The alpha 5 beta 1 integrin fibronectin receptor, but not the alpha 5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly.

C Wu,J.S Bauer,R.L Juliano,J.A Mcdonald

doi:10.1016/s0021-9258(20)80623-1

Abstract

The alpha 5 beta 1 integrin mediates cell adhesion and migration on fibronectin, a glycoprotein critical for normal vertebrate embryonic development. Indirect evidence reported to date suggests that this receptor also functions in the deposition of fibronectin matrices. We used a molecular genetic approach to critically evaluate this role of alpha 5 beta 1 integrins. Mutant Chinese hamster ovary (CHO) cells deficient in alpha 5 integrin expression could not assemble a fibronectin matrix. Reconstituting alpha 5 beta 1 integrin expression by transfecting them with a full-length cDNA encoding the human alpha 5 chain completely restored fibronectin matrix assembly. CHO cells expressing an alpha 5 chain lacking the cytoplasmic domain also assembled a fibronectin matrix. Removing the cytoplasmic domain of alpha 5 appears to increase its activity in fibronectin matrix assembly. In addition to alpha 5 beta 1 integrin binding to fibronectin's RGD-containing domain, cells must bind with high affinity to fibronectin's amino-terminal 29-kDa matrix assembly domain to form a fibronectin matrix. Studies with the alpha 5-deficient CHO cells show that the expression of alpha 5 beta 1 integrin is also necessary for cells to bind fragments containing this distinct site in fibronectin and that a fibronectin matrix increases binding of the 29-kDa fragment. Thus, alpha 5 beta 1 integrins not only mediate cell adhesion to fibronectin, but also play an essential role in the assembly of a fibronectin matrix. This role includes direct binding to fibronectin and modulating a distinct binding event involving the interaction of fibronectin's amino-terminal matrix assembly domain with the cell surface.

Highlights

Pressing an a5 chain lacking the cytoplasmic domain A great deal isknown about the structureof FNs
The first site loicsated within the firfsitve type I repeats matrix assembly domainwith the cell surface
During diverse biological processes including embryonic development and cytodifferentiation, wound healing, and tumor metastasis, cells must interact with components of the extracellular matrix (ECM).’

Summary

EXPERIMENTAL PROCEDURES

(washing buffer), and twice with 0.2% SDS,1%Triton X-100 in 50 m~ Tris-HCI, mM EDTA, 180mM NaCl, pH 7.4, at room temperature. 1 5 2 0 %of the level of a5 expressed by the parent cell line (Table I) and and 10%heat-inactivated fetal bovine serum depleted of FN orcontainwas used as a control for celelxpressing similar levels of the a5 subunit ing 50 pg/ml bovine plasma FN as specified, in 96-well tissue culture with a cytoplasmic domaintruncation (see below).The CHO B2B4 cell plates a t densities which yieldedconfluentmonolayers after 2 days. The line expresses a full length human a5 integrin chain This line was binding of the amino-terminal 29-kDa fragment to the confluent cell obtained by transfecting with a humana expressionvector (pECE-aB), monolayers was measured as previously described(9) using a 1-h incuselection, and screening by ELISA and flow cytometry (25). At the end of the incubation, cells in anti-rabbit IgG antibodiesas previously described(8,lO).The cellswere each well were rinsed five times with a-MEM medium supplemented with 10 mM HEPES and lysed with 0.3 ml of 1%Triton X-100, 180 nM. The amount of human plasma FN 110-kDa fragment in the cell lysate (bound) was determined using the human FN-specific captureELISA as described above

RESULTS

Cultured withFN

Findings

DISCUSSION