The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading.

Abstract

Abstract. Cultured fibroblasts form focal contacts (FCs) associated with actin microfilament bundles (MFBs) during attachment and spreading on serum-or fibronectin (FN)-coated substrates. To determine if the minimum cellular adhesion receptor recognition signal Arg-Gly-Asp-Ser (RGDS) is sufficient to promote FC and MFB formation, rat (NRK), hamster (Nil 8), and mouse (Balb/c 3T3) fibroblasts in serum-free media were plated on substrates derivatized with small synthetic peptides containing RGDS. These cultures were studied with interference reflection microscopy to detect FCs, Normarski optics to identify MFBs, and immunofluorescence microscopy to observe endogenous FN fiber formation. By 1 h, 72-78 % of the NRK and Nil 8 cells plated on RGDS-containing peptide had focal contacts without accompanying FN fibers, while these fibroblasts lacked FCs on control peptide. This early FC formation was followed by the appearance of coincident MFBs and colinear FN fbers forming fibronexuses at 4 h. NRK and Nil 8 cultures on substrates coated with native FN or 75,000-D FN-cell binding fragment showed similar kinetics of FC and MFB formation. In contrast, the Balb/c 3T3 mouse fibroblasts plated on Gly-Arg-Gly-Asp-Ser peptidederivatized substrates, or on coverslips coated with 75,000-D FN cell-binding fragment, were defective in FC formation. These results demonstrate that the apparent binding of substrate-linked RGDS sequences to cell surface adhesion receptors is sufficient to promote early focal contact formation followed by the appearance of fibronexuses in some, but not all, fibroblast lines. F IBRONECTIN (FN) ~ is a large glycoprotein found in plasma and the connective tissue extracellular matrix, which functions primarily in promoting cellular adhesion to solid substrates. It is composed of several protease-resistant functional domains that can bind independently to a number of important substrates, including fibrin, collagen, heparin, and various cell surface components (18,35). The FN cell-attachment domain has recently been shown to bind to a 140-kD FN receptor complex (1, 2, 33) that codistributes with microfilament bundles at cell-substrate adhesion sites (4, 5). Through the use of small synthetic peptides deduced from the primary structure of the FN cellattachment domain, the tetrapeptide Arg-Gly-Asp-Ser has been found to be the minimal FN sequence with cell attachment activity (29-31).Arg-Gly-Asp-Val, a sequence closely related to Arg-GlyAsp-Ser, is present in vitronectin, another protein with potent cell-adhesive properties that is localized with FN in the extracellular matrix (15,16,44). The vitronectin receptor has a different molecular mass than the FN receptor, and specifi-1. Abbreviations used in this paper: DIC, differential interference contrast; FC, focal contact(s); FN, fibronectin; HBSA, heat denatured BSA; IFM, immunofluorescence microscopy; IRM, interference reflection microscopy; SPDP, 3-(2-pyridol-dithio) propionic acid N-hydroxy-succinimide ester.cally binds vitronectin rather than FN. However, bo...

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

“…We tested this possibility by the addition of soluble ligands for either the a5ß, fibronectin receptor or the vitronectin receptor to cells spread on laminin and found that ligand occupancy targeted diffuse receptors to heterologous focal contacts. This redistribution of receptors occurred even with small peptide ligands, ensuring that the striking redistribution observed was not due to receptors binding to ligands absorbing to the substrate, since the use of GRGDS as a substrate requires covalent linkage to a carrier protein (Singer et al ., 1987) . Furthermore, redistribution of receptors occurred both in cells spread for 90 min with newly formed focal contacts and in cells spread for 15 h with more stable and mature focal contacts.…”

Novel UNC‐44 AO13 ankyrin is required for axonal guidance in C. elegans, contains six highly repetitive STEP blocks separated by seven potential transmembrane domains, and is localized to neuronal processes and the periphery of neural cell bodies

“…ALOM also predicts membrane interaction sites at aa 103-119 and 429 -445. AO13 ankyrin contains an RGD sequence that would have an extracellular location in this model, and which acts as a cellbinding site in other proteins (Singer et al, 1987). Alternatively, AO13 ankyrin could interact with membranous organelles within the cell [ Fig.…”

Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts.

“…Under appropriate conditions, many kinds of cells-fibroblast types in particular-show black focal contacts under IRM (Burridge, et al, 1988;Singer et al, 1987Singer et al, , 1988, which represent areas of very close interaction between the cell and the substrate to which it is attached (Izzard and Lochner, 1976). Of the few types of fibroblasts described to translocate in vitro, the best studied example is the fibroblast explanted from the embryonic chick heart (Abercrombie, 1980;Trinkaus, 1984).…”