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Virtually all metazoan cells contain at least one and usually several types of transmembrane proteoglycans. These are varied in protein structure and type of polysaccharide, but the total number of vertebrate genes encoding transmembrane proteoglycan core proteins is less than 10. Some core proteins, including those of the syndecans, always possess covalently coupled glycosaminoglycans; others do not. Syndecan has a long evolutionary history, as it is present in invertebrates, but many other transmembrane proteoglycans are vertebrate inventions. The variety of proteins and their glycosaminoglycan chains is matched by diverse functions. However, all assume roles as coreceptors, often working alongside high-affinity growth factor receptors or adhesion receptors such as integrins. Other common themes are an ability to signal through their cytoplasmic domains, often to the actin cytoskeleton, and linkage to PDZ protein networks. Many transmembrane proteoglycans associate on the cell surface with metzincin proteases and can be shed by them. Work with model systems in vivo and in vitro reveals roles in growth, adhesion, migration, and metabolism. Furthermore, a wide range of phenotypes for the core proteins has been obtained in mouse knockout experiments. Here some of the latest developments in the field are examined in hopes of stimulating further interest in this fascinating group of molecules.

RhoGDI (Rho GDP-dissociation inhibitor) was identified as a down-regulator of Rho family GTPases typified by its ability to prevent nucleotide exchange and membrane association. Structural studies on GTPase-RhoGDI complexes, in combination with biochemical and cell biological results, have provided insight as to how RhoGDI exerts its effects on nucleotide binding, the membrane association-dissociation cycling of the GTPase and how these activities are controlled. Despite the initial negative roles attributed to RhoGDI, recent evidence has come to suggest that it may also act as a positive regulator necessary for the correct targeting and regulation of Rho activities by conferring cues for spatial restriction, guidance and availability to effectors. These potential functions are discussed in the context of RhoGDI-associated multimolecular complexes, the newly emerged shuttling capability and the importance of the particular membrane microenvironment that represents the site of action for GTPases. All these results point to a wider role for RhoGDI than initially perceived, making it a binding partner that can tightly control Rho GTPases, but which also allows them to reach their full spectrum of activities.

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.

The homologous mammalian rho kinases (ROCK I and II) are assumed to be functionally redundant, based largely on kinase construct overexpression. As downstream effectors of Rho GTPases, their major substrates are myosin light chain and myosin phosphatase. Both kinases are implicated in microfilament bundle assembly and smooth muscle contractility. Here, analysis of fibroblast adhesion to fibronectin revealed that although ROCK II was more abundant, its activity was always lower than ROCK I. Specific reduction of ROCK I by siRNA resulted in loss of stress fibers and focal adhesions, despite persistent ROCK II and guanine triphosphate–bound RhoA. In contrast, the microfilament cytoskeleton was enhanced by ROCK II down-regulation. Phagocytic uptake of fibronectin-coated beads was strongly down-regulated in ROCK II–depleted cells but not those lacking ROCK I. These effects originated in part from distinct lipid-binding preferences of ROCK pleckstrin homology domains. ROCK II bound phosphatidylinositol 3,4,5P3 and was sensitive to its levels, properties not shared by ROCK I. Therefore, endogenous ROCKs are distinctly regulated and in turn are involved with different myosin compartments.

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...

Proteolytic processes in the extracellular matrix are a major influence on cell adhesion, migration, survival, differentiation and proliferation. The syndecan cell‐surface proteoglycans are important mediators of cell spreading on extracellular matrix and respond to growth factors and other biologically active polypeptides. The ectodomain of each syndecan is constitutively shed from many cultured cells, but is accelerated in response to wound healing and diverse pathophysiological events. Ectodomain shedding is an important regulatory mechanism, because it rapidly changes surface receptor dynamics and generates soluble ectodomains that can function as paracrine or autocrine effectors, or competitive inhibitors. It is known that the family of syndecans can be shed by a variety of matrix proteinase, including many metzincins. Shedding is particularly active in proliferating and invasive cells, such as cancer cells, where cell‐surface components are continually released. Here, recent research into the shedding of syndecans and its physiological relevance are assessed.

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