Laminin: the crux of basement membrane assembly

Laminin-1 is emerging as the key molecule in early embryonic basement membrane assembly. Here we review recent insights into its functions gained from the synergistic application of genetic and structural methods

Structural and functional analysis of two small leucine-rich repeat proteoglycans, fibromodulin and chondroadherin.

The small leucine-rich proteoglycans (SLRPs) are important regulators of extracellular matrix assembly and cell signalling. We have determined crystal structures at ~2.2Å resolution of human fibromodulin and chondroadherin, two collagen-binding SLRPs. Their overall fold is similar to that of the prototypical SLRP, decorin, but unlike decorin neither fibromodulin nor chondroadherin forms a stable dimer. A previously identified binding site for integrin α2β1 maps to an α-helix in the C-terminal cap region of chondroadherin. Interrogation of the Collagen Toolkits revealed a unique binding site for chondroadherin in collagen II, and no binding to collagen III. A triple-helical peptide containing the sequence GAOGPSGFQGLOGPOGPO (O is hydroxyproline) forms a stable complex with chondroadherin in solution. In fibrillar collagen I and II, this sequence is aligned with the collagen cross-linking site KGHR, suggesting a role for chondroadherin in cross-linking

A peptide from the first fibronectin domain of NCAM acts as an inverse agonist and stimulates FGF receptor activation, neurite outgrowth and survival

Abstract Neural cell adhesion molecule (NCAM) contributes to axon growth and guidance during development and learning and memory in adulthood. Although the Ig domains mediate homophilic binding, outgrowth activity localizes to two membrane proximal fibronectin-like domains. The first of these contains a site identified as a potential FGF receptor (FGFR) activation motif (FRM) important for NCAM stimulation of neurite outgrowth, but its activity has hitherto remained hypothetical. Here, we have tested the effects of a domainspecific antibody and peptides corresponding to the FRM in cellular assays in vitro. The first fibronectin domain antibody inhibited NCAM-stimulated outgrowth, indicating the importance of the domain for NCAM function. Monomeric FRM peptide behaved as an inverse agonist; low concentrations specifically inhibited neurite outgrowth stimulated by NCAM and cellular responses to FGF2, while saturating concentrations stimulated FGFR-dependent neurite outgrowth equivalent to NCAM itself. Dendrimeric FRM peptide was 125-fold more active and stimulated FGFR activation, FGFR-dependent and FGF-mimetic neurite outgrowth and cell survival (but not proliferation). We conclude that the FRM peptide contains NCAM-mimetic bioactivity accounted for by stimulation of FGF signalling pathways at the level of or upstream from FGF receptors, and discuss the possibility that FRM comprises part of an FGFR activation site on NCAM

220th ENMC workshop: Dystroglycan and the dystroglycanopathies Naarden, The Netherlands, 27–29 May 2016

Highlights • Review of clinical phenotypes associated with the dystroglycanopathies. • Discussion of current animal models and their contribution to understanding the disease process. • New insight into the glycosylation of alpha dystroglycan and the role of LARGE. • Structural information on the LARGE glycan

Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity.

Heparin/ heparan sulfate (HS) glycosaminoglycans are required for Slit-Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. Here we extend these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo

A propofol binding site on mammalian GABAA receptors identified by photolabeling

Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABA(A) receptors, but where it binds to this receptor is not known and has been a matter of some controversy. We have synthesized a novel propofol analogue photolabeling reagent that has a biological activity very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin which have been identified using X-ray crystallography. Using a combination of the protiated label and a deuterated version, and mammalian receptors labeled in intact membranes, we have identified a novel binding site for propofol in GABA(A) receptors consisting of both β(3) homopentamers and α(1)β(3) heteropentamers. The binding site is located within the β subunit, at the interface between the transmembrane domains and the extracellular domain, and lies close to known determinants of anesthetic sensitivity in transmembrane segments TM1 and TM2

A molecular mechanism for the heparan sulfate dependence of Slit-Robo signaling

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

Figure_4C.csv

Size exclusion chromatography of 1 mg/ml transmembrane protein 2 ectodomain (sTMEM2) either alone or pre-incubated for 30 minutes with 200 μg/ml high-molecular-weight hyaluronan (HMW-HA, >950 kDa, R&D Systems). The running buffer was 50 mM HEPES, 125 mM NaCl, 2 mM CaCl2, pH 7.5.</p