scholarly journals The two phenylalanines in the GFFKR motif of the integrin α6A subunit are essential for heterodimerization

1997 ◽  
Vol 328 (2) ◽  
pp. 529-537 ◽  
Author(s):  
A. Annemieke DE MELKER ◽  
Duco KRAMER ◽  
Ingrid KUIKMAN ◽  
Arnoud SONNENBERG
Keyword(s):  
Point Mutations ◽  
K562 Cells ◽  
Cytoplasmic Domain ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Α Subunit ◽  
Focal Contacts ◽  
Arginine Residues ◽  
A Site ◽  
Laminin 1

The membrane-proximal domain of the integrin α subunit contains a conserved motif of five amino acid residues, GFFKR. We deleted this motif from the human α6A subunit and found that in COS-7 cells this mutant cannot associate with the β1 subunit and is retained in the endoplasmic reticulum. Point mutations in the GFFKR motif of the glycine residue or the two highly charged amino acids, or deletion of the lysine and arginine residues, had no effect on the ability of α6 to interact with β1 and to be expressed at the cell surface. In contrast, by replacing either of the two phenylalanines with alanine, or by deletion of both of these residues, α6 was incapable of associating with β1. The α6 point mutants that associated with β1 were expressed in K562 cells and their responsiveness to integrin-activating factors was determined. None of these transfectants bound spontaneously to laminin-1, but binding could be induced by either PMA or the stimulating anti-β1 antibody TS2/16 to the same extent as that of the wild-type transfectant. The ability of these mutants to initiate focal-contact formation in CHO cells plated on laminin-1 substrates also appeared to be unaltered. Thus the behaviour of α6 mutants involving the glycine, lysine or arginine residues was indistinguishable from that of wild-type α6 both in inside-out and outside-in signalling. In contrast, deletion of the cytoplasmic domain of α6 C-terminal of the GFFKR motif resulted in a loss of responsiveness of α6β1 to PMA stimulation and formation of focal contacts on laminin-1. However, this mutant was targeted to focal contacts formed by other integrins, even when they had not bound ligand. Together, these results suggest that the two phenylalanine residues of the GFFKR motif provide a site for interaction of the α6A subunit with β1, whereas the cytoplasmic domain C-terminal of this motif is involved in the regulation of bidirectional signalling via α6Aβ1.

scholarly journals Modulation of β1A Integrin Functions by Tyrosine Residues in the β1 Cytoplasmic Domain

1998 ◽  
Vol 141 (2) ◽  
pp. 527-538 ◽  
Author(s):  
Takao Sakai ◽  
Qinghong Zhang ◽  
Reinhard Fässler ◽  
Deane F. Mosher
Keyword(s):  
Stem Cells ◽  
Cell Adhesion ◽  
Point Mutations ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Directed Migration ◽  
Tyrosine Residues ◽  
Impaired Ability ◽  
Focal Contacts ◽  
Activating Mutation

β1A integrin subunits with point mutations of the cytoplasmic domain were expressed in fibroblasts derived from β1-null stem cells. β1A in which one or both of the tyrosines of the two NPXY motifs (Y783, Y795) were changed to phenylalanines formed active α5β1 and α6β1 integrins that mediated cell adhesion and supported assembly of fibronectin. Mutation of the proline in either motif (P781, P793) to an alanine or of a threonine in the inter-motif sequence (T788) to a proline resulted in poorly expressed, inactive β1A. Y783,795F cells developed numerous fine focal contacts and exhibited motility on a surface. When compared with cells expressing wild-type β1A or β1A with the D759A activating mutation of a conserved membrane–proximal aspartate, Y783,795F cells had impaired ability to transverse filters in chemotaxis assays. Analysis of cells expressing β1A with single Tyr to Phe substitutions indicated that both Y783 and Y795 are important for directed migration. Actin-containing microfilaments of Y783,795F cells were shorter and more peripheral than microfilaments of cells expressing wild-type β1A. These results indicate that change of the phenol side chains in the NPXY motifs to phenyl groups (which cannot be phosphorylated) has major effects on the organization of focal contacts and cytoskeleton and on directed cell motility.

scholarly journals Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

1997 ◽  
Vol 109 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Laurent Schild ◽  
Estelle Schneeberger ◽  
Ivan Gautschi ◽  
Dmitri Firsov
Keyword(s):  
Amino Acid ◽  
Ion Selectivity ◽  
Site Directed Mutagenesis ◽  
Ion Permeation ◽  
Amino Acid Residues ◽  
Α Subunit ◽  
Na Ions ◽  
Membrane Spanning ◽  
A Site ◽  
Epithelial Sodium Channel Enac

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (Ki) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn2+ ions, in particular the αS583C mutant showing a Ki for Zn2+of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride Ki, the later mutation generating a Ca2+blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ions at an external Na+binding site preventing ion permeation through the channel pore.

scholarly journals Identification of the cleavage sites in the α6A integrin subunit: structural requirements for cleavage and functional analysis of the uncleaved α6Aβ1 integrin

1997 ◽  
Vol 324 (1) ◽  
pp. 263-272 ◽  
Author(s):  
Gepke O. DELWEL ◽  
Ingrid KUIKMAN ◽  
Roel C. van der SCHORS ◽  
Annemieke A. de MELKER ◽  
Arnoud SONNENBERG
Keyword(s):  
Cleavage Site ◽  
K562 Cells ◽  
Proteolytic Cleavage ◽  
Human Platelets ◽  
Light Chains ◽  
Integrin Subunit ◽  
Cleavage Sites ◽  
Wild Type ◽  
Proper Position ◽  
Arginine Residues

The α6A and α6B integrin subunits are proteolytically cleaved during biosynthesis into a heavy chain (120 kDa) that is disulphide-linked to one of two light chains (31 or 30 kDa). Analysis of the structure of the α6A subunit on the carcinoma cell line T24 and human platelets demonstrated that the two light chains of α6 are not differentially glycosylated products of one polypeptide. Rather they possess different polypeptide backbones, which presumably result from proteolytic cleavage at distinct sites in the α6 precursor. Mutations were introduced in the codons for the R876KKR879, E883K884, R890K891 and R898K899 sequences, the potential proteolytic cleavage sites, and wild-type and mutant α6A cDNAs were transfected into K562 cells. The mutant α6A integrin subunits were expressed in association with endogenous β1 at levels comparable to that of wild-type α6Aβ1. A single α6 polypeptide chain (150 kDa) was precipitated from transfectants expressing α6A with mutations or deletions in the RKKR sequence. Mutations in the EK sequence yielded α6A subunits that were cleaved once into a heavy and a light chain, whereas α6A subunits with mutations in one of the two RK sequences were, like wild-type α6A, cleaved into one heavy and two light chains. Thus a change in the RKKR sequence prevents the cleavage of α6. The EK site is the secondary cleavage site, which is used only when the primary site (RKKR) is intact. Microsequencing of the N-termini of the two α6A light chains from platelets demonstrated that cleavage occurs after Arg879 and Lys884. Because α6RKKG, α6GKKR and α6RGGR subunits were not cleaved it seems that both the arginine residues and the lysine residues are essential for cleavage of RKKR. α6A mutants with the RKKR sequence shifted to the EK site, in such a way that the position of the arginine residue after which cleavage occurs corresponds exactly to Lys884, were partly cleaved, whereas α6A mutants with the RKKR sequence shifted to other positions in the α6A subunit, including one in which it was shifted two residues farther than the EK cleavage site, were not cleaved. In addition, α6A mutants with an α5-like cleavage site, i.e. arginine, lysine and histidine residues at positions -1, -2 and -6, were not cleaved. Thus both an intact RKKR sequence and its proper position are essential. After activation by the anti-β1 stimulatory monoclonal antibody TS2/16, both cleaved and uncleaved α6Aβ1 integrins bound to laminin-1. The phorbol ester PMA, which activates cleaved wild-type and mutant α6Aβ1, did not activate uncleaved α6Aβ1. Thus uncleaved α6Aβ1 is capable of ligand binding, but not of inside-out signalling. Our results suggest that cleavage of α6 is required to generate a proper conformation that enables the affinity modulation of the α6Aβ1 receptor by PMA.

Identification of CD20 Mutations in Malignant Lymphoma: Can They Be Predictors of Response to Rituximab?.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 288-288 ◽  
Author(s):  
Yasuhito Terui ◽  
Takuma Sakurai ◽  
Yuji Mishima ◽  
Yuko Mishima ◽  
Natsuhiko Sugimura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Malignant Lymphoma ◽  
Laser Scanning ◽  
Transmembrane Domain ◽  
Point Mutations ◽  
K562 Cells ◽  
Cytoplasmic Domain ◽  
Isolated Cells ◽  
Mammalian Expression ◽  
Cd20 Expression

Abstract Purpose: Although rituximab is commonly used as induction and maintenance therapy for CD20+ malignant lymphoma, some patients become refractory to treatment and the mechanism of resistance is unclear. The aim of this study was to investigate the relationship between CD20 mutations and rituximab resistance. Methods: To investigate whether CD20 mutations affect the response to rituximab, fresh CD19+ lymphoma cells were isolated from the lymph nodes, peripheral blood or bone marrow of 48 patients with NHL using magnetic activated cell sorting (MACS). CD19+/CD20+ cells were subsequently sorted by flow cytometry. RNA was prepared from the isolated cells and RT-PCR was performed. The resulting PCR products were sequenced, subcloned into the mammalian expression vector pTARGET, transfected into K562 cells and CD20 expression was examined by flow cytometry and laser scanning confocal microscopy. Results: In all 48 patients, overall response rate (CR+CRu+PR) to rituximab was 93.8% (45/48), but two cases became PD after PR. DNA sequence analysis revealed that point mutations were mostly observed in two CD20 domains - the third transmembrane domain and the C-terminal cytoplasmic domain. One patient had point mutations in the transmembrane domain, three cases showed point mutations in the C-terminal cytoplasmic domain and six cases had non-specific CD20 mutations, which did not affect CD20 expression. Thirty-eight patients showed no mutations of CD20 gene. CD20 expression was very weak in patients with point mutations in the C-terminal cytoplasmic domain, whereas expression was increased in patients with point mutations in the transmembrane domain. Conclusions: This study suggests that point mutations in CD20 may cause rituximab resistance and identification of CD20 mutations upon diagnosis may help to predict a patient’s response to rituximab.

Point mutation of C-terminal region of CD20 molecule predicts rituximab-induced complement-dependent cytotoxicity and clinical response to rituximab in non-Hodgkin’s lymphoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7563-7563 ◽  
Author(s):  
Y. Terui ◽  
Y. Mishima ◽  
Y. Mishima ◽  
M. Yokoyama ◽  
K. Hatake ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Laser Scanning ◽  
Transmembrane Domain ◽  
Point Mutations ◽  
K562 Cells ◽  
Cytoplasmic Domain ◽  
Laser Scanning Confocal Microscopy ◽  
Cytoplasmic Domains ◽  
Isolated Cells ◽  
Cd20 Expression

7563 Background: Although rituximab is commonly used as induction and maintenance therapy for malignant lymphoma, some patients become refractory to treatment and the mechanism of resistance is unclear. The aim of this study was to investigate the relationship between CD20 mutations and rituximab resistance. Methods: To investigate whether CD20 mutations affect response to rituximab, fresh CD19+ lymphoma cells were isolated from the lymph nodes, or bone marrow of 68 patients with NHL. The cells were subsequently sorted by flow cytometry. RNA was prepared from the isolated cells and RT-PCR was performed. The PCR products were sequenced, subcloned into an expression vector pTARGET, transfected into K562 cells. CD20 expression was examined by flow cytometry and laser scanning confocal microscopy. Results: In all 68 patients, overall response rate (CR+CRu+PR) to rituximab was 91.2% (62/68), but t four cases became PD after PR. DNA sequence analysis revealed that point mutations were mostly observed in three CD20 domains - extracellular/cytoplasmic domains, the third transmembrane domain and the C-terminal cytoplasmic domain. Two cases had point mutations in extracellular/cytoplasmic domains, one patient had point mutations in the transmembrane domain, four cases showed point mutations in the C-terminal cytoplasmic domain and six cases had non-specific CD20 mutations, which did not affect CD20 expression. 56 patients showed no mutations of CD20 gene. CD20 expression was very weak in patients with point mutations in the C-terminal cytoplasmic domain, whereas expression was increased in patients with point mutations in the transmembrane domain. Conclusions: Point mutations in CD20 may cause rituximab resistance and identification of CD20 mutations upon diagnosis may help to predict a patient’s response to rituximab. No significant financial relationships to disclose.

scholarly journals Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region.

1992 ◽  
Vol 99 (4) ◽  
pp. 545-572 ◽  
Author(s):  
B N Cohen ◽  
C Labarca ◽  
L Czyzyk ◽  
N Davidson ◽  
H A Lester
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Ion Selectivity ◽  
Point Mutations ◽  
Reversal Potential ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Mouse Muscle ◽  
Na Permeability ◽  
Delta Subunit

Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.

scholarly journals Functional Analysis of the Agrobacterium tumefaciens T-DNA Transport Pore Protein VirB8

2001 ◽  
Vol 183 (12) ◽  
pp. 3636-3641 ◽  
Author(s):  
Renu B. Kumar ◽  
Anath Das
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Point Mutations ◽  
Essential Amino Acids ◽  
Integral Membrane Protein ◽  
Tumor Formation ◽  
Dna Transfer ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Protein Protein Interaction ◽  
Pcr Method

ABSTRACT The VirB8 protein of Agrobacterium tumefaciens is essential for DNA transfer to plants. VirB8, a 237-residue polypeptide, is an integral membrane protein with a short N-terminal cytoplasmic domain. It interacts with two transport pore proteins, VirB9 and VirB10, in addition to itself. To study the role of these interactions in DNA transfer and to identify essential amino acids of VirB8, we introduced random mutations in virB8 by the mutagenic PCR method. The putative mutants were tested for VirB8 function by the ability to complement a virB8 deletion mutant in tumor formation assays. After multiple rounds of screening 13 mutants that failed to complement the virB8 deletion mutation were identified. Analysis of the mutant strains by DNA sequence analysis, Western blot assays, and reconstruction of new point mutations led to the identification of five amino acid residues that are essential for VirB8 function. The substitution of glycine-78 to serine, serine-87 to leucine, alanine-100 to valine, arginine-107 to proline or alanine, and threonine-192 to methionine led to the loss of VirB8 activity. When introduced into the wild-type strain, virB8 S87Lpartially suppressed the tumor forming ability of the wild-type protein. Analysis of protein-protein interaction by the yeast two-hybrid assay indicated that VirB8R107P is defective in interactions with both VirB9 and VirB10. A second mutant VirB8S87L is defective in interaction with VirB9.

scholarly journals Point Mutations in Exon I of the Herpes Simplex Virus Putative Terminase Subunit, UL15, Indicate that the Most Conserved Residues Are Essential for Cleavage and Packaging

2003 ◽  
Vol 77 (17) ◽  
pp. 9613-9621 ◽  
Author(s):  
Angela J. Przech ◽  
Dong Yu ◽  
Sandra K. Weller
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Point Mutations ◽  
Mutant Gene ◽  
Vero Cells ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Viral Genomes ◽  
Conserved Regions ◽  
Simplex Virus

ABSTRACT The herpes simplex virus UL15 and UL28 genes are believed to encode two subunits of the terminase involved in cleavage and packaging of viral genomes. Analysis of the UL15 protein sequence and its herpesvirus homologues revealed the presence of 20 conserved regions. Twelve of the twenty regions conserved among herpesviruses are also conserved in terminases from DNA bacteriophage. Point mutations in UL15 were designed in four conserved regions: L120N (CR1), Q205E (CR2), Q251E (CR3), G263A (CR3), and Y285S (CR4). Transfection experiments indicated that each mutant gene could produce stable UL15 protein at wild-type levels; however, only one mutant (Q251E) was able to complement the UL15-null virus. Each mutation was introduced into the viral genome by marker transfer, and all mutants except Q251E were unable to form plaques on Vero cells. Furthermore, failure to form plaques on Vero cells correlated with a defect in cleavage and packaging. Immunofluorescence experiments indicated that in cells infected with all mutant viruses the UL15 protein could be detected and was found to localize to replication compartments. Although wild-type and mutant Q251E were able to produce A, B, and C capsids, the rest of the mutants were only able to produce B capsids, a finding consistent with their defects in cleavage and packaging. In addition, all mutant UL15 proteins retained their ability to interact with B capsids. Therefore, amino acid residues 120, 205, 263, and 285 are essential for the cleavage and packaging process rather than for association with capsids or localization to replication compartments.

Characterization of an amiloride binding region in the α-subunit of ENaC

2003 ◽  
Vol 285 (6) ◽  
pp. F1279-F1290 ◽  
Author(s):  
Ollie Kelly ◽  
Chaomei Lin ◽  
Mohan Ramkumar ◽  
Nina C. Saxena ◽  
Thomas R. Kleyman ◽  
...  
Keyword(s):  
Single Channel ◽  
Point Mutations ◽  
Binding Affinities ◽  
Extracellular Loop ◽  
Wild Type ◽  
Α Subunit ◽  
Open Time ◽  
The Mean ◽  
Α Subunits

One of the defining characteristics of the epithelial sodium channel (ENaC) is its block by the diuretic amiloride. This study investigates the role of the extracellular loop of the α-subunit of ENaC in amiloride binding and stabilization. Mutations were generated in a region of the extracellular loop, residues 278–283. Deletion of this region, WYRFHY, resulted in a loss of amiloride binding to the channel. Channels formed from wild-type α-subunits or α-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities ( Po) of wild-type channels were distributed into two populations: one with a high Po and one with a low Po. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high- Po) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type α-ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel. These data provide new insight into the relative position of the extracellular loop with respect to the pore of ENaC and its role in amiloride binding and channel gating.

scholarly journals The C-Terminal Region of Bacteroides fragilis Toxin Is Essential to Its Biological Activity

2006 ◽  
Vol 74 (10) ◽  
pp. 5595-5601 ◽  
Author(s):  
Cynthia L. Sears ◽  
Simy L. Buckwold ◽  
Jai W. Shin ◽  
Augusto A. Franco
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Point Mutations ◽  
Bacteroides Fragilis ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
C Terminus

ABSTRACT To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.

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