Vascular endothelial cell adhesion and spreading promoted by the peptide REDV of the IIICS region of plasma fibronectin is mediated by integrin α₄β₁

We have recently reported the attachment and spreading of human umbilical vein endothelial cells (HUVECs) upon substrates containing covalently grafted Arg-Glu-Asp-Val (REDV) peptide (Hubbell, J. A., Massia, S. P., Desai, N. P., and Drumheller, P. D. (1991) Bio/Technology 9, 568-572). This peptide has been reported to be the minimal active sequence within the CS5 site of the alternatively spliced type III connecting segment (IIICS) region of fibronectin, and the integrin α₄β₁ has been identified as the receptor on melanoma cells for this site. The integrin α₄β₁ has also been identified as the receptor for the CS1 site in the IIICS region on cells of neural crest origin, melanoma cells, lymphocytes, and hematopoietic stem cells. In this study, we demonstrate that this integrin also serves as a receptor on HUVECs for the peptide REDV from the CS5 site. The α₄ subunit was shown to be expressed upon HUVEC membranes by whole-cell enzyme-linked immunosorbent assay. Antifunctional antibodies directed against integrin subunits α₄ and β₁ inhibited cell adhesion on REDV-grafted substrates, but not on RGD-grafted substrates. The α₄ subunit localized into fibrillar structures within spread cells on the REDV-grafted substrates, but not within spread cells on RGD-grafted substrates. Two proteins (144 and 120 kDa) were isolated from HUVEC extracts by REDV ligand affinity chromatography and were demonstrated by immunoprecipitation and Western blot to be the integrin subunits α₄ (144 kDa) and β₁ (120 kDa); furthermore, the immunoprecipitation analyses demonstrated that the subunits formed a complex. HUVEC binding to REDV-grafted substrates was inhibited by both soluble REDV and RGD, demonstrating that adhesion was biospecific and that the REDV peptide is RGD-like. In this report we demonstrate for the first time that α₄ is present in the endothelial cell membrane, in contrast to previous reports by others, and that integrin α₄β₁ is the receptor for REDV-mediated adhesion to the IIICS region of region of plasma fibronectin.