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.

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.

scholarly journals Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

1995 ◽  
Vol 6 (2) ◽  
pp. 171-183 ◽  
Author(s):  
H Yu ◽  
C V Nicchitta ◽  
J Kumar ◽  
M Becker ◽  
I Toyoshima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Coiled Coil ◽  
Integral Membrane Protein ◽  
In Vitro Translation ◽  
Predicted Amino Acid Sequence ◽  
Hydrophobic Region ◽  
Reading Frame

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.

scholarly journals Identification of amino acid sequences determining interaction between the cucumber mosaic virus-encoded 2a polymerase and 3a movement proteins

2007 ◽  
Vol 88 (12) ◽  
pp. 3445-3451 ◽  
Author(s):  
Min Sook Hwang ◽  
Kyung Nam Kim ◽  
Jeong Hyun Lee ◽  
Young In Park
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Critical Role ◽  
Amino Acid Sequences ◽  
Multiple Sequence ◽  
Movement Proteins ◽  
Gdd Motif

The cucumber mosaic virus (CMV)-encoded 3a movement protein (MP) is indispensable for CMV movement in plants. We have previously shown that MP interacts directly with the CMV-encoded 2a polymerase protein in vitro. Here, we further dissected this interaction and determined the amino acid sequences that are responsible for the MP and 2a polymerase protein interaction. Both the N-terminal 21 amino acids and the central GDD motif of the 2a polymerase protein were important for interacting with the MP. Although each of the regions alone was sufficient for the interaction with MP, quantitative yeast two-hybrid analyses showed that they acted synergistically to enhance the binding affinity. The MP N-terminal 20 amino acids were sufficient for interacting with the 2a polymerase protein, and the serine residue at position 14 played a critical role in the interaction. Multiple sequence alignment showed that the 2a protein interacting regions and the serine at position 14 in the MP are highly conserved among subgroup I and II CMV isolates.

scholarly journals MARCH-II Is a Syntaxin-6–binding Protein Involved in Endosomal Trafficking

2005 ◽  
Vol 16 (4) ◽  
pp. 1696-1710 ◽  
Author(s):  
Nobuhiro Nakamura ◽  
Hidekazu Fukuda ◽  
Akira Kato ◽  
Shigehisa Hirose
Keyword(s):  
Transmembrane Domain ◽  
Retrograde Transport ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Immune Recognition ◽  
Endosomal Trafficking ◽  
Binding Studies ◽  
Recycling Endosomes ◽  
Protein Receptors

Membrane-associated RING-CH (MARCH) is a recently identified member of the mammalian E3 ubiquitin ligase family, some members of which down-regulate the expression of immune recognition molecules. Here, we have identified MARCH-II, which is ubiquitously expressed and localized to endosomal vesicles and the plasma membrane. Immunoprecipitation and in vitro binding studies established that MARCH-II directly associates with syntaxin 6. Overexpression of MARCH-II resulted in redistribution of syntaxin 6 as well as some syntaxin-6–interacting soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) into the MARCH-II–positive vesicles. In addition, the retrograde transport of TGN38 and a chimeric version of furin to trans-Golgi network (TGN) was perturbed—without affecting the endocytic degradative and biosynthetic secretory pathways—similar to effects caused by a syntaxin 6 mutant lacking the transmembrane domain. MARCH-II overexpression markedly reduced the cell surface expression of transferrin (Tf) receptor and Tf uptake and interfered with delivery of internalized Tf to perinuclear recycling endosomes. Depletion of MARCH-II by small interfering RNA perturbed the TGN localization of syntaxin 6 and TGN38/46. MARCH-II is thus likely a regulator of trafficking between the TGN and endosomes, which is a novel function for the MARCH family.

scholarly journals N-terminal binding domain of Gα subunits: involvement of amino acids 11–14 of Gα0 in membrane attachment

1997 ◽  
Vol 323 (1) ◽  
pp. 239-244 ◽  
Author(s):  
Liliana BUSCONI ◽  
Paula M. BOUTIN ◽  
Bradley M. DENKER
Keyword(s):  
Signal Transduction ◽  
Amino Acids ◽  
G Proteins ◽  
Membrane Binding ◽  
Amino Acid Sequences ◽  
Membrane Receptors ◽  
In Vitro Translation ◽  
Membrane Attachment ◽  
Membrane Components

Heterotrimeric guanine nucleotide binding proteins (G-proteins) transmit signals from membrane receptors to a variety of intracellular effectors. G-proteins reversibly associate with components of the signal transduction system, yet remain membrane attached throughout the cycle of activation. The Gα subunits remain attached to the plasma membrane through a combination of factors that are only partially defined. We now demonstrate that amino acids within the N-terminal domain of Gα subunits are involved in membrane binding. We used in vitro translation, a technique widely utilized to characterize functional aspects of G-proteins, and interactions with donor-acceptor membranes to demonstrate that amino acids 11-14 of Gαo contribute to membrane binding. The membrane binding of Gαo lacking amino acids 11-14 (D[11-14]) was significantly reduced at all membrane concentrations in comparison with wild-type Gαo. Several other N-terminal mutants of Gαo were characterized as controls, and these results indicate that differences in myristoylation, palmitoylation and βγ interactions do not account for the reduced membrane binding of D[11-14]. Furthermore, when membrane attachment of Gαo and mutants was characterized in transiently transfected 35S-labelled and [3H]myristate-labelled COS cells, amino acids 11-14 contributed to membrane binding. These studies reveal that membrane binding of Gα subunits occurs by a combination of factors that include lipids and amino acid sequences. These regions may provide novel sites for interaction with membrane components and allow additional modulation of signal transduction.

Bovine thyrotropin receptor cDNA is characterized by full-length and truncated transcripts

1997 ◽  
Vol 18 (2) ◽  
pp. 101-112 ◽  
Author(s):  
D W Silversides ◽  
A Houde ◽  
J-F Ethier ◽  
J G Lussier
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transmembrane Domain ◽  
Full Length ◽  
Tsh Receptor ◽  
Thyrotropin Receptor ◽  
Pcr Cloning ◽  
Specific Expression ◽  
Terminal Domain ◽  
Bovine Tsh

ABSTRACT The complete coding sequence for the bovine thyrotropin (TSH) receptor was derived using a modified PCR cloning strategy. The bovine thyrotropin receptor conforms to the pattern of receptor interacting with membrane-bound G-protein already established in other species for TSH and gonadotropins receptors. The cDNA for the bovine TSH receptor consists of an open reading frame 2289 nucleotides in length, corresponding to a protein of 763 amino acids (estimated molecular mass of 86·4 kDa) which includes a 20 amino acid putative leading signal peptide. The receptor consists of a large NH2-terminal extracellular membrane domain of 417 amino acids with 5 potential N-linked glycosylation sites, a transmembrane domain (265 amino acids) consisting of 7 putative membrane α-helix spanning segments, and an intracytoplasmic COOH-terminal domain (82 amino acids). The bovine TSH receptor is one amino acid less than the corresponding sequence in dog, human, rat and mouse. Cysteine residues (n=22) were conserved when compared with other TSH receptors. Three potential phosphorylation sites were found in the transmembrane domain and the COOH-terminal domain. As with other members of this receptor family, alternative splicing was observed. A transcribed but truncated TSH receptor of 1769 nucleotides was demonstrated, lacking half of the V segment of the transmembrane domain up to the COOH-terminal domain of the full length TSH receptor. Additionally, alternative transcriptional start sites were observed. Northern blot analysis using a probe (1170 bp) spanning part of the extracellular domain up to the first loop of the transmembrane domain showed specific expression in the bovine thyroid gland with major transcripts of 9·3 and 4·3 kb, and a minor transcript of 3·8 kb being detected.

scholarly journals Computational approach for selection of epitope-based dengue vaccine targets

2018 ◽  
Vol 14 (4) ◽  
pp. 605-618
Author(s):  
Phuc Nguyen ◽  
Ly Le
Keyword(s):  
Amino Acids ◽  
T Cell ◽  
Dengue Virus ◽  
Rational Design ◽  
Protein Sequences ◽  
Computational Approach ◽  
Amino Acid Sequences ◽  
Universal Vaccine ◽  
E Protein

High antigenic variability in the envelope (E) protein of different virus strains has been a major obstacle in designing effective vaccines for Dengue virus (DENV). To maintain their biological function, some parts of viral proteins remain stable during evolution thus one possible approach to solve this problem is to recognize specific regions within different protein sequences of E that have the tendency to stay constant through evolution. These regions may possess some special attributes to become a vaccine candidate against dengue virus. In this study, a computational approach was utilized to identify and analyze highly conserved amino acid sequences of the DENV E protein. Sequences of 9 amino acids or more were specifically focused due to their immune-relevant as T-cell determinants. Different bioinformatics tools were responsible for revealing conserved regions in the DENV E protein and constructing the phylogenetic tree from the sequence database. The tools also predicted immunogenicity of the identified vaccine targets. Ultimately, two peptide regions of at least 9 amino acids were chosen due to their high conserved attribute in more than 95% of all collected DENV sequences. Moreover, both of them was found to be immune-relevant by their correspondence to known or putative HLA-restricted T cell determinants. The conserved attribute of these sequences through the entire analysis of this study supports their potential as candidates for further in vitro experiments for rational design a universal vaccine which has longer and broader impact.

scholarly journals Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane domain

2021 ◽  
Author(s):  
James P Bridges ◽  
Caterina Safina ◽  
Bernard Picard ◽  
Kari Brown ◽  
Alyssa Filuta ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Peptide Sequence ◽  
Knock Out ◽  
Autocatalytic Cleavage ◽  
Knock Out Mice

The mechanistic details of the tethered agonist mode of activation for adhesion GPCRs has not been completely deciphered. We set out to investigate the physiologic importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation. To examine this hypothesis, we characterized tethered agonist-mediated activation of GPR116 in vitro and in vivo. A knock-in mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species swapping approaches we identified key conserved amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loops 2/3 (ECL2/3). We further highlight residues in transmembrane7 (TM7) that mediate stronger signaling in mouse versus human GPR116 and recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation.

Phylogenetic and topological analyses of the bovine interferon-induced transmembrane protein (IFITM3)

2021 ◽  
Author(s):  
Yong-Chan Kim ◽  
Byung-Hoon Jeong
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Multiple Sequence Alignment ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Phylogenetic Analyses ◽  
Amino Acid Sequences ◽  
Multiple Sequence ◽  
Interspecific Differences ◽  
Distinct Features

AbstractInterferon-induced transmembrane protein 3 (IFITM3) plays a pivotal role in antiviral capacity in several species. However, to date, investigations of the IFITM3 protein in cattle have been rare. According to recent studies, interspecific differences in the IFITM3 protein result in several unique features of the IFITM3 protein relative to primates and birds. Thus, in the present study, we investigated the bovine IFITM3 protein based on nucleotide and amino acid sequences to find its distinct features. We found that the bovine IFITM3 gene showed a significantly different length and homology relative to other species, including primates, rodents and birds. Phylogenetic analyses indicated that the bovine IFITM3 gene and IFITM3 protein showed closer evolutionary distance with primates than with rodents. However, cattle showed an independent clade among primates, rodents and birds. Multiple sequence alignment of the IFITM3 protein indicated that the bovine IFITM3 protein contains 36 bovine-specific amino acids. Notably, the bovine IFITM3 protein was predicted to prefer inside-to-outside topology of intramembrane domain 1 (IMD1) and inside-to-outside topology of transmembrane domain 2 by TMpred and three membrane embedding domains according to the SOSUI system.

scholarly journals Role of Transmembrane Domain and Cytoplasmic Tail Amino Acid Sequences of Influenza A Virus Neuraminidase in Raft Association and Virus Budding

2004 ◽  
Vol 78 (10) ◽  
pp. 5258-5269 ◽  
Author(s):  
Subrata Barman ◽  
Lopa Adhikary ◽  
Alok K. Chakrabarti ◽  
Carl Bernas ◽  
Yoshihiro Kawaoka ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Influenza A ◽  
Transmembrane Domain ◽  
Amino Acid Sequences ◽  
Type Ii ◽  
Virus Growth ◽  
Transmembrane Glycoprotein ◽  
Virus Life Cycle

ABSTRACT Influenza virus neuraminidase (NA), a type II transmembrane glycoprotein, possesses receptor-destroying activity and thereby facilitates virus release from the cell surface. Among the influenza A viruses, both the cytoplasmic tail (CT) and transmembrane domain (TMD) amino acid sequences of NA are highly conserved, yet their function(s) in virus biology remains unknown. To investigate the role of amino acid sequences of the CT and TMD on the virus life cycle, we systematically mutagenized the entire CT and TMD of NA by converting two to five contiguous amino acids to alanine. In addition, we also made two chimeric NA by replacing the CT proximal one-third amino acids of the NA TMD [NA(1T2N)NA] and the entire NA TMD (NATRNA) with that of human transferrin receptor (TR) (a type II transmembrane glycoprotein). We rescued transfectant mutant viruses by reverse genetics and examined their phenotypes. Our results show that all mutated and chimeric NAs could be rescued into transfectant viruses. Different mutants showed pleiotropic effects on virus growth and replication. Some mutants (NA2A5, NA3A7, and NA4A10) had little effect on virus growth while others (NA3A2, NA5A27, and NA5A31) produced about 50- to 100-fold-less infectious virus and still some others (NA5A14, NA4A19, and NA4A23) exhibited an intermediate phenotype. In general, mutations towards the ectodomain-proximal sequences of TMD progressively caused reduction in NA enzyme activity, affected lipid raft association, and attenuated virus growth. Electron microscopic analysis showed that these mutant viruses remained aggregated and bound to infected cell surfaces and could be released from the infected cells by bacterial NA treatment. Moreover, viruses containing mutations in the extreme N terminus of the CT (NA3A2) as well as chimeric NA containing the TMD replaced partially [NA(1T2N)NA] or fully (NATRNA) with TR TMD caused reduction in virus growth and exhibited the morphological phenotype of elongated particles. These results show that although the sequences of NA CT and TMD per se are not absolutely essential for the virus life cycle, specific amino acid sequences play a critical role in providing structural stability, enzyme activity, and lipid raft association of NA. In addition, aberrant morphogenesis including elongated particle formation of some mutant viruses indicates the involvement of NA in virus morphogenesis and budding.

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