Attachment and neurite extension have been measured when Platt or La-N1 human neuroblastoma cells respond to tissue culture substrata coated with a panel of complementary fragments from the individual chains of human plasma (pFN) or cellular fibronectins (cFN) purified from thermolysin digests. A 110-kD fragment (f110), which contains the Arg-Gly-Asp-Ser sequence (RGDS)-dependent cell-binding domain but no heparin-binding domains and whose sequences are shared in common by both the α- and β-subunits of pFN, facilitated attachment of cells that approached the level observed with either intact pFN or the heparan sulfate-binding platelet factor-4 (PF4). This attachment on f110 was resistant to RGDS-containing peptide in the medium. Neurite outgrowth was also maximal on f110, and half of these neurites were also resistant to soluble RGDS peptide. Treatment of cells with glycosaminoglycan lyases failed to alter these responses on f110. Therefore, there is a second 'cell-binding' domain in the sequences represented by f110 is not RGDS- or heparan sulfate-dependent and that facilitates stable attachment and some neurite outgrowth; this domain appears to be conformation-dependent. Comparisons were also made between two larger fragments generated from the two subunits of pFN-f145 from the α-subunit and f155 from the β-subunit-both of which contain the RGDS-dependent cell-binding domain and the COOH-terminal heparin-binding domain but which differ in the former's containing some IIICS sequence at its COOH terminus and the latter's having an additional type III homology unit. Heparin-binding fragments (with no RGDS activity) of f29 and f38, derived from f145 or f155 of pFN, respectively, and having the same differences in sequence, were also compared with f44+47 having the 'extra domain(a)' characteristic of cFN. Attachment on f145 was slightly sensitive to soluble RGDS peptide; attachment on f155 was much more sensitive. There were also differences in the percentage of cells with neurites on f145 vs. f155 but neurites on either fragment were completely sensitive to RGDS peptide. Mixing of f29, f38, or PF4 with f110 could not reconstitute the activities demonstrated in f145 or f155, demonstrating that covalently linked sequences are critical in modulating these responses. However, mixing of f44+47 from cFN with f110 from pFN increased the sensitivity to RGDS peptide. Attachment on f29 was poor, on f44+47 intermediate, and on f38 or PF4 very good, demonstrating the modulation of responses by the additional type III homology unit (in f38) and by alternately spliced sequences represented in the 'extra domain(a)' (in f44+47). The COOH-terminal heparin-binding domain appears to modulate RGDS-dependent cell-binding activity as well (by comparing f110 with either f145 or f155): (a) better attachment on f110; (b) resistance of attachment on f110 to RGDS peptide; (c) higher percentage of cells with neurites on f110 and their resistance to RGDS peptide; and (d) increased resistance of attachment on f155 when cells were treated with heparitanase. These data suggest that the responses of human neuroblastoma cells are mediated by two different cell-binding domains, as well as by the heparan sulfate-binding domain. Furthermore, the cell-binding responses can be modulated in four different ways: by neighboring sequences found in the IIICS region of pFN or cFN, by 'extradomain(a)', by the additional type III homology unit in the β-subunit of pFN, and by the COOH-terminal heparan sulfate-binding domain.
|Number of pages||13|
|Journal||Journal of Cell Biology|
|Publication status||Published - 1988|
ASJC Scopus subject areas
- Cell Biology