3 treatment significantly enhanced BBR endocytosis (54% by 40 ng/ml IL-3) suggesting that IL-3 signaling pathways play a role in internalization. Conclusions Our data suggests that internalization of BBR by resting and IL-3-activated BMMC utilizes an energy-dependent pathway that is dependent upon glucose metabolism and temperature. Furthermore, this process requires actin polymerization and lysosomal trafficking. These data suggest internalization of benzylisoquinoline compounds is an active and complex process.
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Jochen, Albert, and Paulos Berhanu. "Effects of metalloendoprotease inhibitors on insulin binding, internalization and processing in adipocytes." Biochemical and Biophysical Research Communications 142, no. 1 (January 1987): 205–12. http://dx.doi.org/10.1016/0006-291x(87)90472-4.
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Setiadi, Hendra, and Rodger P. McEver. "Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow." Journal of Cell Biology 163, no. 6 (December 15, 2003): 1385–95. http://dx.doi.org/10.1083/jcb.200307178.
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Flowing leukocytes roll on P-selectin that is mobilized from secretory granules to the surfaces of endothelial cells after stimulation with histamine or thrombin. Before it is internalized, P-selectin clusters in clathrin-coated pits, which enhances its ability to support leukocyte rolling. We found that thrombin and histamine induced comparable exocytosis of P-selectin on endothelial cells. However, compared with histamine, thrombin decreased the recruitment of P-selectin into clathrin-coated pits, slowed the internalization of P-selectin, and reduced the number and stability of neutrophils rolling on P-selectin. Significantly more RhoA was activated in thrombin- than in histamine-stimulated endothelial cells. Inhibitors of RhoA or its effector, Rho kinase, reversed thrombin's ability to inhibit the internalization and adhesive function of P-selectin in endothelial cells. Experiments with transfected cells confirmed that the inhibitory actions of thrombin and Rho kinase on P-selectin required its cytoplasmic domain. Thus, a signaling event affects both the function and clearance of a protein that enters the constitutive clathrin-mediated endocytic pathway.
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Basagiannis, Dimitris, Sofia Zografou, Evangeli Goula, Despoina Gkeka, Evangelos Kolettas, and Savvas Christoforidis. "Chemical Inhibitors of Dynamin Exert Differential Effects in VEGF Signaling." Cells 10, no. 5 (April 23, 2021): 997. http://dx.doi.org/10.3390/cells10050997.
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VEGFR2 is the main receptor and mediator of the vasculogenic and angiogenic activity of VEGF. Activated VEGFR2 internalizes through clathrin-mediated endocytosis and macropinocytosis. As dynamin is a key regulator of the clathrin pathway, chemical inhibitors of dynamin are commonly used to assess the role of the clathrin route in receptor signaling. However, drugs may also exert off-target effects. Here, we compare the effects of three dynamin inhibitors, dynasore, dyngo 4a and dynole, on VEGFR2 internalization and signaling. Although these drugs consistently inhibit clathrin-mediated endocytosis of both transferrin (a typical cargo of this route) and VEGFR2, surprisingly, they exert contradictory effects in receptor signaling. Thus, while dynasore has no effect on phosphorylation of VEGFR2, the other two drugs are strong inhibitors. Furthermore, although dyngo does not interfere with phosphorylation of Akt, dynasore and dynole have a strong inhibitory effect. These inconsistent effects suggest that the above dynamin blockers, besides inhibiting dynamin-dependent endocytosis of VEGFR2, exert additional inhibitory effects on signaling that are independent of endocytosis; i.e., they are due to off-target effects. Using a recently developed protocol, we comparatively validate the specificity of two endocytic inhibitors, dynasore and EIPA. Our findings highlight the importance of assessing whether the effect of an endocytic drug on signaling is specifically due to its interference with endocytosis or due to off-targets.
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Snyers, Luc, Hannes Zwickl, and Dieter Blaas. "Human Rhinovirus Type 2 Is Internalized by Clathrin-Mediated Endocytosis." Journal of Virology 77, no. 9 (May 1, 2003): 5360–69. http://dx.doi.org/10.1128/jvi.77.9.5360-5369.2003.
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ABSTRACT Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K+ depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-β-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.
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Li, Ruifang, Chen Chen, Sha Zhu, Xueqin Wang, Yanhui Yang, Weini Shi, Sijia Chen, Congcong Wang, Lixing Yan, and Jiaofan Shi. "CGA-N9, an antimicrobial peptide derived from chromogranin A: direct cell penetration of and endocytosis by Candida tropicalis." Biochemical Journal 476, no. 3 (February 5, 2019): 483–97. http://dx.doi.org/10.1042/bcj20180801.
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Abstract CGA-N9 is a peptide derived from the N-terminus of human chromogranin A comprising amino acids 47–55. Minimum inhibitory concentration (MIC) assays showed that CGA-N9 had antimicrobial activity and exhibited time-dependent inhibition activity against Candida tropicalis, with high safety in human red blood cells (HRBCs) and mouse brain microvascular endothelial cells (bEnd.3). According to the results of transmission electron microscopy (TEM), flow cytometry and confocal microscopy, CGA-N9 accumulated in cells without destroying the integrity of the cell membrane; the peptide was initially localized to the cell membrane and subsequently internalized into the cytosol. An investigation of the cellular internalization mechanism revealed that most CGA-N9 molecules entered the yeast cells, even at 4°C and in the presence of sodium azide (NaN3), both of which block all energy-dependent transport mechanisms. In addition, peptide internalization was affected by the endocytic inhibitors 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), cytochalasin D (CyD) and heparin; chlorpromazine (CPZ) also had some effect on CGA-N9 internalization. Similar results were obtained in the MIC assays, whereby the anticandidal activity of CGA-N9 was blocked to different degrees in the presence of EIPA, CyD, heparin or CPZ. Therefore, most CGA-N9 passes through the C. tropicalis cell membrane via direct cell penetration, whereas the remainder enters through macropinocytosis and sulfate proteoglycan-mediated endocytosis, with a slight contribution from clathrin-mediated endocytosis.
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Singh, Raman Deep, Vishwajeet Puri, Jacob T. Valiyaveettil, David L. Marks, Robert Bittman, and Richard E. Pagano. "Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids." Molecular Biology of the Cell 14, no. 8 (August 2003): 3254–65. http://dx.doi.org/10.1091/mbc.e02-12-0809.
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We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (>80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80–90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM1, was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM1, normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo “pathway switching” when cav-1 levels are low.
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York, Randall D., Derek C. Molliver, Savraj S. Grewal, Paula E. Stenberg, Edwin W. McCleskey, and Philip J. S. Stork. "Role of Phosphoinositide 3-Kinase and Endocytosis in Nerve Growth Factor-Induced Extracellular Signal-Regulated Kinase Activation via Ras and Rap1." Molecular and Cellular Biology 20, no. 21 (November 1, 2000): 8069–83. http://dx.doi.org/10.1128/mcb.20.21.8069-8083.2000.
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ABSTRACT Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.
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Giorgetti-Peraldi, S., E. Ottinger, G. Wolf, B. Ye, T. R. Burke, and S. E. Shoelson. "Cellular effects of phosphotyrosine-binding domain inhibitors on insulin receptor signaling and trafficking." Molecular and Cellular Biology 17, no. 3 (March 1997): 1180–88. http://dx.doi.org/10.1128/mcb.17.3.1180.
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Shc and insulin receptor substrate 1 (IRS-1) are cytoplasmic substrates of tyrosine kinase receptors that engage, localize, and activate downstream SH2 enzymes. Each contains a phosphotyrosine-binding (PTB) domain that is structurally unrelated to SH2 domains. We have designed high-affinity, cellular inhibitors of the Shc PTB domain by incorporating nonnatural, phosphatase-resistant amino acids into short peptides. None of the inhibitors bind the IRS-1 PTB domain, consistent with distinct specificities for domains. The best inhibitor of the Shc domain was introduced by electroporation into Rat1 fibroblasts that express human insulin receptors. Insulin-stimulated phosphorylation of Shc was inhibited, with no effect on IRS-1, and downstream effects on mitogen-activated protein kinase and DNA synthesis were both inhibited. The PTB domain inhibitor had less influence on epidermal growth factor-induced effects and essentially no impact on serum- or phorbol ester-induced effects. The inhibitor did not affect insulin internalization and its degradation. We conclude that the PTB domain of Shc is critical for its phosphorylation by the insulin receptor, that Shc is an important mediator of insulin's mitogenic effects, and that Shc is not central to insulin receptor cycling in these cells. PTB domains can be inhibited selectively in cells and represent potential targets for drug discovery.
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Kitchens, Kelly M., Rohit B. Kolhatkar, Peter W. Swaan, and Hamidreza Ghandehari. "Endocytosis Inhibitors Prevent Poly(amidoamine) Dendrimer Internalization and Permeability across Caco-2 Cells." Molecular Pharmaceutics 5, no. 2 (January 4, 2008): 364–69. http://dx.doi.org/10.1021/mp700089s.
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Smart, EJ, DC Foster, YS Ying, BA Kamen, and RG Anderson. "Protein kinase C activators inhibit receptor-mediated potocytosis by preventing internalization of caveolae." Journal of Cell Biology 124, no. 3 (February 1, 1994): 307–13. http://dx.doi.org/10.1083/jcb.124.3.307.
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Potocytosis is an endocytic pathway that utilizes glycosylphosphatidylinositol-anchored membrane proteins and caveolae to concentrate and internalize small molecules. We now report that activators of protein kinase C are potent inhibitors of potocytosis. Activators such as phorbol-12-myristate-13-acetate (PMA) inhibit the internalization of receptors for 5-methyltetrahydrofolate but allow the internal receptor pool to return to the cell surface. PMA does not affect the clustering of the folate receptor but instead markedly reduces the number of caveolae. Exposure to PMA totally blocks the intracellular accumulation of 5-methyltetrahydrofolate without affecting receptor-independent uptake or the formation of polyglutamylated species of 5-methyltetrahydrofolate in the cytoplasm. These data suggest that PMA inhibits uptake by inactivating caveolae internalization.