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 Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit.

1991 ◽  
Vol 115 (4) ◽  
pp. 1137-1148 ◽  
Author(s):  
A P Skubitz ◽  
P C Letourneau ◽  
E Wayner ◽  
L T Furcht
Keyword(s):  
Amino Acids ◽  
Cell Adhesion ◽  
Neurite Outgrowth ◽  
Cell Types ◽  
Integrin Subunit ◽  
Globular Domain ◽  
Heparin Binding ◽  
Beta 1 Integrin ◽  
A Chain ◽  
Carboxy Terminal

The large carboxy-terminal globular domain (G domain; residues 2,110-3,060) of the A chain of murine-derived laminin has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. This study was conducted to define the potential sequence(s) originating from the G domain of laminin with any of these functional activities. A series of peptides were synthesized from the G domain, termed GD peptides, each approximately 20 amino acids long and containing multiple positively charged amino acids. In direct 3H-heparin binding assays, peptides GD-1 and GD-2 bound high levels of 3H-heparin, while peptides GD-3 and GD-4 bound lower levels of 3H-heparin, and GD-5 bound essentially no 3H-heparin. The binding of 3H-heparin to peptides GD-1 and GD-2 appeared to be of high affinity, since significant binding of 3H-heparin to these two peptides was still observed even when the NaCl concentration was raised to 1.0 M. Four of the peptides, GD-1, GD-2, GD-3, and GD-4, directly promoted the adhesion and spreading of HT-1080 human fibrosarcoma cells as well as the outgrowth of neurites from chick spinal cord and dorsal root ganglia neurons. In addition, solutions of these peptides or antibodies generated against these peptides inhibited laminin-mediated HT-1080 cell adhesion. Antibodies against the beta 1 integrin subunit inhibited HT-1080 cell adhesion and neurite outgrowth on surfaces adsorbed with peptides GD-3 and GD-4. Therefore, laminin appears to have multiple, independent sequences in the G domain that serve a similar cell adhesion promoting function for different cell types. Furthermore, these results suggest that the sequences comprising peptides GD-3 and GD-4 use an integrin as a receptor, of which the beta 1 integrin subunit is a component for these various cell types.

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 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.

Contrasting migratory response of astrocytoma cells to tenascin mediated by different integrins

1996 ◽  
Vol 109 (8) ◽  
pp. 2161-2168 ◽  
Author(s):  
A. Giese ◽  
M.A. Loo ◽  
S.A. Norman ◽  
S. Treasurywala ◽  
M.E. Berens
Keyword(s):  
Cell Adhesion ◽  
Matrix Protein ◽  
Glioma Cells ◽  
Extracellular Matrix Protein ◽  
Integrin Receptors ◽  
Migration Assay ◽  
Dose Dependent Fashion ◽  
Beta 1 Integrin

Tenascin, an extracellular matrix protein, is expressed in human gliomas in vitro and in vivo. The distribution of tenascin at the invasive edge of these tumors, even surrounding solitary invading cells, suggests a role for this protein as a regulator of glioma cell migration. We tested whether purified tenascin, passively deposited on surfaces, influenced the adhesion or migration of a human gliomaderived cell line, SF-767. Adhesion of glioma cells to tenascin increased in a dose-dependent fashion up to a coating concentration of 10 micrograms/ml. Higher coating concentrations resulted in progressively fewer cells attaching. Cell adhesion could be blocked to basal levels using anti-beta 1 integrin antibodies. In contrast, when anti-alpha v antibodies were added to the medium of cells on tenascin, cell adhesion was enhanced slightly. Using a microliter scale migration assay, we found that cell motility on tenascin was dose dependently stimulated at coating concentrations of 1 and 3 micrograms/ml, but migration was inhibited below levels of non-specific motility when tested at coating concentrations of 30 and 100 micrograms/ml. Migration on permissive concentrations of tenascin could be reversibly inhibited with anti-beta 1, while treatment with anti-alpha v antibodies increased migration rates. We conclude that SF-767 glioma cells express two separate integrin receptors that mediate contrasting adhesive and migratory responses to tenascin.

Effects of polystyrene surface chemistry on the biological activity of solid phase fibronectin and vitronectin, analysed with monoclonal antibodies

1993 ◽  
Vol 104 (3) ◽  
pp. 793-803 ◽  
Author(s):  
P.A. Underwood ◽  
J.G. Steele ◽  
B.A. Dalton
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Solid Phase ◽  
Divalent Cations ◽  
Biological Activities ◽  
Optimal Growth ◽  
Cell Types ◽  
Cell Binding ◽  
Urea Treatment ◽  
Bhk Cells

The conformation and biological activities of fibronectin (FN) and vitronectin (VN) coated on different plastic surfaces were investigated using cell adhesion and a panel of domain-specific monoclonal antibodies (mAbs). The adhesion of BHK fibroblasts was markedly better on FN coated on hydrophilic tissue culture polystyrene (TCPS) than on hydrophobic, untreated polystyrene (PS). mAbs A17 and 3E3, which inhibit the binding of BHK cells to the RGD-containing sequence within the cell binding region of FN, also bound preferentially to FN on TCPS. In contrast, two anti-FN mAbs, which have no effect on cell adhesion (A35 and A3), bound preferentially to the conformation of FN on the more hydrophobic PS. Mouse melanoma cells utilise an additional cell-binding site in the Hep II domain of FN and their preference for FN coated on TCPS was less marked than that of BHK cells. This reduced preference was again mimicked by the binding of a mAb, A32, which inhibits the binding of B16 cells to the Hep II domain of FN. In contrast, BHK cell adhesion to VN did not display a preference for TCPS over PS. The cell-binding activity of adsorbed VN was matched by the binding of a cell adhesion-inhibitory mAb, A18, which, unlike mAbs A17 and A32, displayed slightly increased binding to VN coated on PS, rather than TCPS. When the denaturating effect of coating FN and VN to PS in the presence of urea was investigated, similar correlations between BHK cell adhesion and the binding of inhibitory mAbs were observed. Urea treatment of FN significantly reduced both BHK cell adhesion and the binding of both cell adhesion-inhibitory mAbs, whereas the binding of A35 and A3 was unaffected. There was no significant effect of urea treatment of VN upon either BHK cell adhesion or mAb binding. A larger panel of anti-FN mAbs was used, together with the anti-VN mAbs, to determine whether there were differences in mAb recognition of FN and VN adsorbed on three different brands of TCPS. The mAbs segregated into four reactivity patterns, of which A17, A32, A35 and A18 respectively were representative. Significant differences were observed in mAb recognition of FN and VN adsorbed to different brands of TCPS. These may reflect differences in the ability of these surfaces to support optimal growth of different cell types. The effect of divalent cations upon adsorbed FN and VN was also investigated.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Altered glycosylation and cell surface expression of beta 1 integrin receptors during keratinocyte activation

1992 ◽  
Vol 103 (3) ◽  
pp. 743-753 ◽  
Author(s):  
L.T. Kim ◽  
S. Ishihara ◽  
C.C. Lee ◽  
S.K. Akiyama ◽  
K.M. Yamada ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Surface ◽  
Fold Increase ◽  
Cell Surface Expression ◽  
Activation Process ◽  
Integrin Subunit ◽  
Integrin Receptors ◽  
Serum Factors ◽  
Keratinocyte Proliferation ◽  
Beta 1 Integrin

We studied the mechanism by which cell adhesiveness becomes activated when keratinocytes are removed from skin and placed into cell culture. Our results suggest that activation involves altered beta 1 integrin subunit glycosylation accompanied by an increase in cell surface beta 1 integrin receptors. Activated keratinocytes contained two forms of the beta 1 integrin subunit, approximately 93 kDa and approximately 113 kDa. As shown by pulse-chase experiments, the smaller represented the cytoplasmic precursor of the larger, and only the 113 kDa mature form was detected in integrin receptors expressed at the cell surface. Pre-activated keratinocytes contained beta 1 integrin subunits ranging from approximately 97 to 110 kDa. These beta 1 subunits had been processed through the Golgi, based on resistance to endoglycosidase-H treatment, and were not converted to 113 kDa subunits during subsequent cell culture. Experiments with endoglycosidase-F showed that differences in the apparent sizes of beta 1 integrin subunits observed in pre-activated and activated keratinocytes could be attributed to differences in subunit glycosylation. Smaller beta 1 subunits found in pre-activated keratinocytes, like the precursor beta 1 subunits of activated cells, appeared to be less efficient in reaching the cell surface. Overall, a approximately 10-fold increase in the level of cell surface integrin receptors occurred concomitant with the increased proportion of 113 kDa beta 1 subunits found in activated cells. Endoglycosidase-F experiments also indicated that there were changes in keratinocyte alpha subunits associated with beta 1. In related experiments, keratinocytes cultured in low Ca2+, serum-free MCDB medium for 4 days proliferated but their adhesiveness did not become activated. Therefore, keratinocyte proliferation and activation of adhesion are regulated separately. Finally, substantial activation of keratinocytes was observed when serum was added to cells cultured in MCDB with serum, indicating a role for serum factors in the activation process.

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

Development ◽  
1991 ◽  
Vol 113 (1) ◽  
pp. 327-337 ◽  
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.

Characterization of integrins in cultured human renal cortical tubule epithelial cells

1994 ◽  
Vol 267 (4) ◽  
pp. F612-F623
Author(s):  
E. E. Simon ◽  
C. H. Liu ◽  
M. Das ◽  
S. Nigam ◽  
T. J. Broekelmann ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Epithelial Cells ◽  
Initial Attachment ◽  
Cell Contacts ◽  
Cell Matrix ◽  
Beta 1 Integrin ◽  
Tubule Cells ◽  
Alpha V Beta 3 ◽  
Cell Cell

We have characterized the integrins present on cultured tubule epithelial cells from human renal cortexes, enriched for proximal cells, using fluorescence microscopy, immunoprecipitation, and cell adhesion assays. By immunofluorescence, the alpha 3-integrin subunit stained most intensely and was present on all cells predominantly at cell-cell contacts. The alpha 6-subunit was present on all cells in a pattern consistent with extracellular matrix contacts. The alpha 5-subunit was present on most cells in a cell-matrix contact pattern; alpha V-subunit was weakly positive and occasionally seen in cell-matrix contacts. The alpha 2-subunit was present on clusters of distal tubule cells, predominantly at cell-cell contacts. Immunoprecipitation revealed the predominant integrin to be alpha 3 beta 1 with some alpha 2 beta 1, presumably contributed by distal cells. The alpha 5 beta 1-, alpha 6 beta 1-, alpha 6 beta 4-, and alpha V beta 3-integrins, as well as trace amounts of alpha 1 beta 1-integrins, were also present. The alpha 4 beta 1-integrin was not detected. Initial attachment to fibronectin was mediated by alpha V beta 3- and alpha 5 beta 1-integrins; initial attachment to laminin was mediated by the alpha 6 beta 1- and alpha 3 beta 1- integrins and, in some preparations, by an unidentified integrin; and initial attachment to collagen type IV was mediated by alpha V beta 3-integrin and an unidentified beta 1-integrin. After extensively immunodepleting membrane extracts with anti-alpha 1, -alpha 2, -alpha 3, -alpha 4, -alpha 5, -alpha 6, and -alpha V antibodies, an anti-beta 1 antibody still precipitated an integrin. Its electrophoretic mobility differs from the laminin-binding alpha 7 beta 1-integrin. Thus we have identified many of the integrins on cortical tubule cells and their role in mediating initial attachment to extracellular matrix. However, the cell adhesion assays and immunoprecipitations suggest the presence of an unidentified beta 1-integrin that may mediate renal tubule cell attachment to laminin and collagen.

scholarly journals The size of the intracellular β 1-integrin precursor pool regulates maturation of β 1-integrin subunit and associated α-subunits

1994 ◽  
Vol 300 (3) ◽  
pp. 771-779 ◽  
Author(s):  
L Koivisto ◽  
J Heino ◽  
L Häkkinen ◽  
H Larjava
Keyword(s):  
Cell Surface ◽  
Antisense Rna ◽  
Integrin Subunit ◽  
Integrin Receptors ◽  
Transfected Cells ◽  
Precursor Pool ◽  
Cell Clones ◽  
Beta 1 Integrin ◽  
Α Subunits

A large pool of precursor beta 1-integrin subunits is frequently found intracellularly. During malignant transformation this pool often disappears. Concomitantly, integrin-mediated cell-adhesion functions are disturbed, even though no change in the number of beta 1-integrin receptors on the cell surface can be observed. Here, we have studied the role of an intracellular pre-beta 1-integrin pool by transfecting human MG-63 osteosarcoma cells with plasmid construction producing an antisense RNA for the beta 1-integrin subunit. Stable cell clones expressing beta 1-integrin antisense RNA were shown to have a reduced intracellular pool of pre-beta 1-integrin subunits. In the antisense-transfected cells, the synthesis of the beta 1-integrin chain was reduced by 65% compared with non-transfected or vector-transfected MG-63 cells. The decreased synthesis of the beta 1-integrin chain was associated with accelerated maturation of the beta 1-integrin chain (half-maturation time about 5 h in antisense-transfected cells compared with about 10.5 h in control cells), whereas maturation of the alpha-integrin chain slowed down. The amount of beta 1-integrins on the cell surface, however, remained unaltered. Cell clones with the largest decrease in the relative amount of the pre-beta 1-integrin subunit also showed altered integrin function. They were found to synthesize fibronectin, but were unable to assemble it into a fibronectin matrix on the cell surface. Thus we conclude that the repression of biosynthesis of the beta 1-integrin chain leads to alterations in receptor maturation and may be connected with altered receptor function.

scholarly journals Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors.

1994 ◽  
Vol 125 (1) ◽  
pp. 205-214 ◽  
Author(s):  
P Rousselle ◽  
M Aumailley
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Culture Media ◽  
Human Keratinocytes ◽  
Cell Types ◽  
Heat Denaturation ◽  
Integrin Subunit ◽  
Cellular Interactions ◽  
Primary Human Keratinocytes ◽  
Established Cell

Kalinin was purified from squamous cell carcinoma (SCC25) spent culture media using an immunoaffinity column prepared from the mAb BM165. The affinity-purified material was separated by SDS-PAGE into three bands of 165-155, 140, and 105 kD identical to those obtained from normal human keratinocyte cultures and previously identified as kalinin. Kalinin promoted adhesion of a large number of normal cells and established cell lines with an activity similar to other adhesion molecules such as the laminin-nidogen complex, fibronectin, or collagen IV. However, kalinin was a much better substrate than laminin-nidogen complex for adhesion of cells of epithelial origin including primary human keratinocytes. Adhesion to kalinin was followed by cell shape changes ranging from rounded to fully spread cells depending on the cell types. The adhesion-promoting activity of kalinin was conformation dependent and was abolished by heat denaturation. mAb BM165 prevented cell adhesion to kalinin but not to other extracellular matrix substrates. However, either complete or partial inhibition was observed with different cells suggesting the existence of at least two cell-binding sites on the kalinin molecule. Experiments inhibiting cell adhesion with function-blocking anti-integrin subunit antibodies indicated that both alpha 3 beta 1 and alpha 6 beta 1 integrins are involved in the cellular interactions with kalinin, while for cell adhesion to classical mouse Engelbreth-Holm-Swarm laminin only alpha 6 beta 1 integrins, and not alpha 3 beta 1, appeared to be functional. Altogether, these results suggest that kalinin may fulfill additional functions than laminin, particularly for epithelial cells.

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