The subcellular distribution of the high molecular mass protein, HD1, is determined by the cytoplasmic domain of the integrin beta 4 subunit

1997 ◽  
Vol 110 (2) ◽  
pp. 169-178 ◽  
Author(s):  
P. Sanchez-Aparicio ◽  
A.M. Martinez de Velasco ◽  
C.M. Niessen ◽  
L. Borradori ◽  
I. Kuikman ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Subcellular Distribution ◽  
Cytoplasmic Domain ◽  
High Molecular Mass ◽  
Integrin Subunit ◽  
Type Ii ◽  
Structural Protein ◽  
Focal Contacts ◽  
Cell Substrate ◽  
Beta 1 Integrin

The high molecular mass protein, HD1, is a structural protein present in hemidesmosomes as well as in distinct adhesion structures termed type II hemidesmosomes. We have studied the distribution and expression of HD1 in the GD25 cells, derived from murine embryonal stem cells deficient for the beta 1 integrin subunit. We report here that these cells possess HD1 but not BP230 or BP180; two other hemidesmosomal constituents, and express only traces of the alpha 6 beta 4 integrin. By immunofluorescence and interference reflection microscopy HD1 was found together with vinculin at the end of actin filaments in focal contacts. In OVCAR-4 cells, derived from a human ovarian carcinoma which, like GD25 cells, only weakly express alpha 6 beta 4, HD1 was also localized in focal contacts. Upon transfection of both GD25 and OVCAR-4 cells with cDNA for the human beta 4 subunit the subcellular distribution of HD1 changed significantly. HD1 is then no longer present in focal contacts but in other structures at cell-substrate contacts, colocalized with alpha 6 beta 4. These junctional complexes are probably the equivalent of the type II hemidesmosomes. Transfection of GD25 cells with beta 1 cDNA did not affect the distribution of HD1, which indicates that the localization of HD1 in focal contacts was not due to the absence of beta 1. Moreover, in GD25 cells transfected with cDNA encoding a beta 4/beta 1 chimera, in which the cytoplasmic domain of beta 4 was replaced by that of beta 1, the distribution of HD1 was unaffected. Our findings indicate that the cytoplasmic domain of beta 4 determines the subcellular distribution of HD1 and emphasize the important role of alpha 6 beta 4 in the assembly of hemidesmosomes and other junctional adhesive complexes containing HD1.

Integrin-Mediated Fibroblast Adhesion Strength: Role of the β1 Subunit

1993 ◽  
Vol 331 ◽  
Author(s):  
Kelly A. Ward ◽  
Jun-Lin Guan ◽  
Daniel A. Hammer
Keyword(s):  
Extracellular Matrix ◽  
Adhesion Strength ◽  
Cytoplasmic Domain ◽  
Integrin Receptors ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Cell Substrate ◽  
Extracellular Matrix Molecules ◽  
Integrin Ligand ◽  
Cell Substrate Adhesion

AbstractCell-substratum adhesion is important in wound healing [4], embryogenic development [11], tissue architecture [6], and metastasis [7]. Integrins constitute a major class of heterodimeric cell-surface glycoproteins involved in receptor-mediated adhesion to the extracellular matrix (ECM). Focal contacts are regions of the cell-substratum adhesion in which clusters of integrin receptors connect the cytoskeleton to extracellular matrix molecules such as fibronectin. Focal contacts strengthen cell-substrate adhesion, and are sites of biochemical activity. Since cell adhesion strength in part depends on the cell's ability to cluster receptors and cytoskeleton into focal contacts, the integrity of the focal contact, and hence a cell's adhesive strength, will depend both on integrin-cytoskeletal binding as well as integrin-ligand binding.Using a centrifugation assay, we have quantified cell-substratum adhesion strength of mouse 3T3 cells transfected with the avian β1 integrin receptor (wild type), including various deletion mutants of its cytoplasmic domain, to surfaces containing varying concentrations of CSAT, a monoclonal antibody against the extracellular domain of the avian β1 subunit. For all the transfectants, adhesion strength decreases with decreasing CSAT concentration and increasing centrifugal strength. Different truncations of the cytoplasmic domain lead to different levels of adhesion. There is no simple correlation between the length of the cytoplasmic domain and the strength of adhesion.

Alpha 7 beta 1 integrin is a component of the myotendinous junction on skeletal muscle

1993 ◽  
Vol 106 (2) ◽  
pp. 579-589 ◽  
Author(s):  
Z.Z. Bao ◽  
M. Lakonishok ◽  
S. Kaufman ◽  
A.F. Horwitz
Keyword(s):  
Skeletal Muscle ◽  
Cytoplasmic Domain ◽  
Cross Reactivity ◽  
Myotendinous Junction ◽  
Integrin Subunit ◽  
Adult Skeletal Muscle ◽  
Beta 1 Integrin ◽  
Alpha 7 ◽  
Adhesion Plaque

Immunization against a 70 kDa band that co-purifies with skeletal muscle integrins has resulted in an antibody directed against the avain alpha 7 integrin subunit. The specificity of the antibody was established by patterns of tissue staining and cross-reactivity with antibodies directed against the cytoplasmic domain of the rat alpha 7 cytoplasmic domain. On sections of adult skeletal muscle the alpha 7 integrin was enriched in the myotendinous junction (MTJ). This localization was unique as neither the alpha 1, alpha 3, alpha 5, alpha 6 and alpha v subunit localizes in the myotendinous junction. The distribution of the alpha 7 subunit in the MTJ was examined during embryonic development. alpha 7 expression in the junction is first apparent around embryo day 14 and is almost exclusively at the developing MTJ at this stage. alpha 3 is expressed with distinctive punctate staining around the junctional area in earlier embryos (11-day). The time of appearance of the alpha 7 subunit in the MTJ correlates with the insertion of myofibrils into subsarcolemmal densities and folding of the junctional membrane, suggesting a role of the alpha 7 integrin in this process. Vinculin is present throughout development of the myotendinous junction, suggesting that the alpha 7 integrin recognizes a preformed cytoskeletal structure. The presence of the alpha 7 subunit in the myotendinous junction and the alpha 5 subunit in the adhesion plaque demonstrates a molecular difference between these two adherens junctions. It also points to possible origins of junctional specificity on muscle. Differences between these two junctions were developed further using an antibody against phosphotyrosine (PY20). Phosphotyrosine is thought to participate in the organization and stabilization of adhesions. The focal adhesion and the neuromuscular junction, but not the MTJ, contained proteins phosphorylated on tyrosine.

A chimeric N-cadherin/beta 1-integrin receptor which localizes to both cell-cell and cell-matrix adhesions

1992 ◽  
Vol 103 (4) ◽  
pp. 943-951 ◽  
Author(s):  
B. Geiger ◽  
D. Salomon ◽  
M. Takeichi ◽  
R.O. Hynes
Keyword(s):  
Molecular Mechanisms ◽  
Cytoplasmic Domain ◽  
Cell Junctions ◽  
Integrin Receptor ◽  
Cell Contacts ◽  
Cell Matrix ◽  
Focal Contacts ◽  
Extracellular Region ◽  
Beta 1 Integrin ◽  
Cell Cell

To study the molecular mechanisms involved in formation of cell contacts, we have transfected cultured cells with a chimeric cDNA encoding the cytoplasmic and transmembrane domains of beta 1 integrin and the extracellular region of N-cadherin and determined the subcellular distribution of the chimeric molecule. We show that the chimeric receptor associates preferentially with cell-matrix focal contacts, suggesting that its distribution is directed by its beta 1 integrin segment, presumably via interactions of the cytoplasmic domain with cytoskeletal elements characteristic of focal contacts. Transfected cells which expressed relatively high levels of the cadherin/integrin chimera underwent an apparent epithelialization and contained the molecule both in cell-matrix and cell-cell contacts. Location in cell-cell contacts indicates competence of the cadherin extracellular domain to participate in formation of cell-cell junctions using a foreign cytoplasmic domain. Labeling of these cultures for talin, which is normally associated only with matrix adhesions, revealed specific labeling along the newly formed intercellular junctions. This suggests that the local association of talin with these sites is induced by the cytoplasmic tail of beta 1 integrin receptor presented by the chimeric protein. These results suggest that the formation of adherens-type junctions is driven by the cooperative interactions of the relevant adhesion molecules (cadherins and integrins) both with the respective extracellular ligands and with the cytoskeleton.

scholarly journals Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

1995 ◽  
Vol 6 (2) ◽  
pp. 151-160 ◽  
Author(s):  
J M Lewis ◽  
M A Schwartz
Keyword(s):  
Intracellular Signaling ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Cytoskeletal Proteins ◽  
In Vitro Assays ◽  
Integrin Subunit ◽  
Cytoplasmic Proteins ◽  
C Terminus ◽  
Beta 1 Integrin

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.

scholarly journals Beta 1 integrin-dependent and -independent polymerization of fibronectin.

1996 ◽  
Vol 132 (1) ◽  
pp. 227-238 ◽  
Author(s):  
K Wennerberg ◽  
L Lohikangas ◽  
D Gullberg ◽  
M Pfaff ◽  
S Johansson ◽  
...  
Keyword(s):  
Large Cell ◽  
Mouse Cell ◽  
Integrin Subunit ◽  
Cell Binding ◽  
Matrix Assembly ◽  
Focal Contacts ◽  
Fibronectin Matrix ◽  
Beta 1 Integrin ◽  
Alpha V Beta 3 ◽  
Fibronectin Fibrils

The mouse cell line GD25, which lacks expression of the beta 1 family of integrin heterodimers due to disruption of the beta 1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse beta 1 integrin subunit. In a stably transformed clone (GD25-beta 1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits alpha 3, alpha 5, and alpha 6. Both GD25 and GD25-beta 1A attached to fibronectin and formed focal contacts which contained alpha v beta 3, but no detectable alpha 5 beta 1A. The presence of GRGDS peptide allowed alpha 5 beta 1A to locate to focal contacts of GD25-beta 1A cultured on fibronectin, while the beta 1-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that alpha 5 beta 1A and alpha v beta 3 could bind to a large cell-binding fragment of fibronectin. alpha 5 beta 1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little alpha v beta 3 was colocalized with the fibronectin fibrils in GD25-beta 1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the alpha v beta 3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both alpha 5 beta 1A and alpha v beta 3 are able to support adhesion to fibronectin, alpha v beta 3 dominates in the formation of focal contacts, and alpha 5 beta 1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of beta 1 integrins.

scholarly journals An interaction between alpha-actinin and the beta 1 integrin subunit in vitro.

1990 ◽  
Vol 111 (2) ◽  
pp. 721-729 ◽  
Author(s):  
C A Otey ◽  
F M Pavalko ◽  
K Burridge
Keyword(s):  
Actin Filaments ◽  
Solid Phase ◽  
Immunoblot Analysis ◽  
Cytoplasmic Domain ◽  
Actin Binding ◽  
Integrin Subunit ◽  
Binding Assays ◽  
Beta 1 Integrin ◽  
Domain Peptide

A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1-integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha-actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha-actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.

scholarly journals Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.

1996 ◽  
Vol 7 (11) ◽  
pp. 1737-1748 ◽  
Author(s):  
J T Yang ◽  
R O Hynes
Keyword(s):  
Integrin Subunit ◽  
Embryonic Cells ◽  
Wild Type ◽  
Matrix Assembly ◽  
Fibronectin Receptor ◽  
Focal Contacts ◽  
Genes Encoding ◽  
Beta 1 Integrin

alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo.

scholarly journals The Localization of Bullous Pemphigoid Antigen 180 (BP180) in Hemidesmosomes Is Mediated by Its Cytoplasmic Domain and Seems to be Regulated by the β4 Integrin Subunit

1997 ◽  
Vol 136 (6) ◽  
pp. 1333-1347 ◽  
Author(s):  
Luca Borradori ◽  
Peter J. Koch ◽  
Carien M. Niessen ◽  
Stefan Erkeland ◽  
Manuel R. van Leusden ◽  
...  
Keyword(s):  
Bullous Pemphigoid ◽  
Subcellular Distribution ◽  
Focal Adhesions ◽  
Chimeric Protein ◽  
Cytoplasmic Domain ◽  
Sufficient Information ◽  
Mutant Form ◽  
Integrin Subunit ◽  
Bullous Pemphigoid Antigen ◽  
Mutant Forms

Bullous pemphigoid antigen 180 (BP180) is a component of hemidesmosomes, i.e., cell-substrate adhesion complexes. To determine the function of specific sequences of BP180 to its incorporation in hemidesmosomes, we have transfected 804G cells with cDNA-constructs encoding wild-type and deletion mutant forms of human BP180. The results show that the cytoplasmic domain of BP180 contains sufficient information for the recruitment of the protein into hemidesmosomes because removal of the extracellular and transmembrane domains does not abolish targeting. Expression of chimeric proteins, which consist of the membrane targeting sequence of K-Ras fused to the cytoplasmic domain of BP180 with increasing internal deletions or lacking the NH2 terminus, indicates that the localization of BP180 in hemidesmosomes is mediated by a segment that spans 265 amino acids. This segment comprises two important regions located within the central part and at the NH2 terminus of the cytoplasmic domain of BP180. To investigate the effect of the α6β4 integrin on the subcellular distribution of BP180, we have transfected COS-7 cells, which lack α6β4 and BP180, with cDNAs for BP180 as well as for human α6A and β4. We provide evidence that a mutant form of BP180 lacking the collagenous extracellular domain as well as a chimeric protein, which contains the entire cytoplasmic domain of BP180, are colocalized with α6β4. In contrast, when cells were transfected with cDNAs for α6A and mutant forms of β4, either lacking the cytoplasmic COOH-terminal half or carrying phenylalanine substitutions in the tyrosine activation motif of the cytoplasmic domain, the recombinant BP180 molecules were mostly not colocalized with α6β4, but remained diffusely distributed at the cell surface. Moreover, in cells transfected with cDNAs for α6A and a β4/β1 chimera, in which the cytoplasmic domain of β4 was replaced by that of the β1 integrin subunit, BP180 was not colocalized with the α6β4/β1 chimera in focal adhesions, but remained again diffusely distributed. These results indicate that sequences within the cytoplasmic domain of β4 determine the subcellular distribution of BP180.

scholarly journals Altered localization and cytoplasmic domain-binding properties of tyrosine-phosphorylated beta 1 integrin.

1994 ◽  
Vol 126 (5) ◽  
pp. 1299-1309 ◽  
Author(s):  
M W Johansson ◽  
E Larsson ◽  
B Lüning ◽  
E B Pasquale ◽  
E Ruoslahti
Keyword(s):  
Tyrosine Kinases ◽  
Rous Sarcoma Virus ◽  
Cytoplasmic Domain ◽  
Integrin Subunit ◽  
Sh2 Domains ◽  
Rous Sarcoma ◽  
Phosphorylated Form ◽  
Sarcoma Virus ◽  
Beta 1 Integrin ◽  
Adhesion Plaques

We describe a novel approach to study tyrosine-phosphorylated (PY) integrins in cells transformed by virally encoded tyrosine kinases. We have synthesized a peptide (PY beta 1 peptide) that represents a portion of the cytoplasmic domain of the beta 1 integrin subunit and is phosphorylated on the tyrosine residue known to be the target of oncogenic tyrosine kinases. Antibodies prepared against the PY beta 1 peptide, after removal of cross-reacting antibodies by absorption and affinity purification, recognized the PY beta 1 peptide and the tyrosine-phosphorylated form of the intact beta 1 subunit, but did not bind the nonphosphorylated beta 1 peptide, the nonphosphorylated beta 1 subunit or other unrelated tyrosine-phosphorylated proteins. The anti-PY beta 1 antibodies labeled the podosomes of Rous sarcoma virus-transformed fibroblasts, but did not detectably stain nontransformed fibroblasts. The localization of the tyrosine phosphorylated beta 1 subunits appeared distinct from that of the beta 1 subunit. Adhesion plaques were stained by the anti-beta 1 subunit antibodies in Rous sarcoma virus-transformed fibroblasts plated on fibronectin, whereas neither podosomes nor adhesion plaques were labeled on vitronectin or on uncoated plates. Anti-phosphotyrosine antibodies labeled podosomes, adhesion plaques and cell-cell boundaries regardless of the substratum. One of the SH2 domains of the p85 subunit of phosphatidylinositol-3-kinase bound to the PY beta 1 peptide, but not to the non-phosphorylated beta 1 cytoplasmic peptide. Other SH2 domains did not bind to the PY beta 1 peptide. These results show that the phosphorylated form of the beta 1 integrin subunit is detected in a different subcellular localization than the nonphosphorylated form and suggest that the phosphorylation on tyrosine of the beta 1 subunit cytoplasmic domain may affect cellular signaling pathways.

scholarly journals Lymphocyte migration across high endothelium is associated with increases in alpha 4 beta 1 integrin (VLA-4) affinity

1993 ◽  
Vol 104 (4) ◽  
pp. 1049-1059 ◽  
Author(s):  
H. Hourihan ◽  
T.D. Allen ◽  
A. Ager
Keyword(s):  
Primary Cultures ◽  
Type I ◽  
Integrin Subunit ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lymphocyte Migration ◽  
Endothelial Layer ◽  
Beta 1 Integrin ◽  
Lymphocyte Populations

The constitutive recirculation of lymphocytes between the widely distributed organs of the immune system is essential for host defence. We have developed an in vitro model of lymphocyte migration from the blood into lymph nodes which employs primary cultures of high endothelial cells (HEC). HEC-adherent lymphocytes adopt one of two distinct morphologies which correlates with their position in the endothelial layer; type I cells are bound to the surface of HEC and type II cells are underneath the endothelial layer. In a previous study we reported that the numbers of type I and type II cells are independently regulated, however the relationship between these two lymphocyte populations was not determined. In this study we have carried out detailed kinetic, phenotypic and functional analyses of type I and type II lymphocytes and determined their relationship. Using allotype marked lymphocytes from the PVG.RT7a and PVG.RT7b rat strains in a pulse-chase analysis, type I and type II lymphocytes were found to represent the same population of lymphocytes at different stages of interaction with the endothelial layer, rather than representing two independent lymphocyte populations. Migration was an irreversible event and the efficiency of migration (i.e. transition from type I to type II) was related to the concentration of lymphocytes plated on to the HEC layer. Following transmigration lymphocytes showed an increased ability to migrate across HEC layers and to bind to immobilised CS1 peptide. The increased binding to CS1 peptide was transient and fell to control levels over a 3 hour time period. The expression of alpha 4 integrin subunit on lymphocytes was unchanged following migration which suggests that the affinity of the CS1 receptor, alpha 4 beta 1, is upregulated by interaction with HEC. Together these results suggest that transendothelial migration is regulated by increases in the affinity of alpha 4 beta 1 integrin on lymphocytes following contact with HEC.

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