scholarly journals The role of prolines and glycine in the transmembrane domain of LAT

2020 ◽  
Author(s):  
Daniela Glatzová ◽  
Harsha Mavila ◽  
Maria Chiara Saija ◽  
Tomáš Chum ◽  
Lukasz Cwiklik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Helical Structure ◽  
Surface Expression ◽  
Transmembrane Segment ◽  
Proline Residue ◽  
And Function ◽  
The Impact

ABSTRACTLAT is a critical regulator of T cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and superresolution imaging and flow cytometry we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics is further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of non-palmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

Interactions of mouse glycophorin A with the dRTA-related mutant G719D of the mouse Cl–/HCO3– exchanger Ae1This paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (2) ◽  
pp. 224-235 ◽  
Author(s):  
Andrew K. Stewart ◽  
Fouad T. Chebib ◽  
Syed W. Akbar ◽  
Maria J. Salas ◽  
Rajan A. Sonik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Peer Review Process ◽  
Surface Expression ◽  
Amino Acid Sequences ◽  
Glycophorin A ◽  
Specific Expression ◽  
Health And Disease ◽  
Renal Tubular

The AE1 mutation G701D, associated with recessive distal renal tubular acidosis (dRTA), produces only minimal erythroid phenotype, reflecting erythroid-specific expression of stimulatory AE1 subunit glycophorin A (GPA). GPA transgene expression could theoretically treat recessive dRTA in patients and in mice expressing cognate Ae1 mutation G719D. However, human (h) GPA and mouse (m) Gpa amino acid sequences are widely divergent, and mGpa function in vitro has not been investigated. We therefore studied in Xenopus oocytes the effects of coexpressed mGpa and hGPA on anion transport by erythroid (e) and kidney (k) isoforms of wild-type mAe1 (meAe1, mkAe1) and of mAe1 mutant G719D. Coexpression of hGPA or mGpa enhanced the function of meAe1 and mkAe1 and rescued the nonfunctional meAe1 and mkAe1 G719D mutants through increased surface expression. Progressive N-terminal truncation studies revealed a role for meAe1 amino acids 22–28 in GPA-responsiveness of meAe1 G719D. MouseN-cyto/humanTMD and humanN-cyto/mouseTMD kAE1 chimeras were active and GPA-responsive. In contrast, whereas chimera mkAe1N-cyto/hkAE1 G701DTMD was GPA-responsive, chimera hkAE1N-cyto/mkAe1 G719DTMD was GPA-insensitive. Moreover, whereas the isolated transmembrane domain (TMD) of hAE1 G701D was GPA-responsive, that of mAe1 G719D was GPA-insensitive. Thus, mGpa increases surface expression and activity of meAe1 and mkAe1. However, the G719D mutation renders certain mAe1 mutant constructs GPA-unresponsive and highlights a role for erythroid-specific meAe1 amino acids 22–28 in GPA-responsiveness.

Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5

2001 ◽  
Vol 281 (1) ◽  
pp. C215-C223 ◽  
Author(s):  
Robert T. Watson ◽  
Jeffrey E. Pessin
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Glucose Transporter ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Snare Protein ◽  
Glut 4 ◽  
Golgi Localization ◽  
Syntaxin 3

Insulin recruits glucose transporter 4 (GLUT-4) vesicles from intracellular stores to the plasma membrane in muscle and adipose tissue by specific interactions between the vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) protein VAMP-2 and the target membrane SNARE protein syntaxin 4. Although GLUT-4 vesicle trafficking has been intensely studied, few have focused on the mechanism by which the SNAREs themselves localize to specific membrane compartments. We therefore set out to identify the molecular determinants for localizing several syntaxin isoforms, including syntaxins 3, 4, and 5, to their respective intracellular compartments (plasma membrane for syntaxins 3 and 4; cis-Golgi for syntaxin 5). Analysis of a series of deletion and chimeric syntaxin constructs revealed that the 17-amino acid transmembrane domain of syntaxin 5 was sufficient to direct the cis-Golgi localization of several heterologous reporter constructs. In contrast, the longer 25-amino acid transmembrane domain of syntaxin 3 was sufficient to localize reporter constructs to the plasma membrane. Furthermore, truncation of the syntaxin 3 transmembrane domain to 17 amino acids resulted in a complete conversion to cis-Golgi compartmentalization that was indistinguishable from syntaxin 5. These data support a model wherein short transmembrane domains (≤17 amino acids) direct the cis-Golgi localization of syntaxins, whereas long transmembrane domains (≥23 amino acids) direct plasma membrane localization.

scholarly journals Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase

2020 ◽  
pp. jbc.RA120.015999
Author(s):  
Marius Stopp ◽  
Philipp Aloysius Steinmetz ◽  
Christopher Schubert ◽  
Christian Griesinger ◽  
Dirk Schneider ◽  
...  
Keyword(s):  
Signal Transmission ◽  
Transmembrane Helix ◽  
Helical Structure ◽  
Kinase Domain ◽  
Sensor Kinase ◽  
Transmembrane Signaling ◽  
Activated State ◽  
Sensor Kinases ◽  
And Function ◽  
Cytoplasmic Side

Transmembrane signaling is a key process of membrane bound sensor kinases. The C4-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by transmembrane helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in transmembrane signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable transmembrane homodimer in both the inactive and fumarate-activated state of DcuS. A S186xxxGxxxG194 motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplasmic side into the α6-helix of the sensory PASP domain, and on the cytoplasmic side into the α1-helix of PASC. PASC has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PASC contains a LxxxLxxxL sequence. The dimeric state of the linker was relieved during fumarate activation of DcuS, indicating structural rearrangements in the linker. Thus, DcuS contains a long α-helical structure reaching from the sensory PASP (α6) domain across the membrane to α1(PASC). Taken together, the results suggest piston-type transmembrane signaling by the TM2-homodimer from PASP across the full TM region, whereas the fumarate-destabilized linker dimer converts the signal on the cytoplasmic side for PASC and kinase regulation.

scholarly journals C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

2006 ◽  
Vol 401 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Chiharu Sogawa ◽  
Kei Kumagai ◽  
Norio Sogawa ◽  
Katsuya Morita ◽  
Toshihiro Dohi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Splice Variants ◽  
Functional Expression ◽  
Surface Expression ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
C Terminus ◽  
And Function

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

scholarly journals The CD28-transmembrane domain mediates chimeric antigen receptor heterodimerization with CD28

2020 ◽  
Author(s):  
Yannick D. Muller ◽  
Duy P. Nguyen ◽  
Leonardo M.R. Ferreira ◽  
Patrick Ho ◽  
Caroline Raffin ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Intracellular Signaling ◽  
Transmembrane Domain ◽  
Antigen Receptor ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human T Cells ◽  
Engineered T Cells ◽  
The Impact

AbstractAnti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge (HD) and transmembrane (TMD) domains between the extracellular antigen-targeting and the intracellular signaling modalities of CARs has not been systemically studied. Here, a series of CD19-CARs differing only by their HD (CD8/CD28/IgG4) and TMD (CD8/CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation to anti-CD28 stimulation. This dimerization depended on polar amino-acids in the CD28-TMD. CD28-CAR heterodimerization was more efficient in CARs containing a CD8-HD or CD28-HD as compared to an IgG4-HD. CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but led to a significant reduction of CD28 cell-surface expression. These data unveil a new property of the CD28-TMD and suggest that TMDs can modulate CAR T-cell activities by engaging endogenous partners.Abstract Figure

scholarly journals Conserved Glycines Control Disorder and Function in the Cold-Regulated Protein, COR15A

Biomolecules ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 84 ◽  
Author(s):  
Oluwakemi Sowemimo ◽  
Patrick Knox-Brown ◽  
Wade Borcherds ◽  
Tobias Rindfleisch ◽  
Anja Thalhammer ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Intrinsically Disordered Protein ◽  
Helical Structure ◽  
Cd Spectroscopy ◽  
Protective Function ◽  
Intrinsically Disordered ◽  
Thaw Cycle ◽  
Cellular Dehydration ◽  
And Function ◽  
The Impact

Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly α-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil–helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A mutants on the stability of model membranes during a freeze–thaw cycle was investigated by fluorescence spectroscopy. The results of these experiments showed the mutants had a higher content of α-helical structure and the increased α-helicity improved membrane stabilization during freezing. Comparison of the TFE- and ethylene glycol-induced coil–helix transitions support our conclusion that increasing the transient helicity of COR15A in aqueous solution increases its ability to stabilize membranes during freezing. Altogether, our results suggest the conserved glycine residues are important for maintaining the disordered structure of COR15A but are also compatible with the formation of α-helical structure during freezing induced dehydration.

scholarly journals Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein

2015 ◽  
Vol 467 (1) ◽  
pp. 127-139 ◽  
Author(s):  
Katalin Kiss ◽  
Nora Kucsma ◽  
Anna Brozik ◽  
Gabor E. Tusnady ◽  
Ptissam Bergam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Atp Binding ◽  
Multivesicular Bodies ◽  
Molecular Dissection ◽  
Terminal Domain ◽  
Lysosomal Targeting ◽  
And Function

The intracellular localization of ATP-binding cassette, sub family B (ABCB) 6 is a matter of debate. We show that ABCB6 is internalized from the plasma membrane to multivesicular bodies and lysosomes. Molecular dissection of the ABCB6 protein reveals a role of its N-terminal domain in targeting.

scholarly journals LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor

2016 ◽  
Vol 82 (17) ◽  
pp. 5364-5374 ◽  
Author(s):  
Marija Miljkovic ◽  
Gordana Uzelac ◽  
Nemanja Mirkovic ◽  
Giulia Devescovi ◽  
Dzung B. Diep ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Sugar Transport ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Bacteriocin Activity ◽  
Binding Studies ◽  
Content Type ◽  
The Third

ABSTRACTThe Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. AlthoughyvjBis highly conserved in the genusLactococcus, the bacteriocin appears to be active only against the subspeciesL. lactissubsp.lactis. Comparative analysis of the YvjB proteins of a sensitive strain (YvjBMN) and a resistant strain (YvjBMG) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp25) and terminal alanine (Ala30). It was also shown that the distance between Trp25and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr356) and alanine (Ala353).In vitroexperiments showed that LsbB could interact with both YvjBMNand YvjBMG, but the strength of interaction is significantly less with YvjBMG.In vivoexperiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjBMN.IMPORTANCEThe antimicrobial activity of LsbB bacteriocin depends on the correct interaction with the corresponding receptor in the bacterial membrane of sensitive cells. Membrane-located bacteriocin receptors have essential primary functions, such as cell wall synthesis or sugar transport, and it seems that interaction with bacteriocins is suicidal for cells. This study showed that the C-terminal part of LsbB is crucial for the bacteriocin activity, most probably through adequate interaction with the third transmembrane domain of the YvjB receptor. The conserved Tyr356and Ala353residues of YvjB are essential for the function of this Zn-dependent membrane-located protease as a bacteriocin receptor.

scholarly journals Stac adaptor proteins regulate trafficking and function of muscle and neuronal L-type Ca2+ channels

2014 ◽  
Vol 112 (2) ◽  
pp. 602-606 ◽  
Author(s):  
Alexander Polster ◽  
Stefano Perni ◽  
Hicham Bichraoui ◽  
Kurt G. Beam
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Ryanodine Receptors ◽  
Adaptor Proteins ◽  
Surface Expression ◽  
Ec Coupling ◽  
And Function

Excitation–contraction (EC) coupling in skeletal muscle depends upon trafficking of CaV1.1, the principal subunit of the dihydropyridine receptor (DHPR) (L-type Ca2+ channel), to plasma membrane regions at which the DHPRs interact with type 1 ryanodine receptors (RyR1) in the sarcoplasmic reticulum. A distinctive feature of this trafficking is that CaV1.1 expresses poorly or not at all in mammalian cells that are not of muscle origin (e.g., tsA201 cells), in which all of the other nine CaV isoforms have been successfully expressed. Here, we tested whether plasma membrane trafficking of CaV1.1 in tsA201 cells is promoted by the adapter protein Stac3, because recent work has shown that genetic deletion of Stac3 in skeletal muscle causes the loss of EC coupling. Using fluorescently tagged constructs, we found that Stac3 and CaV1.1 traffic together to the tsA201 plasma membrane, whereas CaV1.1 is retained intracellularly when Stac3 is absent. Moreover, L-type Ca2+ channel function in tsA201 cells coexpressing Stac3 and CaV1.1 is quantitatively similar to that in myotubes, despite the absence of RyR1. Although Stac3 is not required for surface expression of CaV1.2, the principle subunit of the cardiac/brain L-type Ca2+ channel, Stac3 does bind to CaV1.2 and, as a result, greatly slows the rate of current inactivation, with Stac2 acting similarly. Overall, these results indicate that Stac3 is an essential chaperone of CaV1.1 in skeletal muscle and that in the brain, Stac2 and Stac3 may significantly modulate CaV1.2 function.

Identification of a region in glycoprotein IIIa involved in subunit association with glycoprotein IIb: further lessons from Iraqi-Jewish Glanzmann thrombasthenia

Blood ◽  
2001 ◽  
Vol 98 (4) ◽  
pp. 1063-1069 ◽  
Author(s):  
Rivka Yatuv ◽  
Nurit Rosenberg ◽  
Ariella Zivelin ◽  
Hava Peretz ◽  
Rima Dardik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Complex Formation ◽  
Disulfide Bond ◽  
Transmembrane Domain ◽  
Chinese Hamster ◽  
Premature Termination Codon ◽  
Surface Expression ◽  
Lower Efficiency ◽  
Glanzmann Thrombasthenia ◽  
Transfected Cells

The most frequent mutation causing Glanzmann thrombasthenia in Iraqi-Jews (IJ-1) is an 11-bp deletion in exon 13 of the glycoprotein (GP) IIIa gene. This deletion predicts a frameshift that results in the elimination of the C406-C655 disulfide bond and a premature termination codon shortly before the transmembrane domain. To determine the contribution of each of these alterations to the thrombasthenic phenotype, Chinese hamster ovary or baby hamster kidney cells were cotransfected with normal GPIIb complementary DNA (cDNA) and the following GPIIIa cDNAs: normal, cDNA bearing IJ-1 mutation, 2011T>A mutated cDNA predicting C655S (single-letter amino acid codes) substitution, and 2019A>T mutated cDNA predicting Stop657. Elimination of the C406-C655 disulfide bond by C655S substitution did not affect GPIIb/IIIa surface expression or binding of the transfected cells to immobilized fibrinogen, whereas elimination of the transmembrane and cytoplasmic domains in IJ-1 and Stop657 mutants prevented both surface expression and binding of the transfected cells to immobilized fibrinogen. Immunohistochemical staining and immunoprecipitation demonstrated that the elimination of amino acids 657-762 in IJ-1 and Stop657 prevented intracellular GPIIb/IIIa complex formation, and differential immunofluorescence staining of GPIIIa and cellular organelles suggested that the truncated uncomplexed GPIIIa protein was retained in the endoplasmic reticulum. Because the use of GPIIIa Stop693 and normal GPIIb cDNAs yielded GPIIb/IIIa complex formation, though with lower efficiency, it is suggested that amino acids 657-692 of GPIIIa are essential for the intracellular association of GPIIb and GPIIIa.

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