Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation.

Abstract

We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin- coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate- bound vitronectin.

A Novel Mode for Integrin-mediated Signaling: Tethering Is Required for Phosphorylation of FAK Y397

“…The plating of fibroblasts on a fibronectin-coated surface induces not only the binding of ␣5␤1 to fibronectin, but also the progressive organization of ␣5␤1 into focal contacts and focal adhesions during the process of cell spreading (Singer et al, 1988;Pankov et al, 2000). Because the experiments described here do involve the plating of cells on a fibronectin substrate, a reorganization of integrin into focal contacts and focal adhesion did occur.…”

Regulation of fibronectin receptor distribution [published erratum appears in J Cell Biol 1992 Jul;118(2):491]

“…When fibroblasts are plated in serum for 24 h or more on uncoated coverslips, their a5ß, fibronectin receptors become concentrated at sites where extracellular fibronectin fibrils are associated with the plasma membrane, and their vitronectin receptors become concentrated at focal contacts (Singer et al ., 1988) . To determine whether either the a5 or 01 intracellular domain alone was sufficient to target the chimeric receptors to sites of cell contact with fibronectin fibrils, human fibroblasts transiently transfected with the chimeric receptors were plated on coverslips in serumcontaining medium for 36 h .…”

“…The Journal of Cell Biology, Volume 117, 1992 (Singer et al ., 1988). To establish whether either the a2 or 01 intracellular domain alone can determine where the chimeric receptors localize, normal human fibroblasts transiently expressing these chimeras were plated for 2 h on fibronectin-coated coverslips, and the expression of the transfected and endogenous receptors in focal contacts was analyzed by inununofluorescence and interference reflection microscopy.…”

“…To reduce the background of a5ß, fibronecdn receptors in focal contacts in cells spread for short periods of time on laminin (see also Singer et al ., 1988), we preincubated the cells with mAb 333, which binds near the RGD site on fibronectin and inhibits the interaction of fibronectin with the fibronectin receptor (data not shown) . The cells were then washed several times, fresh serum-free medium containing fragment or peptide was added as indicated in the figure legends, and the cells were incubated at 37°C for an additional 60 min .…”

“…For example, the fibronectin receptor, «5ß,, concentrates in focal contacts in cells spread on a substrate of fibronectin ; the vitronectin receptor, aß3, is in focal contacts in cells spread on vitronectin ; and on a collagen substrate, the collagen receptor, a2ß,, is in focal contacts (Singer et al, 1988;Dejana et al ., 1988;Fath et al ., 1989;Carter et al, 1990). However, it is puzzling that the vitronectin receptor, «vß5 , does not localize to focal contacts (Wayner et al, 1991), even though it contains sequence motifs previously demonstrated to be important in receptor localization to these adhesion sites (McLean et al ., 1990;Ramaswamy and Hemler, 1990;Marcantonio et al, 1990) .…”