β1 integrin-dependent and -independent polymerization of fibronectin

Abstract

The mouse cell line GD25, which lacks expression of the β1 family of integrin heterodimers due to disruption of the β1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse β1 integrin subunit. In a stably transformed clone (GD25-β1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits α3, α5, and α6. Both GD25 and GD25-β1A attached to fibronectin and formed focal contacts which contained αvβ3, but no detectable α5β1A. The presence of GRGDS peptide allowed α5β1A to locate to focal contacts of GD25-β1A cultured on fibronectin, while the β1- null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that α5β1A and αvβ3 could bind to a large cell- binding fragment of fibronectin. α5β1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little αvβ3 was colocalized with the fibronectin fibrils in GD25 β1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the αvβ3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both α5β1A and αvβ3 are able to support adhesion to fibronectin, αvβ3 dominates in the formation of focal contacts, and α5β1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of β1 integrins.