Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.Slits are large secreted leucine-rich repeat (LRR) 6 proteins with multiple roles in cell signaling and adhesion. They have well established and evolutionarily conserved functions as guidance cues in the developing nervous system (1, 2), but Slits are also important in the development of the vasculature (3) and other organs (4). The first class of Slit receptors to be identified were Robo family members, which are transmembrane proteins with an extracellular domain resembling cell adhesion molecules and a large cytosolic signaling domain (1, 2). Biochemical studies have defined the domains mediating the SlitRobo interaction (5, 6), as well as some of the components of the signaling cascade downstream of Robo activation (7, 8), but how binding of Slit to Robo receptors conveys a signal across the cell membrane remains unknown.The first indication that there might exist a second Slit receptor came from the observation that heparan sulfate (HS) was required for the repellent activity of Slit in vitro (9) and in vivo (10). The identity of this receptor was revealed by recent genetic studies in invertebrates, which showed that Slit signaling requires Robo to be co-expressed on the same cell with the HS proteoglycan syndecan (11-13). Syndecan is a membranespanning protein to which are covalently attached several HS chains, consisting of repeating sulfated disaccharide units (14). Heparin is a member of the HS family that is more highly and uniformly sulfated than other HS. Johnson et al. (12) showed that both Slit and Robo can be co-immunoprecipitated with syndecan, suggesting the presence of a ternary (or higher order) complex at the neuronal cell membrane. However, the composition and functional relevance of this putative ternary complex was not established. Because the Slit distribution was found to be altered in syndecan-deficient embryos, HS may also be required for...

Determinants of laminin polymerization revealed by the structure of the α5 chain amino-terminal regionLaminin polymers are a key component of all basement membranes. The first crystal structure of a laminin amino-terminal domain identifies a conserved surface loop in laminin alpha chains that is involved in polymer formation.

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The heterotrimeric laminins are a defining component of basement membranes and essential for tissue formation and function in all animals. The three short arms of the cross-shaped laminin molecule are composed of one chain each and their tips mediate the formation of a polymeric network. The structural basis for laminin polymerisation is unknown. We have determined crystal structures of the short-arm tips of the mouse laminin β1 and γ1 chains, which are grossly similar to the previously determined structure of the corresponding α5 chain region. The short-arm tips consist of a laminin N-terminal (LN) domain that is attached like the head of a flower to a rod-like stem formed by tandem laminin-type epidermal growth factor-like (LE) domains. The LN domain is a β-sandwich with elaborate loop regions that differ between chains. The γ1 LN domain uniquely contains a calcium binding site. The LE domains have little regular structure and are stabilised by cysteines that are disulphide-linked 1–3, 2–4, 5–6 and 7–8 in all chains. The LN surface is not conserved across the α, β and γ chains, but within each chain subfamily there is a striking concentration of conserved residues on one face of the β-sandwich, while the opposite face invariably is shielded by glycans. We propose that the extensive conserved patches on the β and γ LN domains mediate the binding of these two chains to each other, and that the α chain LN domain subsequently binds to the composite β-γ surface. Mutations in the laminin β2 LN domain causing Pierson syndrome are likely to impair the folding of the β2 chain or its ability to form network interactions.

SummaryDystroglycan is a ubiquitously expressed cell adhesion protein. Its principal role has been determined as a component of the dystrophinglycoprotein complex of muscle, where it constitutes a key component of the costameric cell adhesion system. To investigate more fundamental aspects of dystroglycan function in cell adhesion, we examined the role of dystroglycan in the dynamics and assembly of cellular adhesions in myoblasts. We show that -dystroglycan is recruited to adhesion structures and, based on staining for vinculin, that overexpression or depletion of dystroglycan affects both size and number of fibrillar adhesions. Knockdown of dystroglycan increases the size and number of adhesions, whereas overexpression decreases the number of adhesions. Dystroglycan knockdown or overexpression affects the ability of cells to adhere to different substrates, and has effects on cell migration that are consistent with effects on the formation of fibrillar adhesions. Using an SH3 domain proteomic screen, we identified vinexin as a binding partner for dystroglycan. Furthermore, we show that dystroglycan can interact indirectly with vinculin by binding to the vinculin-binding protein vinexin, and that this interaction has a role in dystroglycan-mediated cell adhesion and spreading. For the first time, we also demonstrate unequivocally that -dystroglycan is a resident of focal adhesions.