scholarly journals Translocatable voltage-gated Ca2+ channel β subunits in α1–β complexes reveal competitive replacement yet no spontaneous dissociation

2018 ◽  
Vol 115 (42) ◽  
pp. E9934-E9943 ◽  
Author(s):  
Jun-Hee Yeon ◽  
Cheon-Gyu Park ◽  
Bertil Hille ◽  
Byung-Chang Suh
Keyword(s):  
Plasma Membrane ◽  
Channel Gating ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Loop ◽  
Drug Induced ◽  
Interaction Domain ◽  
Intact Cells ◽  
Voltage Gated ◽  
The Stability

β subunits of high voltage-gated Ca2+ (CaV) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of CaV α1B–β interactions by rapamycin-translocatable CaV β subunits that allow drug-induced sequestration and uncoupling of the β subunit from CaV2.2 channel complexes in intact cells. Without CaV α1B/α2δ1, all modified β subunits, except membrane-tethered β2a and β2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing CaV α1B/α2δ1 subunits, the translocatable β subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound β subunits are very stable. However, the interaction becomes dynamic when other competing β isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipid. Thus, expression of free β isoforms around the channel reveals a dynamic aspect to the α1B–β interaction. On the other hand, translocatable β subunits with AID-binding site mutations are easily dissociated from CaV α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P2 sensitivity. Furthermore, the mutations slow CaV2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable β subunits work similarly in CaV2.3 channels. In sum, the strong interaction of CaV α1B–β subunits can be overcome by other free β isoforms, permitting dynamic changes in channel properties in intact cells.

scholarly journals Cell surface expression of murine, rat, and human Fc receptors by Xenopus oocytes.

1984 ◽  
Vol 160 (2) ◽  
pp. 606-611 ◽  
Author(s):  
E Pure ◽  
A D Luster ◽  
J C Unkeless
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Surface Expression ◽  
Gamma Interferon ◽  
Membrane Receptors ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes ◽  
High Avidity ◽  
U937 Cells ◽  
Mouse Igg2a

We report that Xenopus laevis oocytes can efficiently translate and insert heterologous membrane receptors into the oocyte plasma membrane, where they can be detected by the binding of either monoclonal antibodies or ligands. Thus, oocytes injected with mRNA from the mouse J774 macrophage-like cell line, the rat RBL-1 basophilic leukemia, and the U937 promonocyte cell line, bound 2.4G2 Fab, rat IgE, and mouse IgG2a, respectively. The increase in the high avidity Fc gamma R observed after gamma-interferon induction of U937 cells was also observed after injection of mRNA from gamma-interferon-induced U937 cells into oocytes. This suggests either much greater message stability or a greater rate of transcription of Fc gamma Rhi mRNA in the gamma-interferon-induced cells. The assay affords a sensitive method for the detection of rare mRNA species that code for plasma membrane proteins.

Effect of aging on CD11b and CD69 surface expression by vesicular insertion in human polymorphonuclear leucocytes

1999 ◽  
Vol 97 (3) ◽  
pp. 323-329 ◽  
Author(s):  
J. M. NOBLE ◽  
G. A. FORD ◽  
T. H. THOMAS
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Elderly Subjects ◽  
Polymorphonuclear Leucocytes ◽  
Cd11b Expression ◽  
Cd69 Expression ◽  
Vesicle Exocytosis

The exocytosis of intracellular vesicles is an important function of the plasma membrane, which is responsible for hormone secretion, cell surface expression of antigens, ion transporters and receptors, and intracellular and intercellular signalling. Human aging is associated with many physiological and cellular changes, many of which are due to alterations in plasma membrane functioning. Alterations in vesicle externalization with age could account for many of these changes. We investigated whether alterations in vesicle exocytosis occur with increasing age by flow-cytometric determination of CD11b and CD69 expression on the surface of human polymorphonuclear leucocytes (PMN) stimulated with phorbol myristate acetate (PMA), a tumour promoter which binds to and activates protein kinase C (PKC) directly, or with formyl-Met-Leu-Phe (fMLP), which activates PKC indirectly via interactions with a cell surface receptor and G-protein, and subsequent inositol phosphate hydrolysis. Following stimulation with PMA, a decrease in the proportion of PMN expressing CD69 at high levels was observed in elderly compared with young subjects (young, 55.3%; elderly, 43.9%; P = 0.01). No aging-related differences in the proportion of PMN expressing CD11b (young, 73.7%; elderly, 68.4%; P = 0.15), or in the number of molecules of CD69 or CD11b expressed per cell, were observed. Stimulation with fMLP or low PMA concentrations resulted in full CD11b expression but minimal CD69 expression in both young and elderly subjects. Cells which expressed CD69 had no CD11b expression, while those cells expressing CD11b had minimal CD69 expression. Thus the PMA-induced expression of CD11b and CD69 in human PMN represents two separate processes, only one of which is affected in aging. CD11b expression appears to require a lesser degree of PKC stimulation compared with that required for CD69 expression. The age-associated reduction in PMA-stimulated CD69 expression may occur either at or distal to PKC activation. Such a decrease may contribute to the age-associated impairments in PMN function that contribute, in turn, to immunosenescence.

scholarly journals GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

2016 ◽  
Vol 36 (7) ◽  
pp. 1152-1163 ◽  
Author(s):  
Maoxiang Zhang ◽  
Jason E. Davis ◽  
Chunman Li ◽  
Jie Gao ◽  
Wei Huang ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Molecular Mechanisms ◽  
Specific Interaction ◽  
Adaptor Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Loop ◽  
Interaction Sites ◽  
G Protein Coupled

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α2-adrenergic receptors (α2-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at thetrans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.

Mapping the Assembly of Band 3 and Rhesus Multi-Protein Complexes During Erythropoiesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 812-812
Author(s):  
Emile van den Akker ◽  
Timothy J. Satchwell ◽  
Geoff Daniels ◽  
Ashley M Toye
Keyword(s):  
Plasma Membrane ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Surface Expression ◽  
Band 3 ◽  
Membrane Expression ◽  
Intact Cells ◽  
Cell Lysates ◽  
Protein 4.2 ◽  
Pronase Treatment

Abstract Abstract 812 Band 3 forms the core of a large multiprotein complex in the erythrocyte membrane, the Band 3 macrocomplex, which also includes proteins of the Rhesus complex (Rh and RhAG). Mutations in genes encoding proteins within this complex can result in hereditary spherocytosis with varying severity. The effect of distinct mutations and deficiencies in proteins of the Band 3 macrocomplex has been studied in detail in mature erythrocytes. This revealed important functional and structural properties of individual proteins and their relationships with other proteins within the Band 3 macrocomplex. Nevertheless, considerably less is know about the spatio-temporal mechanisms that direct the formation of the Band 3 macrocomplex, and that may explain the aberrations in the complex observed in spherocytosis. Therefore, we studied expression and mutual interactions of proteins of the band3 macrocomplex during development of proerythroblasts to reticulocytes. Using confocal microscopy and western blotting, significant pools of intracellular Band 3 and RhAG were found in the basophilic normoblast. These intracellular pools gradually decreased in the polychromatic normoblast and were absent or low in the orthochromatic normoblast and reticulocytes, while surface expression increased. We used pronase treatment of intact cells to remove extracellular epitopes of BRIC 6 (Band 3 antibody) and LA1818 (RhAG antibody) to study the mechanism by which the intracellular pool of Band 3 and RhAG contributes to formation of the Band 3 complex on the cell surface. Pronase treatment of cells incubated with cycloheximide to block protein synthesis resulted in a reduced but still significant reappearance of BRIC6 (Band 3) and LA1818 (RhAG) epitopes on the plasma membrane confirming the presence of intracellular Band 3 and RhAG pools. It also showed that the bulk of Band 3 and RhAG is synthesized and trafficked to the membrane between the early basophilic and polychromatic stage. Immuneprecipitation of Band 3 from cell lysates of pronase treated cells pre-treated with brefeldin A to collapse the Golgi showed no increase in co-immuneprecipitated protein 4.2 albeit an increase in intracellular Band 3 expression. This suggests that protein 4.2 and Band 3 interact in the first Golgi compartment or late ER. In addition, pre-treatment of cells with cycloheximide prior to pronase treatment resulted in depletion of the intracellular Band 3 and co-immuneprecipitated protein 4.2 pool indicating that Band 3 and protein 4.2 traffic as a complex to the plasma-membrane. We were unable to co-immuneprecipitate Rh or Band 3 with intracellular pools of RhAG, whereas Rh was co-immuneprecipitated with RhAG from the plasma-membrane and from total cell lysates. Knockdown of RhAG in differentiating erythroblasts revealed a concomitant drop in membrane expression of Rh, leaving Band 3 unaffected, indicating that plasma-membrane expression of Rh but not Band 3 is dependent on RhAG. In conclusion, despite the described association between the RhAG complex and the Band 3 complex in erythrocytes, the data suggest that the Band 3-protein 4.2 complex traffics and assembles independently from Rh and RhAG during erythroid differentiation. The experiments suggest that Rh and RhAG do not traffic as a complex to the plasma-membrane but probably assemble in the plasma-membrane. The RhAG knockdown experiments suggest that the dependency of Rh on RhAG as observed in Rhnull syndrome erythrocytes (“Rh regulator type”) originates early during erythropoiesis. Band3 surface expression was not affected upon RhAG knock down, which re-produced the unperturbed Band 3 levels seen in these patients. Disclosures: No relevant conflicts of interest to declare.

Evidence that the C Terminus of the A Subunit Suppresses Thyrotropin Receptor Constitutive Activity

Endocrinology ◽  
2003 ◽  
Vol 144 (9) ◽  
pp. 3821-3827 ◽  
Author(s):  
Chun-Rong Chen ◽  
Gregorio D. Chazenbalk ◽  
Sandra M. McLachlan ◽  
Basil Rapoport
Keyword(s):  
Surface Expression ◽  
Structural Effect ◽  
Cell Surface Expression ◽  
Constitutive Activity ◽  
Intact Cells ◽  
Lh Receptor ◽  
B Subunit ◽  
C Terminus ◽  
Tryptic Cleavage ◽  
A Subunit

Abstract The TSH receptor (TSHR), unlike the LH receptor (LHR), has considerable ligand-independent adenylyl cyclase activity, a feature of pathophysiological importance. The TSHR ectodomain partially suppresses constitutive activity, an effect reversed by trypsin treatment of intact cells. Localizing the functional site of trypsin action would provide insight into how the TSHR ectodomain exerts its constraint. For this purpose, we examined the effect of trypsin on intact cells expressing a series of modified TSHR. Trypsin did not increase cAMP production by a chimeric TSH-LH receptor involving substitution of TSHR residues 261–418 (the ectodomain C terminus). In contrast, with the wild-type TSHR, trypsin enhanced constitutive activity despite mutation of the following potential tryptic cleavage sites [arginine (R) and lysine (K) residues]: 1) K565, K651, K660 in the extracellular loops of the serpentine region; 2) B subunit juxtamembrane residues K371, K401, K415; 3) A subunit residues R310, R312, K313. We previously excluded K337 and K339 from being implicated in TSHR tryptic activation. By exclusion, only one R/K cluster remains as a possible target for the functional effect of trypsin, namely K287, K290, K291, and R293. Mutation of this cluster is incompatible with TSHR cell surface expression. However, tryptic clipping at this locus would reproduce a previously demonstrated structural effect of trypsin on the TSHR, removal of about a 2-kDa polypeptide fragment extending downstream from the locus to the C terminus of the A subunit. Taken together, these data suggest that the C terminus of the A subunit functions as a suppressor of TSHR constitutive activity.

scholarly journals BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis

2014 ◽  
Vol 205 (2) ◽  
pp. 233-249 ◽  
Author(s):  
Pascal Béguin ◽  
Kazuaki Nagashima ◽  
Ramasubbu N. Mahalakshmi ◽  
Réjan Vigot ◽  
Atsuko Matsunaga ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Functional Characterization ◽  
Regulatory Protein ◽  
Binding Pocket ◽  
Neuroendocrine Cells ◽  
Cell Surface Expression ◽  
Channel Activity ◽  
Interaction Domain ◽  
Voltage Gated ◽  
Domain I

Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca2+-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca2+ overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain–binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca2+ channel activity at the plasma membrane, resulting in the inhibition of Ca2+-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity.

scholarly journals GLUT8 Subcellular Localization and Absence of Translocation to the Plasma Membrane in PC12 Cells and Hippocampal Neurons

Endocrinology ◽  
2005 ◽  
Vol 146 (11) ◽  
pp. 4727-4736 ◽  
Author(s):  
Mathieu Widmer ◽  
Marc Uldry ◽  
Bernard Thorens
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Cell Surface ◽  
Pc12 Cells ◽  
Hippocampal Neurons ◽  
Osmotic Shock ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Compartment ◽  
Early Endosomes

GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.

Sign in / Sign up

Export Citation Format

Share Document