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Adhesion to fibronectin's EDbdomain induces tyrosine phosphorylation of focal adhesion proteins in Balb/c 3T3 cells (1998)

Chen, Wensheng ; Culp, Lloyd A.

Springer

Clinical & experimental metastasis 16 (1998), S. 30-42

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ISSN: 1573-7276

Keywords: alternative splicing ; fibronectin ; focal adhesion ; focal adhesion kinase ; tyrosine phosphorylation

Source: Springer Online Journal Archives 1860-2000

Topics: Medicine

Notes: Abstract Balb/c 3T3 cell adhesion on substrata coated with fibronectin's (FN) alternatively-spliced EDb, implicated in some tumor cell systems, and its neighboring type III repeats (III7 and III8) induced intracellular signaling coincident with morphological responses. These events were analysed using minigene-expressed proteins containing various permutations of type III repeats of FN. Cells adherent to the tri-repeat protein 7-EDb-8 were compared to those attached to the tri-repeat 8-9-10 which can interact with integrins through RGD and its synergistic sequences. Cell adhesion to 7-EDb-8 generated rapid tyrosine phosphorylation of several intra-cellular proteins (particularly the complex at 120-130 kD), with the overall phosphorylation level and its sensitivity to tyrosine kinase inhibitors similar to responses on the 8-9-10 tri-repeat. This similarity contrasted with the differential morphological responses of cells mediated by these proteins. Further analysis of the kinetics of pho sphorylation through immunoblotting of two focal adhesion proteins, p125FAK and p130cas, revealed a profile for Balb/c 3T3 adhesion to 7-EDb-8 distinct from that on 8-9-10. EDb mono-repeat was also more potent for inducing both limited cell spreading and FAK tyrosine phosphorylation than its neigh-boring repeats III7 or III8. Examination of cellular localization of FAK and vinculin showed that cells spread on the 7-EDb-8 substratum displayed vinculin-positive focal complex-like structures at the cell periphery, in contrast to the classical focal adhesions seen in 8-9-10-adherent cells. These results suggest that EDb induces cell signaling events, leading to tyrosine phosphorylation of focal adhesion proteins, through a mechanism different from that mediated by integrins recognizing sequences in III8-9-10. EDb-dependent signaling may have special significance in some tumor systems. © Rapid Science 1998

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1006507917700

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Molecular composition and origin of substrate-attached material from normal and virus-transformed cells (1976)

Culp, Lloyd A.

New York, N.Y. : Wiley-Blackwell

Journal of Supramolecular Structure 5 (1976), S. 239-255

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ISSN: 0091-7419

Keywords: substrate ; adhesion ; footpad ; microfilaments ; protoglycans ; glycoprotein ; Life Sciences ; Molecular Cell Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: The proteins and polysaccharides which are left adherent to the tissue culture substrate after EGTA-mediated removal of normal, virus-transformed, and revertant mouse cells (so-called SAM, or substrate-attached material), and which have been implicated in the cell-substrate adhesion process, have been characterized by SDS-PAGE and other types of analyses under various conditions of cell growth and attachment. The following components have been identified in SAM: 3 size classes of hyaluronate proteoglycans; glycoprotein Co (the LETS glycoprotein); protein Ca (a myosin-like protein); protein Cb (MW 85,000); protein C1 (MW 56,000, which is apparently not tubulin); protein C2 (actin); proteins C3-C5 (histones) which are artifactually bound to the substrate as a result of EGTA-mediated leaching from the cell; and proteins Cc, Cd, Ce, and Cf. The LETS glycoprotein (Co) and Cd appear in newly-synthesized SAM (which is probably enriched in “footpad” material - “footpads” being focal areas of subsurface membranous contact with the substrate) in greater relative quantities than in the SAM accumulated over a long period of time (which is probably enriched in “footprint” material - remnants of footpads left behind as cells move across the substrate). Co and Cd turn over very rapidly following short radiolabeling periods during chase analysis. The SAM's deposited during a wide variety of cellular attachment and growth conditions contained the same components in similar relative proportions. This may indicate well-controlled and coordinate deposition of a cell “surface” complex involving the hyaluronate proteoglycans, the LETS glycoprotein, actin-containing microfilaments with associated proteins, and a limited number of additional proteins in the substrate adhesion site. Evidence indicates that SAM is the remnant of “footpad” vesicles by which the cell adheres to the substrate and that EGTA treatment weakens the subsurface cytoskeleton, allowing these footpad vesicles to be pinched off from the rest of the cell. Three different models of cell-substrate adhesion are presented and discussed.

Additional Material: 4 Ill.