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Adhesion activity in fibronectin's alternatively spliced domain EDa (EIIIA) and its neighboring type III repeats:
1Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106.
Abstract:
EDa (EIIIA) is one of two alternatively spliced type III repeats in cellular fibronectin (cFN) lacking in plasma fibronectin (pFN). Previous studies using proteolytic fragments of cFN suggested that EDa may harbor adhesion activity for various Balb/c 3T3 cell derivatives. This putative adhesion activity has now been analyzed more directly. EDa and neighboring type III repeats III11 and III12 from human cFN cDNA were subcloned in various permutations and recombinant minigenes expressed in Escherichia coli. Purified recombinant polypeptide corresponding to EDa type III repeat alone is capable of promoting 3T3 cell attachment and limited cytoplasmic spreading, as are neighboring repeats III11 and III12 when tested as single repeats. While EDa alone exhibited 40-60% of the attachment activity of human pFN depending upon cell type, EDa with both neighboring repeats displayed 70-90% of pFN activity; furthermore, cell spreading was more extensive with the three-repeat molecule. Two experimental approaches indicated that cell surface proteoglycans do not participate in these adhesion processes. Finally, the effects of various oncogenes upon transformation of Balb/c 3T3 cells were investigated. Adhesion activity to all three repeats is completely abrogated by two different ras oncogenes, unaffected by the sis oncogene, and elevated by the src oncogene. Furthermore, ras- revertants of ras-transformed cells had reacquired adhesion activity for EDa and its neighboring repeats. Comparison of individual repeats confirmed oncogene-dependent regulation of receptor activity to these sequences--for 3T3 cells, EDa > III11 = III12, but for src-transformed cells III12 >> EDa > III11. These studies reveal a new adhesion-promoting activity in alternatively spliced EDa and in neighboring repeats III11 and III12; this receptor activity is regulated either positively or negatively subsequent to transformation by specific oncogenes.