The alpha 5 beta 1 integrin fibronectin receptor, but not the alpha 5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly.

The fibronectin receptor, alpha 5 beta 1, has been shown to be required for fibronectin matrix assembly and plays an important role in cell migration on fibronectin. However, it is not clear whether other fibronectin binding integrins can take the place of alpha 5 beta 1 during matrix assembly and cell migration. To test this, we expressed the human alpha v subunit in the CHO cell line CHO-B2 that lacks the alpha 5 subunit. We found that the human alpha v combined with CHO cell beta 1 to form the integrin alpha v beta 1. Cells that expressed alpha v beta 1 attached to and spread well on fibronectin-coated dishes, but did so less well on vitronectin-coated dishes. This, along with other data, indicated that alpha v beta 1 functions as a fibronectin receptor in CHO-B2 cells. The alpha v beta 1-expressing cells failed to produce a fibronectin matrix or to migrate on fibronectin, although the same cells transfected with alpha 5 do produce a matrix and migrate on fibronectin. The affinity of the alpha v beta 1-expressing cells for fibronectin was fourfold lower than that of the alpha 5 beta 1-expressing cells. In addition, alpha v beta 1 was distributed diffusely throughout the cell surface, whereas alpha 5 beta 1 was localized to focal adhesions when cells were seeded onto fibronectin-coated surfaces. Thus, of the two fibronectin receptors, alpha v beta 1 and alpha 5 beta 1, only alpha 5 beta 1 supports fibronectin matrix assembly and promotes cell migration on fibronectin in the CHO-B2 cells. Possible reasons for this difference in the activities of alpha v beta 1 and alpha 5 beta 1 include the lower affinity of alpha v beta 1 for fibronectin and the failure of this integrin to localize in adhesion plaques on a fibronectin substrate. These results show that two integrins with similar ligand specificities and cell attachment functions may be quite different in their ability to support fibronectin matrix assembly and cell motility on fibronectin.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.133.2.431 ◽

1996 ◽

Vol 133 (2) ◽

pp. 431-444 ◽

Cited By ~ 97

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.

Download Full-text

Laminins promote the locomotion of skeletal myoblasts via the alpha 7 integrin receptor

Journal of Cell Science ◽

10.1242/jcs.109.13.3139 ◽

1996 ◽

Vol 109 (13) ◽

pp. 3139-3150 ◽

Cited By ~ 4

Author(s):

C.C. Yao ◽

B.L. Ziober ◽

A.E. Sutherland ◽

D.L. Mendrick ◽

R.H. Kramer

Keyword(s):

Cytoplasmic Domain ◽

Matrix Assembly ◽

Developmentally Regulated ◽

Laminin 5 ◽

Alternative Mrna Splicing ◽

Beta 1 Integrin ◽

Alpha 7 ◽

Myoblast Cell ◽

And Migration

The alpha 7 beta 1 integrin is specifically expressed by skeletal and cardiac muscles, and its expression and alternative mRNA splicing at the cytoplasmic domain are developmentally regulated. We analyzed the role of alpha 7 integrin in mediating myoblast adhesion and motility on different laminin isoforms. Mouse C2C12 and MM14 myoblast cell lines were found by flow cytometry and immunoprecipitation to express high levels of the alpha 7 integrin. Overall expression of alpha 7 increased as the C2C12 myoblasts differentiated; myoblasts expressed only the alpha 7B cytoplasmic variant whereas in differentiating myotubes alpha 7A increased markedly. Function-perturbing monoclonal antibodies generated to alpha 7 integrin efficiently blocked both adhesion and migration of MM14 and C2C12 mouse myoblasts on laminin 1. Other studies with MM14 myoblasts showed that alpha 7 is also a receptor for laminin 2/4 (human placental merosins) but not for epithelial-cell-specific laminin 5. Blocking antibody to alpha 7 only partially inhibited adhesion to laminin 2/4 but almost completely blocked motility on this substrate. Finally, to assess the potential role of the alpha 7 cytoplasmic domain, CHO cells were stably transfected to expressed chimeric alpha 5 cDNA constructs containing the wild-type alpha 5 or the alpha 7A or alpha 7B cytoplasmic domain; all forms of the integrin showed identical activities for adhesion, migration, proliferation, and matrix assembly on fibronectin substrates. These results established that alpha 7 beta 1 receptor can promote myoblast adhesion and motility on a restricted number of laminin isoforms and may be important in myogenic precursor recruitment during regeneration and differentiation.

Download Full-text

Down-regulation of the chicken alpha 5 beta 1 integrin fibronectin receptor during development

Development ◽

10.1242/dev.113.1.327 ◽

1991 ◽

Vol 113 (1) ◽

pp. 327-337 ◽

Cited By ~ 10

Author(s):

J.L. Muschler ◽

A.F. Horwitz

Keyword(s):

Cell Types ◽

Embryonic Cell ◽

Alpha Subunit ◽

Down Regulation ◽

Fibronectin Receptor ◽

Alpha Subunits ◽

Adult Tissues ◽

Beta 1 Integrin ◽

Integrin Fibronectin Receptor ◽

Integrin Family

We have characterized the diversity of the chicken beta 1 integrin family and studied the expression of individual receptors during development. The diversity of the beta 1 integrin family was investigated by affinity purifying the beta 1 integrins from a variety of adult and embryonic tissues. These purifications reveal the relative levels of expression and also the differential expression of the alpha subunits in those tissues. Monoclonal antibodies were generated against the prominent ‘band 1’ of the embryonic chicken integrins and used to characterize the expression of this alpha subunit in embryonic and adult tissues. This alpha subunit is shown to be the chicken homologue of human alpha 5 fibronectin receptor. The chicken alpha 5 beta 1 integrin is the most prominent beta 1 integrin in the embryo and is expressed on the majority of cell types through the day 17 stage. The distribution of this receptor in the embryo closely parallels the distribution of its ligand, fibronectin. In adult tissues, expression of this receptor is greatly diminished relative to the expression of other alpha subunits. The cell type distribution is highly restricted: limited primarily to the vasculature and to connective tissue regions. These studies reveal a prominent role for the alpha 5 beta 1 integrin in embryonic cell types and a down-regulation of this receptor on many cell types during development.

Download Full-text

β1 Integrin Cytoplasmic Domain Residues Selectively Modulate Fibronectin Matrix Assembly and Cell Spreading through Talin and Akt-1

Journal of Biological Chemistry ◽

10.1074/jbc.m805934200 ◽

2009 ◽

Vol 284 (12) ◽

pp. 8148-8159 ◽

Cited By ~ 26

Author(s):

J. Angelo Green ◽

Allison L. Berrier ◽

Roumen Pankov ◽

Kenneth M. Yamada

Keyword(s):

Cell Spreading ◽

Cytoplasmic Domain ◽

Β1 Integrin ◽

Matrix Assembly ◽

Fibronectin Matrix

Download Full-text

Temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction.

The Journal of Cell Biology ◽

10.1083/jcb.110.1.133 ◽

1990 ◽

Vol 110 (1) ◽

pp. 133-145 ◽

Cited By ~ 228

Author(s):

M P Welch ◽

G F Odland ◽

R A Clark

Keyword(s):

Granulation Tissue ◽

Receptor Expression ◽

Wound Contraction ◽

Tissue Loss ◽

Matrix Assembly ◽

Fibronectin Receptor ◽

Actin Bundle ◽

Actin Bundles ◽

Fibronectin Matrix ◽

Temporal Relationships

Wound contraction can substantially reduce the amount of new tissue needed to reestablish organ integrity after tissue loss. Fibroblasts, rich in F-actin bundles, generate the force of wound contraction. Fibronectin-containing microfibrils link fibroblasts to each other and to collagen bundles and thereby provide transduction cables across the wound for contraction. The temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction have not been determined. To establish these relationships, we used a cutaneous gaping wound model in outbred Yorkshire pigs. Granulation tissue filled approximately 80% of the wound space by day 5 after injury while wound contraction was first apparent at day 10. Neither actin bundles nor fibronectin receptors were observed in 5-d wound fibroblasts. Although fibronectin fibrils were assembled on the surfaces of 5-d fibroblasts, few fibrils coursed between cells. Day-7 fibroblasts stained strongly for nonmuscle-type F-actin bundles consistent with a contractile fibroblast phenotype. These cells expressed fibronectin receptors, were embedded in a fibronectin matrix that appeared to connect fibroblasts to the matrix and to each other, and were coaligned across the wound. Transmission EM confirmed the presence of microfilament bundles, cell-cell and cell-matrix linkages at day 7. Fibroblast coalignment, matrix interconnections, and actin bundles became more pronounced at days 10 and 14 coinciding with tissue contraction. These findings demonstrate that granulation tissue formation, F-actin bundle and fibronectin receptor expression in wound fibroblasts, and fibroblast-matrix linkage precede wound contraction.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.132.1.227 ◽

1996 ◽

Vol 132 (1) ◽

pp. 227-238 ◽

Cited By ~ 201

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.

Download Full-text

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

Molecular Biology of the Cell ◽

10.1091/mbc.7.11.1737 ◽

1996 ◽

Vol 7 (11) ◽

pp. 1737-1748 ◽

Cited By ~ 93

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.

Download Full-text

Identification and characterization of a dimeric chicken fibronectin receptor. Subunit-specific monoclonal antibodies to the putative chicken alpha 5 beta 1 integrin.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)77339-0 ◽

1990 ◽

Vol 265 (24) ◽

pp. 14561-14565

Author(s):

U. Hofer ◽

J. Syfrig ◽

R. Chiquet-Ehrismann

Keyword(s):

Monoclonal Antibodies ◽

Receptor Subunit ◽

Fibronectin Receptor ◽

Beta 1 Integrin ◽

Identification And Characterization

Download Full-text

Sphingosine 1-Phosphate Stimulates Fibronectin Matrix Assembly Through a Rho-Dependent Signal Pathway

Blood ◽

10.1182/blood.v93.9.2984 ◽

1999 ◽

Vol 93 (9) ◽

pp. 2984-2990 ◽

Cited By ~ 49

Author(s):

Qinghong Zhang ◽

Olivier Peyruchaud ◽

Kelly J. French ◽

Magnus K. Magnusson ◽

Deane F. Mosher

Keyword(s):

Binding Sites ◽

Signal Pathway ◽

Bioactive Lipids ◽

Surface Binding ◽

Matrix Assembly ◽

Fibronectin Matrix ◽

Mg63 Cells ◽

Sphingosine 1 Phosphate ◽

Common Signal ◽

Stimulation Of

Abstract Fibronectin matrix assembly is a cell-dependent process mediated by cell surface binding sites for the 70-kD N-terminal portion of fibronectin. We have shown that Rho-dependent cytoskeleton reorganization induced by lysophosphatidic acid (LPA) or the microtubule-disrupting agent nocodazole increases fibronectin binding (Zhang et al, Mol Biol Cell 8:1415, 1997). Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in mitogenesis and cytoskeletal remodelling. Both LPA and S1P are present in increased amounts in serum as compared with plasma as a result of platelet activation. Addition of S1P to human osteosarcoma MG63 cells or human foreskin fibroblasts increased cell-mediated binding and assembly of fibronectin. MG63 cells expressed the Edg-2 and Edg-4 G-protein–coupled receptors for bioactive lipids, whereas foreskin fibroblasts expressed Edg-2, Edg-3, and Edg-4. The stimulatory effect of S1P on the binding of fibronectin or the N-terminal 70-kD fragment of fibronectin was dynamic and due to increases in both the number and affinity of binding sites. The stimulation of 70-kD fragment binding by nanomolar S1P, like stimulation of binding by LPA or nocodazole, was blocked by inactivation of Rho with C3 exotoxin but not by pertussis toxin-mediated inactivation of Gi. These results indicate a common signal pathway leading to control of cellular fibronectin matrix assembly by bioactive lipids generated during blood coagulation.

Download Full-text