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 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 Integrin alpha 2 cytoplasmic domain deletion effects: loss of adhesive activity parallels ligand-independent recruitment into focal adhesions.

1994 ◽  
Vol 5 (9) ◽  
pp. 977-988 ◽  
Author(s):  
S Kawaguchi ◽  
J M Bergelson ◽  
R W Finberg ◽  
M E Hemler
Keyword(s):  
Amino Acids ◽  
Cell Adhesion ◽  
Focal Adhesion ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Inverse Correlation ◽  
Cytoplasmic Domain ◽  
Negative Regulator ◽  
Wild Type

Chinese hamster ovary (CHO) cells transfected with the integrin alpha 2 subunit formed a stable VLA-2 heterodimer that mediated cell adhesion to collagen. Within CHO cells spread on collagen, but not fibronectin, wild-type alpha 2 subunit localized into focal adhesion complexes (FACs). In contrast, alpha 2 with a deleted cytoplasmic domain was recruited into FACs whether CHO cells were spread on collagen or fibronectin. Thus, as previously seen for other integrins, the alpha 2 cytoplasmic domain acts as a negative regulator, preventing indiscriminate integrin recruitment into FACs. Notably, ligand-independent localization of the VLA-2 alpha 2 subunit into FACs was partially prevented if only one or two amino acids were present in the alpha 2 cytoplasmic domain (beyond the conserved GFFKR motif) and was completely prevented by four to seven amino acids. The addition of two alanine residues (added to GFFKR) also partially prevented ligand-independent localization. In a striking inverse correlation, the same mutants showing increased ligand-independent recruitment into FACs exhibited diminished alpha 2-dependent adhesion to collagen. Thus, control of VLA-2 localization may be closely related to the suppression of cell adhesion to collagen. In contrast to FAC localization and collagen adhesion results, VLA-2-dependent binding and infection by echovirus were unaffected by either alpha 2 cytoplasmic domain deletion or exchange with other cytoplasmic domains.

scholarly journals Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization.

1992 ◽  
Vol 116 (4) ◽  
pp. 875-888 ◽  
Author(s):  
H M Miettinen ◽  
K Matter ◽  
W Hunziker ◽  
J K Rose ◽  
I Mellman
Keyword(s):  
Cytoplasmic Domain ◽  
Insoluble Fraction ◽  
Fc Receptor ◽  
Proximal Region ◽  
Wild Type ◽  
Coated Pits ◽  
Tyrosine Residues ◽  
Receptor Endocytosis ◽  
Alternative Mrna Splicing ◽  
Membrane Proximal Region

Macrophages and B-lymphocytes express two major isoforms of Fc receptor (FcRII-B2 and FcRII-B1) that exhibit distinct capacities for endocytosis. This difference in function reflects the presence of an in-frame insertion of 47 amino acids in the cytoplasmic domain of the lymphocyte isoform (FcRII-B1) due to alternative mRNA splicing. By expressing wild type and mutant FcRII cDNAs in fibroblasts, we have now examined the mechanism by which the insertion acts to prevent coated pit localization and endocytosis. We first identified the region of the FcRII-B2 cytoplasmic domain that is required for rapid internalization. Using a biochemical assay for endocytosis and an immuno-EM assay to determine coated pit localization directly, we found that the distal half of the cytoplasmic domain, particularly a region including residues 18-31, as needed for coated pit-mediated endocytosis. Elimination of the tyrosine residues at position 26 and 43, separately or together, had little effect on coated pit localization and a partial effect on endocytosis of ligand. Since the FcRII-B1 insertion occurs in the membrane-proximal region of the cytoplasmic domain (residue 6) not required for internalization, it is unlikely to act by physically disrupting the coated pit localization determinant. In fact, the insertion was found to prevent endocytosis irrespective of its position in the cytoplasmic tail and appeared to selectively exclude the receptor from coated regions. Moreover, receptors bearing the insertion exhibited a temperature- and ligand-dependent association with a detergent-insoluble fraction and with actin filaments, perhaps in part explaining the inability of FcRII-B1 to enter coated pits.

scholarly journals The Nonreceptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-Cadherin and Regulates the Cadherin–Actin Linkage

1998 ◽  
Vol 143 (2) ◽  
pp. 523-532 ◽  
Author(s):  
Janne Balsamo ◽  
Carlos Arregui ◽  
TinChung Leung ◽  
Jack Lilien
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Cytoplasmic Domain ◽  
Dominant Negative ◽  
Wild Type ◽  
Protein Tyrosine ◽  
Tyrosine Residues ◽  
Cytoplasmic Proteins ◽  
Inactive Form

Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: α-and β- or γ- catenin. Phosphorylation of β-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from β-catenin, thus maintaining the cadherin–actin connection (Balsamo et al., 1996). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and β-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion.

scholarly journals Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung

2016 ◽  
Vol 39 (2) ◽  
pp. 544-553 ◽  
Author(s):  
Sabrina V. Martini ◽  
Adriana L. Silva ◽  
Debora Ferreira ◽  
Rafael Rabelo ◽  
Felipe M. Ornellas ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Point Mutations ◽  
Lung Mechanics ◽  
Mouse Lung ◽  
Control Group ◽  
Polymorphonuclear Cells ◽  
Wild Type ◽  
Tyrosine Residues

Background/Aims: Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. Results: No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Conclusion: Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.

scholarly journals Mapping of the functional determinants of the integrin beta 1 cytoplasmic domain by site-directed mutagenesis.

1990 ◽  
Vol 1 (8) ◽  
pp. 597-604 ◽  
Author(s):  
E E Marcantonio ◽  
J L Guan ◽  
J E Trevithick ◽  
R O Hynes
Keyword(s):  
Consensus Sequence ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Focal Contact ◽  
Wild Type ◽  
3T3 Cells ◽  
Transmembrane Region ◽  
High Level Expression ◽  
Focal Contacts ◽  
High Level

We describe here the expression of deletion mutants of the cytoplasmic domain of the avian integrin beta 1 subunit. These mutants, which contain termination codons at positions 767, 776, 791, and 800, were transfected into mouse 3T3 cells to determine which sequences were essential for localization of integrins into focal contact sites. In all cases, high-level expression of the truncated avian integrins was obtained. Heterodimers were formed between the exogenous truncated avian beta 1 subunits and endogenous mouse alpha subunits, and these heterodimers were efficiently exported to the cell surface. The longest truncated beta 1 subunit tested, which is only four amino acids shorter than the wild type, does localize to focal contacts. In contrast, beta 1 subunits with moderately long truncations of the cytoplasmic domain failed to localize to focal contacts, including one which contains the consensus sequence for tyrosine phosphorylation. Surprisingly, a mutant subunit in which the bulk of the cytoplasmic domain was missing (but the segment nearest the membrane including the dibasic residues (RR) remained) did localize weakly to focal contacts. These results implicate the peptide segment nearest to the transmembrane region in focal contact localization. In addition, mutant subunits that included this segment together with a larger portion of the cytoplasmic domain did not localize as well as the shorter form, suggesting that these cytoplasmic domain segments are defective, presumably because of abnormal folding.

scholarly journals Deletions in the cytoplasmic domain of platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) result in changes in ligand binding properties

1994 ◽  
Vol 124 (1) ◽  
pp. 195-203 ◽  
Author(s):  
HM DeLisser ◽  
J Chilkotowsky ◽  
HC Yan ◽  
ML Daise ◽  
CA Buck ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Platelet Endothelial Cell Adhesion ◽  
Cell Adhesion Molecule 1 ◽  
Endothelial Cell Adhesion ◽  
Cell Cell

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a member of the immunoglobulin superfamily present on platelets, endothelial cells, and leukocytes that may function as a vascular cell adhesion molecule. The purpose of this study was to examine the role of the cytoplasmic domain in PECAM-1 function. To accomplish this, wild-type and mutated forms of PECAM-1 cDNA were transfected into murine fibroblasts and the functional characteristics of the cells analyzed. Wild-type PECAM-1 localized to the cell-cell borders of adjacently transfected cells and mediated heterophilic, calcium-dependent L-cell aggregation that was inhibitable by a polyclonal and two monoclonal anti-PECAM-1 antibodies. A mutant protein lacking the entire cytoplasmic domain did not support aggregation or move to cell-cell borders. In contrast, both forms of PECAM-1 with partially truncated cytoplasmic domains (missing either the COOH-terminal third or two thirds of the cytoplasmic domain) localized to cell-cell borders in 3T3 cells in a manner analogous to the distribution seen in cultured endothelial cells. L-cells expressing these mutants demonstrated homophilic, calcium-independent aggregation that was blocked by the polyclonal anti-PECAM-1 antibody, but not by the two bioactive monoclonal antibodies. Although changes in the cytoplasmic domain of other receptors have been shown to alter ligand-binding affinity, to our knowledge, PECAM-1 is the first example of a cell adhesion molecule where changes in the cytoplasmic domain result in a switch in the basic mechanism of adhesion leading to different ligand-binding specificity. Variations in the cytoplasmic domain could thus be a potential mechanism for regulating PECAM-1 activity in vivo.

alpha 3A beta 1 integrin localizes to focal contacts in response to diverse extracellular matrix proteins

1995 ◽  
Vol 108 (6) ◽  
pp. 2321-2336 ◽  
Author(s):  
C.M. DiPersio ◽  
S. Shah ◽  
R.O. Hynes
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Broad Spectrum ◽  
Time Course ◽  
Cell Attachment ◽  
Cell Types ◽  
Cytoplasmic Domain ◽  
Laminin 5 ◽  
Focal Contacts

In vitro binding assays and inhibition of cell adhesion with monoclonal antibodies have implicated the integrin alpha 3 beta 1 as a receptor for a variety of extracellular ligands. However, reports of alpha 3 beta 1-ligand interactions are inconsistent, and transfection studies have suggested that alpha 3 beta 1 is not sufficient for cell attachment to ligands other than kalinin/laminin 5. We used immunofluorescence to study subcellular localization of the alpha 3A cytoplasmic domain variant in different cultured cell types. Using standard fixation and permeabilization methods, antibodies specific for alpha 3A stained most cell types in a diffuse pattern, consistent with previous reports. Surprisingly, however, chemical cross-linking of integrins to the extracellular matrix and extraction of the cytoskeleton prior to immunofluorescence revealed alpha 3A in focal contacts of most cells tested, suggesting that the cytoplasmic domain was concealed in intact focal contacts by cytoskeletal or other cytoplasmic proteins. The alpha 3A subunit localized to focal contacts in several cell types cultured on fibronectin, kalinin/laminin 5, EHS-laminin/laminin 1, type IV collagen, or vitronectin. In contrast, alpha 5 and alpha V integrins were detected in focal contacts only in cells grown on their known ligands (fibronectin, and fibronectin or vitronectin, respectively). Therefore, our results show that alpha 3A beta 1 responds to a broad spectrum of extracellular ligands. Time course comparisons of the recruitment of alpha subunits from different fibronectin receptors suggested that localization of alpha 3A beta 1 to fibronectin-induced focal contacts was independent of the recruitment of alpha 5 and alpha 4 integrins. However, other studies have shown that alpha 3A beta 1 does not mediate initial cell adhesion to many of the ligands that induced its focal contact localization, including fibronectin. Therefore, we suggest that alpha 3A beta 1 may be a secondary receptor with post-cell-adhesion functions for a broad spectrum of extracellular matrices.

scholarly journals Interchangeable alpha chain cytoplasmic domains play a positive role in control of cell adhesion mediated by VLA-4, a beta 1 integrin.

1993 ◽  
Vol 178 (2) ◽  
pp. 649-660 ◽  
Author(s):  
P D Kassner ◽  
M E Hemler
Keyword(s):  
Cell Adhesion ◽  
Cell Lines ◽  
Deletion Mutant ◽  
K562 Cells ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Cytoplasmic Domains ◽  
Positive Role ◽  
Alpha Chain ◽  
Poor Adhesion

Integrins can exist in a range of functional states, depending on the cell type and its state of activation. Although the mechanism that controls activity is unknown, it has been suggested that for some integrins, alpha chain cytoplasmic domains may exert either a negative effect or no effect on adhesion function. To address this issue for VLA-4 (an alpha 4 beta 1 heterodimer), we constructed an alpha 4 cytoplasmic deletion mutant and chimeric alpha chains composed of the extracellular domains of alpha 4 and the cytoplasmic domains of alpha 2, alpha 4, or alpha 5. Upon stable transfection of wild-type alpha 4, VLA-4 heterodimer was obtained that mediated (a) poor adhesion to CS1 peptide, fibronectin, or vascular cell adhesion molecule 1 (VCAM-1) (in K562 cells); (b) poor adhesion to CS1 peptide but moderate adhesion to VCAM-1 (in MIP101 cells); and (c) moderate adhesion to both CS1 peptide and VCAM-1 (in PMWK cells). Chimeric alpha 4 constructs and wild-type alpha 4 yielded similar results in these cell lines. In contrast, truncation of the alpha 4 cytoplasmic domain (after the conserved GFFKR motif) caused an almost complete loss of adhesive activity in all three cell lines. Thus, several interchangeable alpha chain cytoplasmic domains play a fundamentally positive role in determining the state of constitutive activity for VLA-4. The alpha chain cytoplasmic domain is also required for agonist-stimulated adhesion, since phorbol ester stimulated the cell adhesion mediated by wild-type and chimeric alpha chains, but not by the cytoplasmic deletion mutant. The inactivity of both wild-type VLA-4 (in K562 cells), and truncated VLA-4 (in all three cell lines) was overcome by the addition of a stimulatory anti-beta 1 monoclonal antibody. Thus, the alpha cytoplasmic domain-dependent cellular mechanism controlling both constitutive and agonist-stimulated VLA-4 activity could be bypassed by external manipulation of the integrin.

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