Fibronectin has been shown previously to promote complete cell adhesion in the absence of other serum components or de novo protein synthesis. Recently a sequence of four amino acids from the cell‐binding domain of fibronectin has been termed the ‘cell recognition site’ of this multidomain molecule since it mediates cell attachment and inhibits cell adhesion to intact fibronectin. We show here, however, that substrata coated with an isolated cell‐binding domain of fibronectin are not sufficient for complete cell adhesion; cells attach and spread but, unlike those adhering to intact fibronectin, they do not form stress fibres terminating in focal adhesions. An additional external stimulus is needed for this cytoskeletal reorganisation and may be provided by one of two heparin‐binding fragments of fibronectin. The two ‘signals’ required for complete adhesion need not be provided simultaneously since focal adhesion formation can be promoted by stimulating cells pre‐spread on a cell‐binding fragment of fibronectin with a soluble heparin‐binding fragment. This second stimulation may involve cell membrane heparan sulphate proteoglycans.

During cell-matrix adhesion, both tyrosine and serine/ threonine kinases are activated. Integrin ligation correlates with tyrosine phosphorylation, whereas the later stages of spreading and focal adhesion and stress fiber formation in primary fibroblasts requires interactions of cell surface proteoglycan with heparin-binding moieties. This correlates with protein kinase C (PKC) activation, and PKC␣ can become localized to focal adhesions in normal, but not transformed, cells. PKC activation has been thought to be downstream of initial receptor-ligand interactions. We now show, however, that syndecan-4 transmembrane heparan sulfate proteoglycan and PKC co-immunoprecipitate and co-patch in vivo. The core protein of syndecan-4 can directly bind the catalytic domain of PKC␣ and potentiate its activation by phospholipid mediators. It can also directly activate PKC␣ in the absence of other mediators. This activity resides in the sequence LGKKPIYKK in the center of the short cytoplasmic domain, and other syndecans lack this sequence and PKC regulatory properties. Syndecan-4 is a focal adhesion component, and this interaction may both localize PKC and amplify its activity at sites of forming adhesions. This represents the first report of direct transmembrane signaling through cell surface proteoglycans.In cell-matrix interactions, as with many cellular responses to ligands, both tyrosine and serine/threonine kinases participate. During adhesion to fibronectin, integrin clustering activates tyrosine kinases (1-3), but we, and others, have noted that full spreading (4) and the formation of stress fibers and focal adhesions requires additional signals (3,(5)(6)(7)(8)(9)(10)(11)(12). Adhesion to the "cell-binding" 105-kDa fragment of fibronectin through ligation of integrin ␣ 5 ␤ 1 is sufficient only for attachment and spreading in anchorage-dependent primary fibroblasts in the absence of serum and protein synthesis (8 -11). To drive the cytoskeletal and receptor clustering that accompany the formation of stress fibers and focal adhesions, an additional activation of protein kinase C (PKC) 1 is needed (2,8,9). This can be provided by addition of heparin-binding fibronectin moieties (8 -11), either of the whole heparin-binding domain of fibronectin or of a peptide PRARI from this domain. These agents appear to signal through a cell surface heparan sulfate proteoglycan, since treatment with heparinase prevents the response (11), and mutant cells lacking (13), or having undersulfated cell surface heparan sulfate proteoglycans (14), have reduced capacity to form focal adhesions or stress fibers. Similarly, addition of PRARI peptides to synovial fibroblasts prespread on substrates of 105-kDa fragment markedly increased the size of vinculin-positive adhesion plaques (12). The need for addition of heparin-binding agents can be circumvented by treatment of cells with active, but not inactive, phorbol esters (8). In addition, there is downstream activation of the RhoA G-protein (15), which may initiate a contractile response (3).Syn...

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Focal adhesion formation in fibroblasts results from complex transmembrane signaling processes initiated by extracellular matrix molecules. Although a role for integrins with attendant tyrosine kinases has been established, there is evidence that cell surface heparan sulfate proteoglycans (HSPGs) are also involved with an associated role of protein kinase C. The identity of the proteoglycan has remained elusive, but we now report that syndecan 4 (ryudocan/amphiglycan) is present in focal adhesions of a number of cell types. Affinitypurified antibodies raised against a unique portion of the cytoplasmic domain of syndecan 4 core protein recognized an HSPG of similar characteristics to those of syndecan 4. These antibodies stained focal adhesions only after cell permeabilization and recognized differing mammalian species. Syndecan 4 was associated with focal adhesions that contained either i1 or f3 integrin subunits and those that formed on substrates of fibronectin, laminin, vitronectin, or type I collagen. No focal adhesions were found that were vinculin-containing but lacked syndecan 4. In contrast, syndecan 2, whose cytoplasmic domain is closely homologous to syndecan 4, does not appear to be a focal adhesion component. Thus, syndecan 4 represents a new transmembrane focal adhesion component, probably involved in their assembly.

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(Ruoslahti, 1988;Hynes, 1990) of fibronectin interacts with RGD-dependent integrins (e.g.,

The transmembrane proteoglycan syndecan-4, which is a coreceptor with integrins in cytoskeleton-matrix interactions, appears to be multimerized in vivo. Both purified and recombinant core proteins form sodium dodecyl sulfate-resistant oligomers, and we now report that a synthetic peptide corresponding to the central region of syndecan-4 cytoplasmic domain (4V) also oligomerizes. The degree of oligomerization correlates with the previously reported ability to bind protein kinase C (PKC) and regulate its activity. Only multimeric recombinant syndecan-4 core protein, but not the monomeric protein, potentiated the activity of PKC␣, and only oligomeric syndecan-4 cytoplasmic peptides were active. Changes in peptide sequence caused parallel loss of stable oligomeric status and ability to regulate a mixture of PKC␣␤␥ activity. A synthetic peptide encompassing the whole cytoplasmic domain of syndecan-4 (4L) containing a membrane-proximal basic sequence did not form higher order oligomers and could not regulate the activity of PKC␣␤␥ unless induced to aggregate by phosphatidylinositol 4,5-bisphosphate. Oligomerization and PKC regulatory activity of the 4V peptide were both increased by addition of N-terminal cysteine and reduced by phosphorylation of the cysteine thiol group. Concentration of syndecan-4 at sites of focal adhesion formation may enhance multimerization and both localize PKC and potentiate its activity to induce stable complex formation.Extracellular matrix molecules such as fibronectin regulate many cellular processes through information encoded in the ligand-receptor interaction. Fibronectin has at least two distinct classes of cell-surface receptors: integrins and heparan sulfate proteoglycans. Integrins bind at several sites, but adhesion for many cell types is primarily through the classical RGD sequence in the tenth type III repeat of the molecule (1, 2). Clustering of specific integrins by either immobilized extracellular molecules or anti-integrin antibodies has many biological effects (reviewed in Refs. 3-6). It stimulates tyrosine phosphorylation (7, 8), elevates intracellular calcium (9), activates the Na ϩ /H ϩ antiporter (10), and activates phosphatidylinositol 4-phosphate 5-kinase (11) with cytoskeletal rearrangement (3, 12). Ligand-induced dimerization or oligomerization is a key event in transmembrane signaling by hormone or growth factor receptors with tyrosine kinase activity. This leads to an increase in tyrosine kinase activity, autophosphorylation of receptors, and the induction of diverse biological responses (13,14). Although integrins have no intrinsic tyrosine kinase activity, it is clear that their clustering is needed prior to subsequent tyrosine phosphorylation events (reviewed in Refs. 3-6). In addition, integrin-associated kinase(s) has been identified (15,16).Interactions between integrins and the cell-binding domain of fibronectin are only sufficient for attachment and spreading in normal primary fibroblasts (17), and the integrin ␣5␤1, which is the primary ligand for adhesio...

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