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.

scholarly journals Analysis of the N-terminal binding domain of Goα

1997 ◽  
Vol 328 (1) ◽  
pp. 23-31 ◽  
Author(s):  
Liliana BUSCONI ◽  
M. Bradley DENKER
Keyword(s):  
Amino Acids ◽  
Cultured Cells ◽  
Membrane Binding ◽  
Receptor Activation ◽  
Binding Domain ◽  
Membrane Receptors ◽  
Membrane Interactions ◽  
Cos Cells ◽  
Vitro Analysis

Signalling from membrane receptors through heterotrimeric G-proteins (Gα and Gβγ) to intracellular effectors is a highly regulated process. Receptor activation causes exchange of GTP for GDP on Gα and dissociation of Gα from Gβγ. Both subunits remain membrane-associated and interact with a series of other molecules throughout the cycle of activation. The N-terminal binding domain of Gα subunits interacts with the membrane by several partially defined mechanisms: the anchoring of Gα to the more hydrophobic Gβγ subunits, the interaction of N-terminal lipids (palmitate and/or myristate) with the membrane, and attachment of amino acid regions to the membrane {amino acids 11-14 of Goα (D[11-14]); Busconi, Boutin and Denker (1997) Biochem. J. 323, 239-244}. We characterized N-terminal mutants of Goα with known Gβγ-binding properties for the ability to interact with phospholipid vesicles and membranes prepared from cultured cells (acceptor membranes). In vitro analysis allows membrane interactions that are important to the activated and depalmitoylated state of Gα to be characterized. Subcellular localization was also determined in transiently transfected COS cells. All of the mutant proteins are myristoylated, and differences in myristoylation do not account for changes in membrane binding. Disrupting the N-terminal α-helix of Goα with a proline point mutation at Arg-9 (R9P) does not affect interactions with Gβγ on sucrose-density gradients but significantly reduces acceptor membrane binding. Deletion of amino acids 6-15 (D[6-15]; reduced Gβγ binding) or deletion of amino acids 3-21 (D[3-21]); no detectable Gβγ binding) further reduces acceptor membrane binding. When expressed in COS cells, R9P and D[6-15] are localized in the membrane similar to wild-type Goα as a result of the contribution from palmitoylation. In contrast, D[3-21] is completely soluble in COS cells, and no palmitoylation is detected. The binding of Goα and mutants translated in vitro to liposomes indicates that Goα preferentially binds to neutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to phosphatidylcholine liposomes like Goα, but D[6-15] exhibits no detectable binding. Taken together, these studies suggest that interactions of the N-terminus of Gα subunits with the membrane may be affected by both membrane proteins and lipids. A detailed understanding of Gα-membrane interactions may reveal unique mechanisms for regulating signal transduction.

scholarly journals Every Detail Matters. That Is, How the Interaction between Gα Proteins and Membrane Affects Their Function

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 222
Author(s):  
Agnieszka Polit ◽  
Paweł Mystek ◽  
Ewa Błasiak
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
Lipid Membranes ◽  
Signaling Proteins ◽  
Heterotrimeric G Proteins ◽  
Membrane Attachment ◽  
The Individual ◽  
Multicellular Organisms ◽  
G Protein Coupled

In highly organized multicellular organisms such as humans, the functions of an individual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increasing interest in studying the accompanying protein–lipid interactions, which have been demonstrated to not only provide the environment but also regulate proper and efficient signal transduction. The mode of interaction between the cell membrane and G proteins is well known. Despite this, the recognition mechanisms at the molecular level and how the individual G protein-membrane attachment signals are interrelated in the process of the complex control of membrane targeting of G proteins remain unelucidated. This review focuses on the mechanisms by which mammalian Gα subunits of G proteins interact with lipids and the factors responsible for the specificity of membrane association. We summarize recent data on how these signaling proteins are precisely targeted to a specific site in the membrane region by introducing well-defined modifications as well as through the presence of polybasic regions within these proteins and interactions with other components of the heterocomplex.

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 Translational and Structural Requirements of the Early Nodulin Gene enod40, a Short-Open Reading Frame-Containing RNA, for Elicitation of a Cell-Specific Growth Response in the Alfalfa Root Cortex

2001 ◽  
Vol 21 (1) ◽  
pp. 354-366 ◽  
Author(s):  
Carolina Sousa ◽  
Christina Johansson ◽  
Celine Charon ◽  
Hamid Manyani ◽  
Christof Sautter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Rna Structure ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Cortical Cells ◽  
Root Cortex ◽  
Reading Frame ◽  
Early Nodulin

ABSTRACT A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin geneenod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5′ and 3′ regions ofenod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

scholarly journals Structural analysis of a mouse mammary tumor virus superantigen.

1992 ◽  
Vol 175 (3) ◽  
pp. 847-852 ◽  
Author(s):  
Y Choi ◽  
P Marrack ◽  
J W Kappler
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
Mammary Tumor ◽  
Integral Membrane Protein ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Type Ii ◽  
Long Terminal Repeats ◽  
Mouse Mammary Tumor

It has recently been shown that the minor lymphocyte stimulating-like products expressed by some mice are actually encoded by open reading frames in the 3' long terminal repeats of mouse mammary tumor viruses. These products act as viral superantigens (vSAGs). That is, they stimulate most T cells bearing particular V beta s almost regardless of the rest of the variable components of the T cell receptors expressed by those cells. To find out more about the structure of these vSAGs, a set of truncated vSAG genes was used in transfection and in vitro translation experiments to show that the functional vSAG is a type II integral membrane protein with a large glycosylated extracellular COOH-terminal domain and a small, nonessential, intracellular NH2-terminal cytoplasmic domain. These results are consistent with the fact that the vSAGs must be expressed on the cell surface in order to interact with T cells and class II major histocompatibility complex proteins. They also account for the finding that much of the V beta specificity of the vSAGs is controlled by amino acids at the COOH-terminal end of the vSAG proteins, amino acids that will be extracellular in type II proteins.

scholarly journals Identification of Mimotope Peptides Which Bind to the Mycotoxin Deoxynivalenol-Specific Monoclonal Antibody

1999 ◽  
Vol 65 (8) ◽  
pp. 3279-3286 ◽  
Author(s):  
Qiaoping Yuan ◽  
James J. Pestka ◽  
Brandon M. Hespenheide ◽  
Leslie A. Kuhn ◽  
John E. Linz ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Synthetic Peptide ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Translation System

ABSTRACT Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.

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 Differential Rescue of Poliovirus RNA Replication Functions by Genetically Modified RNA Polymerase Precursors

2004 ◽  
Vol 78 (23) ◽  
pp. 13007-13018 ◽  
Author(s):  
Christopher T. Cornell ◽  
Jo Ellen Brunner ◽  
Bert L. Semler
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Genetically Modified ◽  
Rna Replication ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Wild Type ◽  
Positive Strand ◽  
Positive Strand Rna

ABSTRACT We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

scholarly journals Ribosomal protein L7a is encoded by a gene (Surf-3) within the tightly clustered mouse surfeit locus.

1989 ◽  
Vol 9 (1) ◽  
pp. 224-231 ◽  
Author(s):  
A Giallongo ◽  
J Yon ◽  
M Fried
Keyword(s):  
Amino Acids ◽  
Ribosomal Protein ◽  
Ribosomal Subunit ◽  
Ribosomal Protein Gene ◽  
Mouse Cell ◽  
Nucleic Acid Hybridization ◽  
Peptide Sequence ◽  
In Vitro Translation ◽  
Cell Components

The mouse Surfeit locus, which contains a cluster of at least four genes (Surf-1 to Surf-4), is unusual in that adjacent genes are separated by no more than 73 base pairs (bp). The heterogeneous 5' ends of Surf-1 and Surf-2 are separated by only 15 to 73 bp, the 3' ends of Surf-1 and Surf-3 are only 70 bp apart, and the 3' ends of Surf-2 and Surf-4 overlap by 133 bp. This very tight clustering suggests a cis interaction between adjacent Surfeit genes. The Surf-3 gene (which could code for a basic polypeptide of 266 amino acids) is a highly expressed member of a pseudogene-containing multigene family. By use of an anti-peptide serum (against the C-terminal nine amino acids of the putative Surf-3 protein) for immunofluorescence and immunoblotting of mouse cell components and by in vitro translation of Surf-3 cDNA hybrid-selected mRNA, the Surf-3 gene product was identified as a 32-kilodalton ribosomal protein located in the 60S ribosomal subunit. From its subunit location, gel migration, and homology with a limited rat ribosomal peptide sequence, the Surf-3 gene was shown to encode the mouse L7a ribosomal protein. The Surf-3 gene is highly conserved through evolution and was detected by nucleic acid hybridization as existing in multiple copies (multigene families) in other mammals and as one or a few copies in birds, Xenopus, Drosophila, and Schizosaccharomyces pombe. The Surf-3 C-terminal anti-peptide serum detects a 32-kilodalton protein in other mammals, birds, and Xenopus but not in Drosophila and S. pombe. The possible effect of interaction of the Surf-3 ribosomal protein gene with adjacent genes in the Surfeit locus at the transcriptional or posttranscriptional level or both levels is discussed.

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