scholarly journals Translocation of TMEM175 Lysosomal Potassium Channel to the Plasma Membrane by Dynasore Compounds

2021 ◽  
Vol 22 (19) ◽  
pp. 10515
Author(s):  
Enikő Pergel ◽  
Irén Veres ◽  
Gergely Imre Csigi ◽  
Gábor Czirják
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Protein ◽  
Intracellular Localization ◽  
Growth Factor Receptor ◽  
Direct Interaction ◽  
Dominant Negative ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Increased Risk ◽  
Inhibitor Compounds

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson’s disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.

scholarly journals Ezrin Phosphorylation at T567 Modulates Cell Migration, Mechanical Properties, and Cytoskeletal Organization

2020 ◽  
Vol 21 (2) ◽  
pp. 435 ◽  
Author(s):  
Xiaoli Zhang ◽  
Luis R. Flores ◽  
Michael C. Keeling ◽  
Kristina Sliogeryte ◽  
Núria Gavara
Keyword(s):  
Mechanical Properties ◽  
Plasma Membrane ◽  
Cell Migration ◽  
Intracellular Localization ◽  
Dominant Negative ◽  
Cell Stiffness ◽  
Cell Cortex ◽  
Image Quantification ◽  
Cytoskeleton Organization ◽  
Nuclear Changes

Ezrin, a member of the ERM (ezrin/radixin/moesin) family of proteins, serves as a crosslinker between the plasma membrane and the actin cytoskeleton. By doing so, it provides structural links to strengthen the connection between the cell cortex and the plasma membrane, acting also as a signal transducer in multiple pathways during migration, proliferation, and endocytosis. In this study, we investigated the role of ezrin phosphorylation and its intracellular localization on cell motility, cytoskeleton organization, and cell stiffness, using fluorescence live-cell imaging, image quantification, and atomic force microscopy (AFM). Our results show that cells expressing constitutively active ezrin T567D (phosphomimetic) migrate faster and in a more directional manner, especially when ezrin accumulates at the cell rear. Similarly, image quantification results reveal that transfection with ezrin T567D alters the cell’s gross morphology and decreases cortical stiffness. In contrast, constitutively inactive ezrin T567A accumulates around the nucleus, and although it does not impair cell migration, it leads to a significant buildup of actin fibers, a decrease in nuclear volume, and an increase in cytoskeletal stiffness. Finally, cell transfection with the dominant negative ezrin FERM domain induces significant morphological and nuclear changes and affects actin, microtubules, and the intermediate filament vimentin, resulting in cytoskeletal fibers that are longer, thicker, and more aligned. Collectively, our results suggest that ezrin’s phosphorylation state and its intracellular localization plays a pivotal role in cell migration, modulating also biophysical properties, such as membrane–cortex linkage, cytoskeletal and nuclear organization, and the mechanical properties of cells.

scholarly journals Direct Interactions between Calcitonin-Like Receptor (CLR) and CGRP-Receptor Component Protein (RCP) Regulate CGRP Receptor Signaling

Endocrinology ◽  
2012 ◽  
Vol 153 (4) ◽  
pp. 1850-1860 ◽  
Author(s):  
Sophie C. Egea ◽  
Ian M. Dickerson
Keyword(s):  
Cell Surface ◽  
Transmembrane Protein ◽  
Direct Interaction ◽  
Dominant Negative ◽  
In Vivo Studies ◽  
Cgrp Receptor ◽  
Receptor Component ◽  
Soluble Fusion Protein ◽  
Component Protein

Calcitonin gene-related peptide (CGRP) is a neuropeptide with multiple neuroendocrine roles, including vasodilation, migraine, and pain. The receptor for CGRP is a G protein-coupled receptor (GPCR) that requires three proteins for function. CGRP binds to a heterodimer composed of the GPCR calcitonin-like receptor (CLR) and receptor activity-modifying protein (RAMP1), a single transmembrane protein required for pharmacological specificity and trafficking of the CLR/RAMP1 complex to the cell surface. In addition, the CLR/RAMP1 complex requires a third protein named CGRP-receptor component protein (RCP) for signaling. Previous studies have demonstrated that depletion of RCP from cells inhibits CLR signaling, and in vivo studies have demonstrated that expression of RCP correlates with CLR signaling and CGRP efficacy. It is not known whether RCP interacts directly with CLR to exert its effect. The current studies identified a direct interaction between RCP and an intracellular domain of CLR using yeast two-hybrid analysis and coimmunoprecipitation. When this interacting domain of CLR was expressed as a soluble fusion protein, it coimmunoprecipitated with RCP and inhibited signaling from endogenous CLR. Expression of this dominant-negative domain of CLR did not significantly inhibit trafficking of CLR to the cell surface, and thus RCP may not have a chaperone function for CLR. Instead, RCP may regulate CLR signaling in the cell membrane, and direct interaction between RCP and CLR is required for CLR activation. To date, RCP has been found to interact only with CLR and represents a novel neuroendocrine regulatory step in GPCR signaling.

scholarly journals Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways

2001 ◽  
Vol 154 (3) ◽  
pp. 535-548 ◽  
Author(s):  
Vishwajeet Puri ◽  
Rikio Watanabe ◽  
Raman Deep Singh ◽  
Michel Dominguez ◽  
Jennifer C. Brown ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Human Skin ◽  
Growth Factor Receptor ◽  
Dominant Negative ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Cellular Functions ◽  
Intracellular Targeting ◽  
Direct Demonstration ◽  
Early Endosomes

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent (“caveolar-like”) mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a “vital marker” for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.

Functional characterization of an insulin-responsive glucose transporter (GLUT4) from fish adipose tissue

2004 ◽  
Vol 287 (2) ◽  
pp. E348-E357 ◽  
Author(s):  
Encarnación Capilla ◽  
Mònica Díaz ◽  
Amaya Albalat ◽  
Isabel Navarro ◽  
Jeffrey E. Pessin ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Plasma Membrane ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Intracellular Localization ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes ◽  
Native Form ◽  
Protein Motifs ◽  
Glucose Transporter Glut4

Glucose transport across the plasma membrane is mediated by a family of glucose transporter proteins (GLUTs), several of which have been identified in mammalian, avian, and, more recently, in fish species. Here, we report on the cloning of a salmon GLUT from adipose tissue with a high sequence homology to mammalian GLUT4 that has been named okGLUT4. Kinetic analysis of glucose transport following expression in Xenopus laevis oocytes demonstrated a 7.6 ± 1.4 mM Km for 2-deoxyglucose (2-DG) transport measured under zero- trans conditions and 14.4 ± 1.5 mM by equilibrium exchange of 3- O-methylglucose. Transport of 2-DG by okGLUT4-injected oocytes was stereospecific and was competed by d-glucose, d-mannose, and, to a lesser extent, d-galactose and d-fructose. In addition, 2-DG uptake was inhibited by cytochalasin B and ethylidene glucose. Moreover, insulin stimulated glucose uptake in Xenopus oocytes expressing okGLUT4 and in isolated trout adipocytes, which contain the native form of okGLUT4. Despite differences in protein motifs important for insulin-stimulated translocation of mammalian GLUT4, okGLUT4 was able to translocate to the plasma membrane from intracellular localization sites in response to insulin when expressed in 3T3-L1 adipocytes. These data demonstrate that okGLUT4 is a structural and functional fish homolog of mammalian GLUT4 but with a lower affinity for glucose, which could in part explain the lower ability of fish to clear a glucose load.

scholarly journals With-No-Lysine Kinase 1 (WNK1) Augments TRPV4 Function in the Aldosterone-Sensitive Distal Nephron

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1482
Author(s):  
Viktor N. Tomilin ◽  
Kyrylo Pyrshev ◽  
Naghmeh Hassanzadeh Khayyat ◽  
Oleg Zaika ◽  
Oleh Pochynyuk
Keyword(s):  
Collecting Duct ◽  
Chinese Hamster ◽  
Dominant Negative ◽  
K Channel ◽  
Apical Plasma Membrane ◽  
Dependent Manner ◽  
Distal Nephron ◽  
Potassium Homeostasis ◽  
Wnk Kinases ◽  
K Secretion

Kidneys play a central role in regulation of potassium homeostasis and maintenance of plasma K+ levels within a narrow physiological range. With-no-lysine (WNK) kinases, specifically WNK1 and WNK4, have been recognized to regulate K+ balance, in part, by orchestrating maxi K+ channel (BK)-dependent K+ secretion in the aldosterone-sensitive distal nephron (ASDN), which includes the connecting tubule and collecting duct. We recently demonstrated that the Ca2+-permeable TRPV4 channel is essential for BK activation in the ASDN. Furthermore, high K+ diet increases TRPV4 activity and expression largely in an aldosterone-dependent manner. In the current study, we aimed to test whether WNK kinases contribute to regulation of TRPV4 activity and its stimulation by aldosterone. Systemic inhibition of WNK with WNK463 (1 mg/kgBW for 3 days) markedly decreased TRPV4-dependent Ca2+ influx in freshly isolated split-opened collecting ducts. Aldosterone greatly increased TRPV4 activity and expression in cultured mpkCCDc14 cells and this effect was abolished in the presence of WNK463. Selective inhibition of WNK1 with WNK-in-11 (400 nM, 24 h) recapitulated the effects of WNK463 on TRPV4-dependent Ca2+ influx. Interestingly, WNK-in-11 did not interfere with up-regulation of TRPV4 expression by aldosterone, but prevented translocation of the channel to the apical plasma membrane. Furthermore, co-expression of TRPV4 and WNK1 into Chinese hamster ovary (CHO) cells increased the macroscopic TRPV4-dependent cation currents. In contrast, over-expression of TRPV4 with a dominant negative WNK1 variant (K233M) decreased the whole-cell currents, suggesting both stimulatory and permissive roles of WNK1 in regulation of TRPV4 activity. Overall, we show that WNK1 is essential for setting functional TRPV4 expression in the ASDN at the baseline and in response to aldosterone. We propose that this new mechanism contributes to regulation of K+ secretion and, by extension, urinary K+ levels to maintain systemic potassium homeostasis.

scholarly journals Interaction of the Putative Human Cytomegalovirus Portal Protein pUL104 with the Large Terminase Subunit pUL56 and Its Inhibition by Benzimidazole-d-Ribonucleosides

2005 ◽  
Vol 79 (23) ◽  
pp. 14660-14667 ◽  
Author(s):  
Alexandra Dittmer ◽  
John C. Drach ◽  
Leroy B. Townsend ◽  
Anke Fischer ◽  
Elke Bogner
Keyword(s):  
Dna Replication ◽  
Human Cytomegalovirus ◽  
Unit Length ◽  
Large Subunit ◽  
Intracellular Localization ◽  
Direct Interaction ◽  
Portal Protein ◽  
Specific Association ◽  
Carboxy Terminal

ABSTRACT Herpesvirus DNA replication leads to unit length genomes that are translocated into preformed procapsids through a unique portal vertex. The translocation is performed by the terminase that cleaves the DNA and powers the insertion by its ATPase activity. Recently, we demonstrated that the putative human cytomegalovirus (HCMV) portal protein, pUL104, also forms high-molecular-weight complexes. Analyses now have been performed to determine the intracellular localization and identification of interaction partners of pUL104. In infected cells, HCMV pUL104 was found to be predominantly localized throughout the nucleus as well as in cytoplasmic clusters at late times of infection. The latter localization was abolished by phosphonoacetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL104 colocalized with pUL56, the large subunit of the HCMV terminase. Specific association of in vitro translated pUL104 with the carboxy-terminal half of GST-UL56C was detected. By using coimmunoprecipitations a direct interaction with pUL56 was confirmed. In addition, this interaction was no longer detected when the benzimidazole-d-nucleosides BDCRB or Cl4RB were added, thus indicating that these HCMV inhibitors block the insertion of the DNA into the capsid by preventing a necessary interaction of pUL56 with the portal. Electron microscopy revealed that in the presence of Cl4RB DNA is not packaged into capsids and these capsids failed to egress from the nucleus. Furthermore, pulsed-field gel electrophoresis showed that DNA concatemers synthesized in the presence of the compound failed to be processed.

scholarly journals Epidermal Growth Factor Receptor Distribution during Chemotactic Responses

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

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

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