scholarly journals Characteristics of the tyrosine recognition signal for internalization of transmembrane surface glycoproteins.

1990 ◽  
Vol 111 (4) ◽  
pp. 1393-1407 ◽  
Author(s):  
N T Ktistakis ◽  
D Thomas ◽  
M G Roth
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Glycophorin A ◽  
Coated Pits ◽  
Surface Receptors ◽  
Influenza Virus Hemagglutinin ◽  
Short Amino Acid Sequence ◽  
Human Glycophorin

A tyrosine residue in the cytoplasmic domain of a class of cell surface receptors is necessary, but not sufficient, for internalization through coated pits. To identify the amino acid context enabling a tyrosine to serve as a signal for endocytosis, we mutated the short cytoplasmic domain of a mutant influenza virus hemagglutinin that is competent for internalization, HA-Y543, and determined the effect of each change on internalization. From these results and a comparison of sequences of other proteins recognized by coated pits, a "tyrosine internalization signal" was proposed. Site-directed mutagenesis was employed to insert complete, or incomplete "tyrosine internalization signals" into the cytoplasmic domain of a protein normally not endocytosed, human glycophorin A. Only the complete signal caused internalization of mutant glycophorins by coated pits. The signal is formed by a short amino acid sequence, with polar or basic residues preferred at certain positions on either side of the tyrosine. Amino acids, which in proteins of known structure are frequently found in turns, are clustered near the tyrosine on the side of the signal nearest the transmembrane domain.

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

1999 ◽  
Vol 112 (11) ◽  
pp. 1721-1732 ◽  
Author(s):  
M.J. Francis ◽  
E.E. Jones ◽  
E.R. Levy ◽  
R.L. Martin ◽  
S. Ponnambalam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Domain ◽  
Menkes Disease ◽  
Cytoplasmic Domain ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Golgi Network

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

R to Q Amino Acid Substitution in the GFFKR Sequence of the Cytoplasmic Domain of the Integrin IIb Subunit in a Patient With a Glanzmann’s Thrombasthenia-Like Syndrome

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4178-4187 ◽  
Author(s):  
O. Peyruchaud ◽  
A.T. Nurden ◽  
S. Milet ◽  
L. Macchi ◽  
A. Pannochia ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Heterozygous Mutation ◽  
Polymorphism Analysis ◽  
Wild Type ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

Abstract The integrin IIbβ3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of IIb and β3. We now report the molecular analysis of IIbβ3 from a patient with a Glanzmann’s thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of IIbβ3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of IIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the IIb gene and a single allele of the β3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated IIb together with wild-type β3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type IIb and β3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary–transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the IIb cytoplasmic domain.

scholarly journals Two novel mutations of the calcium-sensing receptor gene affecting the same amino acid position lead to opposite phenotypes and reveal the importance of p.N802 on receptor activity

2013 ◽  
Vol 168 (2) ◽  
pp. K27-K34 ◽  
Author(s):  
Anne-Sophie Lia-Baldini ◽  
Corinne Magdelaine ◽  
Angélique Nizou ◽  
Coraline Airault ◽  
Jean-Pierre Salles ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Direct Sequencing ◽  
Receptor Gene ◽  
Amino Acid Position ◽  
Receptor Activity ◽  
Site Directed Mutagenesis ◽  
Missense Mutations ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing

ObjectiveGain-of-function mutations of the calcium-sensing receptor (CASR) gene have been identified in patients with sporadic or familial autosomal dominant hypocalcemia (ADH). Inactivating mutations of the CASR gene cause familial hypocalciuric hypercalcemia (FHH). Here, we report two novel CASR mutations affecting the same amino acid (p.N802); one causes ADH and the other atypical FHH.Patients and methodsThe first patient, an 11-year-old girl suffering from hypocalcemia, developed nephrocalcinosis when she was only 5 years old. The second patient is a 30-year-old woman who presented with mild hypercalcemia. PCR amplification of CASR coding exons and direct sequencing of PCR products were used to identify mutations. Site-directed mutagenesis was used to generate mutated CASR cDNAs in an expression plasmid. Using the MAPK assay system and transient transfection of Cos-7 cells with wild-type (WT) and mutated CASR, we studied the responses of these mutated receptors to extracellular Ca2+ and to the negative allosteric CASR modulator, NPS2143.ResultsTwo heterozygous missense mutations (p.N802I and p.N802S) affecting a residue in the sixth transmembrane domain of CASR were identified. In functional tests, the response of the p.N802S mutant to calcium was typical of an inactivating mutation. However, the p.N802I mutant had 70% of the maximally stimulated WT receptor activity even in the absence of extracellular calcium. This constitutive activity was only partially inhibited by the inhibitor, NPS2143.ConclusionsThe asparagine at amino acid position 802 appears to be essential for the activity of the CASR protein and is implicated in the mechanism of CASR signaling.

R to Q Amino Acid Substitution in the GFFKR Sequence of the Cytoplasmic Domain of the Integrin IIb Subunit in a Patient With a Glanzmann’s Thrombasthenia-Like Syndrome

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4178-4187 ◽  
Author(s):  
O. Peyruchaud ◽  
A.T. Nurden ◽  
S. Milet ◽  
L. Macchi ◽  
A. Pannochia ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Heterozygous Mutation ◽  
Polymorphism Analysis ◽  
Wild Type ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

The integrin IIbβ3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of IIb and β3. We now report the molecular analysis of IIbβ3 from a patient with a Glanzmann’s thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of IIbβ3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of IIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the IIb gene and a single allele of the β3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated IIb together with wild-type β3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type IIb and β3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary–transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the IIb cytoplasmic domain.

scholarly journals A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin.

1991 ◽  
Vol 114 (3) ◽  
pp. 413-421 ◽  
Author(s):  
C B Brewer ◽  
M G Roth
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Amino Acid Change ◽  
Mdck Cells ◽  
Single Amino Acid ◽  
Apical Surface ◽  
Membrane Glycoproteins ◽  
Coated Pits ◽  
Influenza Virus Hemagglutinin ◽  
Single Amino Acid Change

In the polarized kidney cell line MDCK, the influenza virus hemagglutinin (HA) has been well characterized as a model for apically sorted membrane glycoproteins. Previous work from our laboratory has shown that a single amino acid change in the cytoplasmic sequence of HA converts it from a protein that is excluded from coated pits to one that is efficiently internalized. Using trypsin or antibodies to mark protein on the surface, we have shown in MDCK cells that HA containing this mutation is no longer transported to the apical surface but instead is delivered directly to the basolateral plasma membrane. We propose that a cytoplasmic feature similar to an endocytosis signal can cause exclusive basolateral delivery.

Glycophorin A dimerization and band 3 interaction during erythroid membrane biogenesis: in vivo studies in human glycophorin A transgenic mice

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2872-2878 ◽  
Author(s):  
Isabelle Auffray ◽  
Shirin Marfatia ◽  
Kitty de Jong ◽  
Gloria Lee ◽  
Cheng-Han Huang ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Erythrocyte Membrane ◽  
Transmembrane Domain ◽  
Band 3 ◽  
In Vivo Studies ◽  
Glycophorin A ◽  
Membrane Biogenesis ◽  
Mature Erythrocyte ◽  
Human Glycophorin

Abstract Band 3 and glycophorin A (GPA) are the 2 most abundant integral proteins in the human erythrocyte membrane. Earlier studies suggested that the 2 proteins may associate not only in the mature erythrocyte membrane, but also during their posttranslational processing and intracellular trafficking. The purpose of this study was to directly examine the GPA–band 3 interaction in vivo and determine the nature of this association during erythroid membrane biogenesis. Transgenic mice were generated expressing the human glycophorin A gene and were used to examine how the induction of human GPA expression affected the levels of murine GPA and band 3 expression in the red cell membrane. Murine GPA expression was reduced in erythrocytes expressing human GPA, whereas the level of band 3 expression remained constant, implying a tight coupling of band 3 and GPA expression in the membrane of mature red cells. In vivo GPA dimerization was not modulated solely by the GPA transmembrane motif, but the distance between this motif and the basic residues on the cytoplasmic side of the transmembrane domain may also be important. In addition, GPA monomers with varying degrees of glycosylation dimerized, providing clear evidence that carbohydrate structures on the extracellular domain do not affect dimerization. The association between the multiple transmembrane-spanning protein, band 3, and the single transmembrane-spanning sialoglycoprotein, GPA, may serve as a model for interactions of other multi-pass and single-pass polypeptides during membrane biogenesis.

scholarly journals Cooperation of the Conserved Aspartate 439 and Bound Amino Acid Substrate Is Important for High-Affinity Na+ Binding to the Glutamate Transporter EAAC1

2007 ◽  
Vol 129 (4) ◽  
pp. 331-344 ◽  
Author(s):  
Zhen Tao ◽  
Christof Grewer
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Glutamate Transporter ◽  
Site Directed Mutagenesis ◽  
Amino Acid Side Chain ◽  
Kinetics And Thermodynamics ◽  
Anion Conductance ◽  
Glutamate Binding ◽  
Fold Reduction

The neuronal glutamate transporter EAAC1 contains several conserved acidic amino acids in its transmembrane domain, which are possibly important in catalyzing transport and/or binding of co/countertransported cations. Here, we have studied the effects of neutralization by site-directed mutagenesis of three of these amino acid side chains, glutamate 373, aspartate 439, and aspartate 454, on the functional properties of the transporter. Transport was analyzed by whole-cell current recording from EAAC1-expressing mammalian cells after applying jumps in voltage, substrate, or cation concentration. Neutralization mutations in positions 373 and 454, although eliminating steady-state glutamate transport, have little effect on the kinetics and thermodynamics of Na+ and glutamate binding, suggesting that these two positions do not constitute the sites of Na+ and glutamate association with EAAC1. In contrast, the D439N mutation resulted in an approximately 10-fold decrease of apparent affinity of the glutamate-bound transporter form for Na+, and an ∼2,000-fold reduction in the rate of Na+ binding, whereas the kinetics and thermodynamics of Na+ binding to the glutamate-free transporter were almost unchanged compared to EAAC1WT. Furthermore, the D439N mutation converted l-glutamate, THA, and PDC, which are activating substrates for the wild-type anion conductance, but not l-aspartate, into transient inhibitors of the EAAC1D439 anion conductance. Activation of the anion conductance by l-glutamate was biphasic, allowing us to directly analyze binding of two of the three cotransported Na+ ions as a function of time and [Na+]. The data can be explained with a model in which the D439N mutation results in a dramatic slowing of Na+ binding and a reduced affinity of the substrate-bound EAAC1 for Na+. We propose that the bound substrate controls the rate and the extent of Na+ interaction with the transporter, depending on the amino acid side chain in position 439.

scholarly journals Formation of bovine viral diarrhea virus E1–E2 heterodimers is essential for virus entry and depends on charged residues in the transmembrane domains

2008 ◽  
Vol 89 (9) ◽  
pp. 2114-2121 ◽  
Author(s):  
Saskia Ronecker ◽  
Gert Zimmer ◽  
Georg Herrler ◽  
Irene Greiser-Wilke ◽  
Beatrice Grummer
Keyword(s):  
Amino Acid ◽  
Bovine Viral Diarrhea Virus ◽  
Transmembrane Domain ◽  
Virus Entry ◽  
Transmembrane Domains ◽  
Site Directed Mutagenesis ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

The envelope of bovine viral diarrhea virus (BVDV) contains the glycoproteins Erns, E1 and E2. Complementation of a recombinant vesicular stomatitis virus (VSV) with BVDV glycoproteins resulted in infectious pseudotyped viruses. To elucidate the specific role of each of the single envelope glycoproteins during viral entry, pseudotypes were generated bearing the BVDV envelope proteins in different combinations. Pseudoviruses that contained E1 and E2 but not Erns were infectious, indicating that Erns is dispensable for virus entry. VSV/BVDV pseudotypes with chimeric proteins (the ectodomain of the BVDV glycoprotein and the transmembrane domain of the VSV-G protein) were not infectious. The fact that E1–E2 heterodimers were not detected if one of the proteins was chimeric indicated that the heterodimers are crucial for BVDV entry. It was shown by site-directed mutagenesis that the charged amino acids in the transmembrane domains of BVDV E1 (lysine and arginine) and the charged amino acid in the transmembrane domain of E2 (arginine) play a key role in heterodimer formation. Pseudoviruses bearing the mutation E2-R/A, where the charged amino acid was substituted by alanine, were not infectious, supporting the hypothesis that E1–E2 heterodimers are essential for BVDV entry.

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