Role of FcαR EC2 region in extracellular membrane localization

A fraction of the nuclear estrogen receptor α (ERα) is localized to the plasma membrane region of 17β-estradiol (E2) target cells. We previously reported that ERα is a palmitoylated protein. To gain insight into the molecular mechanism of ERα residence at the plasma membrane, we tested both the role of palmitoylation and the impact of E2 stimulation on ERα membrane localization. The cancer cell lines expressing transfected or endogenous human ERα (HeLa and HepG2, respectively) or the ERα nonpalmitoylable Cys447Ala mutant transfected in HeLa cells were used as experimental models. We found that palmitoylation of ERα enacts ERα association with the plasma membrane, interaction with the membrane protein caveolin-1, and nongenomic activities, including activation of signaling pathways and cell proliferation (i.e., ERK and AKT activation, cyclin D1 promoter activity, DNA synthesis). Moreover, E2 reduces both ERα palmitoylation and its interaction with caveolin-1, in a time- and dose-dependent manner. These data point to the physiological role of ERα palmitoylation in the receptor localization to the cell membrane and in the regulation of the E2-induced cell proliferation.

Download Full-text

Optogenetic inhibition and activation of Rac and Rap1 using a modified iLID system

10.1101/2020.12.11.421990 ◽

2020 ◽

Author(s):

Nick R. Elston ◽

Michael Pablo ◽

Fred Pimenta ◽

Klaus M. Hahn ◽

Takashi Watanabe

Keyword(s):

Plasma Membrane ◽

Living Cells ◽

Small Gtpases ◽

Spatiotemporal Dynamics ◽

Membrane Localization ◽

Cellular Functions ◽

Activation Kinetics ◽

System 1 ◽

Control Plasma

The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatiotemporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light induced dimerization (iLID) system [1] was used to control plasma membrane localization of protein domains that specifically activate or inactivate Rap1 and Rac (Tiam1 and Chimerin for Rac, RasGRP2 and Rap1GAP for Rap1 [2, 3, 4, 5]). Irradiation yielded a 50% to 230% increase in the concentration of these domains at the membrane, leading to effects on cell morphodynamics consistent with the known roles of Rac1 and Rap1.

Download Full-text

Role of N-Linked Glycosylation in PKR2 Trafficking and Signaling

Frontiers in Neuroscience ◽

10.3389/fnins.2021.730417 ◽

2021 ◽

Vol 15 ◽

Author(s):

Jissele A. Verdinez ◽

Julien A. Sebag

Keyword(s):

Plasma Membrane ◽

Energy Homeostasis ◽

Reproductive Function ◽

Receptor Trafficking ◽

Membrane Localization ◽

Accessory Protein ◽

Post Translational Modification ◽

Physiological Processes ◽

Glycosylation Sites

Prokineticin receptors are GPCRs involved in several physiological processes including the regulation of energy homeostasis, nociception, and reproductive function. PKRs are inhibited by the endogenous accessory protein MRAP2 which prevents them from trafficking to the plasma membrane. Very little is known about the importance of post-translational modification of PKRs and their role in receptor trafficking and signaling. Here we identify 2 N-linked glycosylation sites within the N-terminal region of PKR2 and demonstrate that glycosylation of PKR2 at position 27 is important for its plasma membrane localization and signaling. Additionally, we show that glycosylation at position 7 results in a decrease in PKR2 signaling through Gαs without impairing Gαq/11 signaling.

Download Full-text

Mutation of Hydrophobic Residues in the C-Terminal Domain of the Marburg Virus Matrix Protein VP40 Disrupts Trafficking to the Plasma Membrane

Viruses ◽

10.3390/v12040482 ◽

2020 ◽

Vol 12 (4) ◽

pp. 482 ◽

Cited By ~ 2

Author(s):

Kaveesha J. Wijesinghe ◽

Luke McVeigh ◽

Monica L. Husby ◽

Nisha Bhattarai ◽

Jia Ma ◽

...

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Matrix Protein ◽

Marburg Virus ◽

Membrane Localization ◽

Hydrophobic Residues ◽

Plasma Membrane Localization ◽

Terminal Domain ◽

Loop Region

Marburg virus (MARV) is a lipid-enveloped negative sense single stranded RNA virus, which can cause a deadly hemorrhagic fever. MARV encodes seven proteins, including VP40 (mVP40), a matrix protein that interacts with the cytoplasmic leaflet of the host cell plasma membrane. VP40 traffics to the plasma membrane inner leaflet, where it assembles to facilitate the budding of viral particles. VP40 is a multifunctional protein that interacts with several host proteins and lipids to complete the viral replication cycle, but many of these host interactions remain unknown or are poorly characterized. In this study, we investigated the role of a hydrophobic loop region in the carboxy-terminal domain (CTD) of mVP40 that shares sequence similarity with the CTD of Ebola virus VP40 (eVP40). These conserved hydrophobic residues in eVP40 have been previously shown to be critical to plasma membrane localization and membrane insertion. An array of cellular experiments and confirmatory in vitro work strongly suggests proper orientation and hydrophobic residues (Phe281, Leu283, and Phe286) in the mVP40 CTD are critical to plasma membrane localization. In line with the different functions proposed for eVP40 and mVP40 CTD hydrophobic residues, molecular dynamics simulations demonstrate large flexibility of residues in the EBOV CTD whereas conserved mVP40 hydrophobic residues are more restricted in their flexibility. This study sheds further light on important amino acids and structural features in mVP40 required for its plasma membrane localization as well as differences in the functional role of CTD amino acids in eVP40 and mVP40.

Download Full-text

A Conserved Tryptophan in the Ebola Virus Matrix Protein C-Terminal Domain Is Required for Efficient Virus-Like Particle Formation

Pathogens ◽

10.3390/pathogens9050402 ◽

2020 ◽

Vol 9 (5) ◽

pp. 402 ◽

Cited By ~ 1

Author(s):

Kristen A. Johnson ◽

Rudramani Pokhrel ◽

Melissa R. Budicini ◽

Bernard S. Gerstman ◽

Prem P. Chapagain ◽

...

Keyword(s):

Plasma Membrane ◽

Matrix Protein ◽

Ebola Virus ◽

Membrane Localization ◽

Virus Like Particles ◽

Plasma Membrane Localization ◽

The Matrix ◽

Dynamics Simulations

The Ebola virus (EBOV) harbors seven genes, one of which is the matrix protein eVP40, a peripheral protein that is sufficient to induce the formation of virus-like particles from the host cell plasma membrane. eVP40 can form different structures to fulfil different functions during the viral life cycle, although the structural dynamics of eVP40 that warrant dimer, hexamer, and octamer formation are still poorly understood. eVP40 has two conserved Trp residues at positions 95 and 191. The role of Trp95 has been characterized in depth as it serves as an important residue in eVP40 oligomer formation. To gain insight into the functional role of Trp191 in eVP40, we prepared mutations of Trp191 (W191A or W191F) to determine the effects of mutation on eVP40 plasma membrane localization and budding as well as eVP40 oligomerization. These in vitro and cellular experiments were complemented by molecular dynamics simulations of the wild-type (WT) eVP40 structure versus that of W191A. Taken together, Trp is shown to be a critical amino acid at position 191 as mutation to Ala reduces the ability of VP40 to localize to the plasma membrane inner leaflet and form new virus-like particles. Further, mutation of Trp191 to Ala or Phe shifted the in vitro equilibrium to the octamer form by destabilizing Trp191 interactions with nearby residues. This study has shed new light on the importance of interdomain interactions in stability of the eVP40 structure and the critical nature of timing of eVP40 oligomerization for plasma membrane localization and viral budding.

Download Full-text

Presentation of Integrins on Leukocyte Microvilli: A Role for the Extracellular Domain in Determining Membrane Localization

The Journal of Cell Biology ◽

10.1083/jcb.139.2.563 ◽

1997 ◽

Vol 139 (2) ◽

pp. 563-571 ◽

Cited By ~ 65

Author(s):

M. Abi Abitorabi ◽

Russell K. Pachynski ◽

Ronald E. Ferrando ◽

Mark Tidswell ◽

David J. Erle

Keyword(s):

Cell Body ◽

K562 Cells ◽

Membrane Localization ◽

Cytoplasmic Domains ◽

Blood Leukocytes ◽

Extracellular Domains ◽

Associated Proteins ◽

Cell Adhesion Molecule 1 ◽

Β2 Integrins

Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins α4β7, αLβ2, and αMβ2 were heterologously expressed in K562 cells. α4β7 initiated adhesion under flow and localized to microvilli, whereas β2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the α4β7 cytoplasmic and/or transmembrane domains with the homologous domains of αLβ2 or αMβ2. Unexpectedly, these chimeras efficiently mediated adhesion to the α4β7 ligand mucosal addressin cell adhesion molecule–1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of α4 and β2 integrins do not account for differences in ability to support attachment under flow or in membrane localization. Integrins α4β1, α5β1, α6Aβ1, αvβ3, and αEβ7 also localized to microvilli. Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well. These findings suggest that interactions between the extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli.

Download Full-text

Role of the Membrane Localization Domain of the Pseudomonas aeruginosa Effector Protein ExoU in Cytotoxicity

Infection and Immunity ◽

10.1128/iai.00223-10 ◽

2010 ◽

Vol 78 (8) ◽

pp. 3346-3357 ◽

Cited By ~ 27

Author(s):

Jeff L. Veesenmeyer ◽

Heather Howell ◽

Andrei S. Halavaty ◽

Sebastian Ahrens ◽

Wayne F. Anderson ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Host Cell ◽

Membrane Localization ◽

Effector Protein ◽

Insertion And Deletion ◽

Cell Extracts ◽

Localization Domain ◽

Host Cell Death ◽

Membrane Localization Domain

ABSTRACT ExoU is a potent effector protein that causes rapid host cell death upon injection by the type III secretion system of Pseudomonas aeruginosa. The N-terminal half of ExoU contains a patatin-like phospholipase A2 (PLA2) domain that requires the host cell cofactor superoxide dismutase 1 (SOD1) for activation, while the C-terminal 137 amino acids constitute a membrane localization domain (MLD). Previous studies had utilized insertion and deletion mutations to show that portions of the MLD are required for membrane localization and catalytic activity. Here we further characterize this domain by identifying six residues that are essential for ExoU activity. Substitutions at each of these positions resulted in abrogation of membrane targeting, decreased ExoU-mediated cytotoxicity, and reductions in PLA2 activity. Likewise, each of the six MLD residues was necessary for full virulence in cell culture and murine models of acute pneumonia. Purified recombinant ExoU proteins with substitutions at five of the six residues were not activated by SOD1, suggesting that these five residues are critical for activation by this cofactor. Interestingly, these same five ExoU proteins were partially activated by HeLa cell extracts, suggesting that a host cell cofactor other than SOD1 is capable of modulating the activity of ExoU. These findings add to our understanding of the role of the MLD in ExoU-mediated virulence.

Download Full-text