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 Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8.

1990 ◽  
Vol 110 (6) ◽  
pp. 2145-2155 ◽  
Author(s):  
A Sonnenberg ◽  
C J Linders ◽  
P W Modderman ◽  
C H Damsky ◽  
M Aumailley ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Types ◽  
Integrin Subunit ◽  
Integrin Receptors ◽  
Cell Binding ◽  
Vitronectin Receptor ◽  
Beta 1 Integrin ◽  
Integrin Alpha 6 ◽  
Alpha V Beta 3 ◽  
Integrin Family

The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.

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 Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

1994 ◽  
Vol 124 (3) ◽  
pp. 373-380 ◽  
Author(s):  
E Koivunen ◽  
B Wang ◽  
E Ruoslahti
Keyword(s):  
Phage Display ◽  
Cyclic Peptide ◽  
Cell Attachment ◽  
Phage Display Library ◽  
Cell Binding ◽  
Rgd Peptides ◽  
Beta 1 Integrin ◽  
Alpha V Beta 3 ◽  
Integrin Ligand ◽  
Specific Ligand

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.

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.

scholarly journals A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

1996 ◽  
Vol 133 (2) ◽  
pp. 431-444 ◽  
Author(s):  
D C Hocking ◽  
R K Smith ◽  
P J McKeown-Longo
Keyword(s):  
Binding Site ◽  
Wound Repair ◽  
Solid Phase ◽  
Focal Adhesions ◽  
Amino Terminus ◽  
Matrix Assembly ◽  
Amino Terminal ◽  
Fibronectin Matrix ◽  
Beta 1 Integrin ◽  
Rgd Site

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

scholarly journals TSG-6 binds via its CUB_C domain to the cell-binding domain of fibronectin and increases fibronectin matrix assembly

2008 ◽  
Vol 27 (3) ◽  
pp. 201-210 ◽  
Author(s):  
S KUZNETSOVA ◽  
D MAHONEY ◽  
G MARTINMANSO ◽  
T ALI ◽  
H NENTWICH ◽  
...  
Keyword(s):  
Binding Domain ◽  
Cell Binding ◽  
Matrix Assembly ◽  
Fibronectin Matrix ◽  
Cell Binding Domain

scholarly journals Requirement of the integrin beta 3 subunit for carcinoma cell spreading or migration on vitronectin and fibrinogen

1992 ◽  
Vol 117 (5) ◽  
pp. 1101-1107 ◽  
Author(s):  
DI Leavesley ◽  
GD Ferguson ◽  
EA Wayner ◽  
DA Cheresh
Keyword(s):  
Surface Expression ◽  
Porous Membrane ◽  
Biological Properties ◽  
Carcinoma Cells ◽  
Dependent Manner ◽  
Focal Contacts ◽  
Beta 1 Integrin ◽  
Alpha V Beta 3 ◽  
Integrin Beta 3 ◽  
Extracellular Environment

FG human pancreatic carcinoma cells use integrin alpha v beta 5 as their primary vitronectin receptor since they fail to express integrin alpha v beta 3. These cells are unable to form focal contacts, spread, or migrate on vitronectin but readily do so on collagen in a beta 1 integrin-dependent manner. Transfection of FG cells with a cDNA encoding the integrin beta 3 subunit results in the surface expression of a functional integrin alpha v beta 3 heterodimer providing these cells with novel adhesive and biological properties. Specifically, FG cells expressing beta 3 acquire the capacity to attach and spread on vitronectin as well as fibrinogen with beta 3 localization to focal contacts. Moreover, these cells gain the capacity to migrate through a porous membrane in response to either vitronectin or fibrinogen. These results demonstrate that the beta 3 and beta 5 integrin subunits when associated with alpha v, promote distinct cellular responses to a vitronectin extracellular environment.

A novel role for alpha 3 beta 1 integrins in extracellular matrix assembly

1995 ◽  
Vol 108 (6) ◽  
pp. 2511-2523 ◽  
Author(s):  
C. Wu ◽  
A.E. Chung ◽  
J.A. McDonald
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Binding Activity ◽  
Integrin Subunit ◽  
Pericellular Matrix ◽  
Matrix Assembly ◽  
Amino Terminal ◽  
Beta 1 Integrin ◽  
Fibronectin Deposition

To study the biological role of alpha 3 beta 1 integrins in cell adhesion, migration, and in the deposition of extracellular matrix, we stably expressed the human alpha 3 integrin subunit in the alpha 4, alpha 5 integrin deficient CHO cell line B2. The expression of alpha 3 beta 1 integrins enhanced cell adhesion on entactin (also known as nidogen), but not on fibronectin. Using recombinant GST-fusion proteins that span the entire length of the entactin molecule, we located cell adhesive activity to the G2 domain of entactin. These results suggest that the alpha 3 beta 1 integrin functions as an adhesion receptor interacting with the G2 domain of entactin. On the other hand, the expression of alpha 3 beta 1 integrins did not confer the ability to migrate on entactin. Strikingly, the expression of alpha 3 beta 1 dramatically increased the deposition of entactin and fibronectin into the pericellular matrix. This was accompanied by increased binding activity of the 29 kDa amino-terminal domain of fibronectin. Thus, similar to alpha 5 beta 1 integrins, alpha 3 beta 1 integrins can play an important role in modulating the assembly of pericellular matrices. However, unlike fibronectin deposition supported by alpha 5 beta 1, alpha 3 beta 1 supported fibronectin deposition into pericellular matrix was not inhibited by antibodies binding to the RGD containing cell adhesion domain of fibronectin, demonstrating that the two processes are mechanistically distinct. The role of alpha 3 beta 1 in pericellular matrix assembly potentially implicates this receptor in the assembly and/or recognition of entactin-containing pericellular matrices, an observation consistent with its apparent role in the renal glomerulus of the mammalian kidney.

Immuno-EM localization of the beta 1 integrin subunit in wet-cleaved fibronectin-adherent fibroblasts

1994 ◽  
Vol 107 (5) ◽  
pp. 1229-1239 ◽  
Author(s):  
A.M. Meijne ◽  
D.M. Casey ◽  
C.A. Feltkamp ◽  
E. Roos
Keyword(s):  
Plasma Membranes ◽  
Matrix Proteins ◽  
Integrin Subunit ◽  
Beta 1 Integrin ◽  
Coated Surface ◽  
20 Nm ◽  
Fibronectin Fibrils ◽  
Adhesion Plaque ◽  
Punctate Staining ◽  
Chicken Embryo Fibroblasts

Using immuno-EM, we have studied the distribution of the beta 1 integrin subunit in chicken embryo fibroblasts allowed to adhere and spread for 3 hours on a fibronectin-coated surface in serum-free medium. The cells were wet-cleaved, which removed most of the cell body, yielding ventral plasma membranes with little, and sometimes virtually no, associated cytoskeleton. The beta 1 integrin subunit was detected with antibodies against the cytoplasmic domain. In immune fluorescence, it colocalized with adhesion plaques, in a punctate staining pattern, and often seemed to be at the periphery of the plaque. By immuno-EM, beta 1 was in fact found in discrete clusters, not throughout the plaque. In deep-cleaved cells from which virtually all cytoskeleton was removed, clusters could often be seen to be located on fibronectin fibrils. Furthermore, beta 1 was present in clusters at the cell margins, and isolated or in small groups at the very edge of the cell. When fibronectin synthesis, and consequently fibril formation, was inhibited by cycloheximide, large adhesion plaque-like structures were formed at the cell margin. This phenotype was reversed by addition of soluble fibronectin, which was incorporated into fibrils. As in normal plaques, talin and vinculin were present, the plasma membrane was very close (10-20 nm) to the substratum and the fibronectin layer underneath was removed. These plaques did contain beta 1 integrins but they were not in clusters. These observations indicate that the talin-vinculin network of an adhesion plaque is normally anchored to the substratum at discrete beta 1 integrin clusters that may be located on fibronectin fibrils, and that elsewhere the plaque is not necessarily attached to the substratum by interaction of integrins with matrix proteins. In the absence of fibronectin fibrils, adhesion plaque-like structures can be formed, but these are aberrant in size, location and fine structure.

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