The alternatively spliced domains EIIIB and EIIIA of human fibronectin affect cell adhesion and spreading

Abstract

Fibronectin has a complex pattern of alternative splicing at the pre-mRNA level leading to the expression of different isoforms. The alternatively spliced domains EIIIB and EIIIA are known to be prominently expressed during development and wound healing. While the other spliced domain (CS-segment) is known to promote cell adhesion in a cell type specific manner, the biological functions of the spliced domains EIIIB and EIIIA are not well understood. In the present study, we have prepared expression proteins of specific domains of human fibronectin using a prokaryotic expression system and used the purified fragments to test their ability to support adhesion and spreading of cultured cells. Fragments from type-III domains #7 to #12 were prepared in various combinations to include or exclude the spliced domains EIIIB and EIIIA. The results indicate that cultured NIL fibroblasts adhere to many of the fragments tested. However, the cell adhesion and spreading are enhanced, especially at lower concentrations, to fragments including the domain EIIIB. The inclusion of domain EIIIA led to a decrease in the adhesion of cells and those that adhered did not spread well. When tested in a centrifugal cell adhesion assay, fragments including domain EIIIB resisted the detaching forces and stayed adhered. Fragments that included domain EIIIA were unable to resist the detaching centrifugal forces to the same extent as the fragments that included domain EIIIB alone. These results suggest that the spliced domain EIIIB may be serving important biological functions in enhancing cell adhesion and spreading. This is likely to be mediated by conformational effects because domain EIIIB alone neither exhibited any adhesive activity nor competed in inhibiting adhesion to fragments #7-10.

Purification of Fibronectin

“…Cellular fibronectin contains up to three inserted sequences not present in all chains of plasma fibronectin. The functional differences between plasma and cellular fibronectins are most striking in assays that measure morphological effects on cells, with the latter being more effective in certain adhesion and cell morphology assays (Yamada and Olden, 1978;Hashimoto-Uoshima et al, 1997;Manabe et al, 1997).…”

Purification of Fibronectin

“…Cellular fibronectin contains up to three inserted sequences not present in all chains of plasma fibronectin. The functional differences between plasma and cellular fibronectins are most striking in assays that measure morphological effects on cells, with the latter being more effective in certain adhesion and cell morphology assays (Yamada and Olden, , Hashimoto‐Uoshima et al, ; Manabe et al, ).…”