Biomedical subjects

Erhard Hohenester

Publications and source records attributed to Erhard Hohenester.

17 recordsLinked to original sources

PubMed2006

Mammalian collagen receptors.

Collagen-rich extracellular matrices are abundant and ubiquitous in the mammalian body. Collagens are not only essential for the mechanical stability of tissues, but are also intimately involved in controlling cell behaviour. The hallmark of collagens is a triple helix made up of polypeptide chains containing glycine-X-Y repeats. A structurally and functionally diverse group of cell surface receptors mediates the recognition of triple-helical collagen: integrins, discoidin domain receptors, glycoprotein VI, leukocyte-associated IG-like receptor-1, and members of the mannose receptor family. In this review, we discuss the structure and function of these receptors, focussing on the principles involved in collagen recognition.

Animals↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

A transmembrane leucine zipper is required for activation of the dimeric receptor tyrosine kinase DDR1.

Receptor tyrosine kinases of the discoidin domain family, DDR1 and DDR2, are activated by different types of collagen and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. In a previous study, we found that collagen binding by the discoidin domain receptors (DDRs) requires dimerization of their extracellular domains (Leitinger, B. (2003) J. Biol. Chem. 278, 16761-16769), indicating that the paradigm of ligand-induced receptor dimerization may not apply to the DDRs. Using chemical cross-linking and co-immunoprecipitation of differently tagged DDRs, we now show that the DDRs form ligand-independent dimers in the biosynthetic pathway and on the cell surface. We further show that both the extracellular and the cytoplasmic domains are individually dispensable for DDR1 dimerization. The DDR1 transmembrane domain contains two putative dimerization motifs, a leucine zipper and a GXXXG motif. Mutations disrupting the leucine zipper strongly impaired collagen-induced transmembrane signaling, although the mutant DDR1 proteins were still able to dimerize, whereas mutation of the GXXXG motif had no effect. A bacterial reporter assay (named TOXCAT) showed that the DDR1 transmembrane domain has a strong potential for self-association in a biological membrane and that this interaction occurs via the leucine zipper and not the GXXXG motif. Our results demonstrate that the DDRs exist as stable dimers in the absence of ligand and that receptor activation requires specific interactions made by the transmembrane leucine zipper.

Amino Acid Motifs↗

PubMed2005

Structural basis for Gas6-Axl signalling.

Receptor tyrosine kinases of the Axl family are activated by the vitamin K-dependent protein Gas6. Axl signalling plays important roles in cancer, spermatogenesis, immunity, and platelet function. The crystal structure at 3.3 A resolution of a minimal human Gas6/Axl complex reveals an assembly of 2:2 stoichiometry, in which the two immunoglobulin-like domains of the Axl ectodomain are crosslinked by the first laminin G-like domain of Gas6, with no direct Axl/Axl or Gas6/Gas6 contacts. There are two distinct Gas6/Axl contacts of very different size, both featuring interactions between edge beta-strands. Structure-based mutagenesis, protein binding assays and receptor activation experiments demonstrate that both the major and minor Gas6 binding sites are required for productive transmembrane signalling. Gas6-mediated Axl dimerisation is likely to occur in two steps, with a high-affinity 1:1 Gas6/Axl complex forming first. Only the minor Gas6 binding site is highly conserved in the other Axl family receptors, Sky/Tyro3 and Mer. Specificity at the major contact is suggested to result from the segregation of charged and apolar residues to opposite faces of the newly formed beta-sheet.

Amino Acid Sequence↗

PubMed2005

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

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 domain-specific 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.

3T3 Cells↗

PubMed2005

A simplified laminin nomenclature.

A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered.

Animals↗

PubMed2004

Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit.

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit-Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.

Amino Acid Sequence↗

PubMed2004

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.

Animals↗

PubMed2004

Structure of a thrombospondin C-terminal fragment reveals a novel calcium core in the type 3 repeats.

Thrombospondins (TSPs) are extracellular regulators of cell-matrix interactions and cell phenotype. The most highly conserved region of all TSPs are the calcium-binding type 3 (T3) repeats and the C-terminal globular domain (CTD). The crystal structure of a cell-binding TSP-1 fragment, spanning three T3 repeats and the CTD, reveals a compact assembly. The T3 repeats lack secondary structure and are organised around a core of calcium ions; two DxDxDGxxDxxD motifs per repeat each encapsulate two calcium ions in a novel arrangement. The CTD forms a lectin-like beta-sandwich and contains four strictly conserved calcium-binding sites. Disruption of the hairpin structure of T3 repeats 6 and 7 decreases protein secretion and stability. The availability for cell attachment of an RGD motif in T3 repeat 7 is modulated by calcium loading. The central architectural role of calcium explains how it is critical for the functions of the TSP C-terminal region. Mutations in the T3 repeats of TSP-5/COMP, which cause two human skeletal disorders, are predicted to disrupt the tertiary structure of the T3-CTD assembly.

Amino Acid Sequence↗

PubMed2003

Distinct requirements for heparin and alpha-dystroglycan binding revealed by structure-based mutagenesis of the laminin alpha2 LG4-LG5 domain pair.

Laminin-2 (alpha2beta1gamma1) is found in basement membranes surrounding muscle and peripheral nerve cells. Several types of cellular receptors bind to the laminin G-like (LG) domains at the C terminus of the alpha2 chain, the interaction with alpha-dystroglycan (alpha-DG) being particularly important in muscle. We have used site-directed mutagenesis and in vitro binding assays to map the binding sites on the laminin alpha2 chain LG4-LG5 domain pair for alpha-DG, heparin and sulfatides. Calcium-dependent alpha-DG recognition requires the calcium ion in LG4, but not the one in LG5, as well as basic residues in both LG domains. Heparin and sulfatides also bind to basic residues in both LG domains, but there is little overlap in the binding sites for alpha-DG and heparin/sulfatides. The results should prove useful for the molecular dissection of laminin-receptor interactions in vivo.

Animals↗

PubMed2003

A model of FAS1 domain 4 of the corneal protein beta(ig)-h3 gives a clearer view on corneal dystrophies.

PURPOSE: A progressive alteration of the cornea resulting in loss of transparency occurs in a set of hereditary diseases known as corneal dystrophies. A number of these dystrophies have been linked to mutations in the 5q31-linked gene product beta(ig)-h3 (TGFBIP, kerato-epithelin, MP78/70, RGD-CAP) although the mechanism by which the mutations cause disease remains unknown. Here we investigate the structural basis for the different corneal dystrophies caused by mutations of the beta(ig)-h3 protein. The integrin binding properties of beta(ig)-h3, described in several recent studies, have been analysed with respect to the beta(ig)-h3 structure. METHODS: The recently determined structure of a FAS1 domain pair from fasciclin I, an insect cell adhesion molecule and beta(ig)-h3 homologue, was used to generate a homology model of the beta(ig)-h3 FAS1 domain 4. RESULTS: The structural analysis of FAS1 domain 4 of beta(ig)-h3 predicts that the common mutations at positions 124 and 555 do not substantially alter the beta(ig)-h3 structure. In contrast, the rare missense mutations appear incompatible with the FAS1 fold. A number of residues implicated in integrin binding by previous mutagenesis are mostly buried and appear to have important structural roles. CONCLUSIONS: The common mutations of beta(ig)-h3 at positions 124 and 555 are likely to affect protein-protein interactions directly, whereas the rare mutations are likely to cause misfolding of the protein within the cell. Previously identified integrin binding residues are unlikely to be directly involved in receptor binding.

Amino Acid Sequence↗

PubMed2003

Crystal structure of the collagen alpha1(VIII) NC1 trimer.

Collagen VIII is a major component of Descemet's membrane and is also found in vascular subendothelial matrices. The C-terminal non-collagenous domain (NC1) domain of collagen VIII, which is a member of the C1q-like protein family, forms a stable trimer and is thought to direct the assembly of the collagen triple helix, as well as polygonal supramolecular structures. We have solved the crystal structure of the mouse alpha1(VIII)(3) NC1 domain trimer at 1.9 A resolution. Each subunit of the intimate NC1 trimer consists of a ten-stranded beta-sandwich. The surface of the collagen VIII NC1 trimer presents three strips of partially exposed aromatic residues shown to interact with the non-ionic detergent CHAPS, which are likely to be involved in supramolecular assemblies. Equivalent strips exist in the NC1 domain of the closely related collagen X, suggesting a conserved assembly mechanism. Surprisingly, the collagen VIII NC1 trimer lacks the buried calcium cluster of the collagen X NC1 trimer. The mouse alpha1(VIII) and alpha2(VIII) NC1 domains are 71.5% identical in sequence, with the differences being concentrated on the NC1 trimer surface. A few non-conservative substitutions map to the subunit interfaces near the surface, but it is not obvious from the structure to what extent they determine the preferred assembly of collagen VIII alpha1 and alpha2 chains into homotrimers.

Amino Acid Sequence↗

PubMed2003

Novel fold revealed by the structure of a FAS1 domain pair from the insect cell adhesion molecule fasciclin I.

Fasciclin I is an insect neural cell adhesion molecule consisting of four FAS1 domains, homologs of which are present in many bacterial, plant, and animal proteins. The crystal structure of FAS1 domains 3 and 4 of Drosophila fasciclin I reveals a novel domain fold, consisting of a seven-stranded beta wedge and a number of alpha helices. The two domains are arranged in a linear fashion and interact through a substantial polar interface. Missense mutations in the FAS1 domains of the human protein betaig-h3 cause corneal dystrophies. Many mutations alter highly conserved core residues, but the two most common mutations, affecting Arg-124 and Arg-555, map to exposed alpha-helical regions, suggesting reduced protein solubility as the disease mechanism.

Amino Acid Sequence↗

PubMed2002

Crystal structure of a C-terminal fragment of growth arrest-specific protein Gas6. Receptor tyrosine kinase activation by laminin G-like domains.

Receptor tyrosine kinases of the Axl family are activated by Gas6, the product of growth arrest-specific gene 6. Gas6-Axl signaling is implicated in cell survival, adhesion, and migration. The receptor-binding site of Gas6 is located within a C-terminal pair of laminin G-like (LG) domains that do not resemble any other receptor tyrosine kinase ligand. We report the crystal structure at 2.2-A resolution of a Gas6 fragment spanning both LG domains (Gas6-LG). The structure reveals a V-shaped arrangement of LG domains strengthened by an interdomain calcium-binding site. LG2 of Gas6-LG contains two unusual features: an alpha-helix cradled by one edge of the LG beta-sandwich and a conspicuous patch of surface-exposed hydrophobic residues. Mutagenesis of some residues in this patch reduces Gas6-LG binding to the extracellular domain of Axl as well as Axl activation in glioblastoma cells, identifying a component of the receptor-binding site of Gas6.

Amino Acid Sequence↗

PubMed2002

Domain structure and organisation in extracellular matrix proteins.

Extracellular matrix (ECM) proteins are large modular molecules built up from a limited set of modules, or domains. The basic folds of many domains have now been determined by crystallography or NMR spectroscopy. Recent structures of domain pairs and larger tandem arrays, as well as of oligomerisation domains, have begun to reveal the principles underlying the higher order architecture of ECM proteins. Structural information, coupled with site-directed mutagenesis, has been instrumental in showing how adjacent domains can co-operate in ligand binding. Very recently, the first heterotypic ECM protein complexes have become available. Here, we review the advances of the last 5 years in understanding ECM protein structure, with special emphasis on those structures that have given insight into the biological functions of ECM proteins.

Activins↗

PubMed2002

Insight into Schmid metaphyseal chondrodysplasia from the crystal structure of the collagen X NC1 domain trimer.

Collagen X is expressed specifically in the growth plate of long bones. Its C1q-like C-terminal NC1 domain forms a stable homotrimer and is crucial for collagen X assembly. Mutations in the NC1 domain cause Schmid metaphyseal chondrodysplasia (SMCD). The crystal structure at 2.0 A resolution of the human collagen X NC1 domain reveals an intimate trimeric assembly strengthened by a buried cluster of calcium ions. Three strips of exposed aromatic residues on the surface of NC1 trimer are likely to be involved in the supramolecular assembly of collagen X. Most internal SMCD mutations probably prevent protein folding, whereas mutations of surface residues may affect the collagen X suprastructure in a dominant-negative manner.

Amino Acid Sequence↗

PubMed2002

Structure and function of collagen-derived endostatin inhibitors of angiogenesis.

Endostatins are inhibitors of endothelial cell migration and angiogenesis and have been shown to reduce tumor growth in animal models. They are derived from the nontriplehelical C-terminal NC1 domains of collagens XV and XVIII, which are released proteolytically in trimeric form and further converted to monomeric endostatins of about 20 kDa. Both endostatin isoforms share a compact globular fold, but differ in certain binding properties for proteins and cells, as well as in tissue distribution. Differences in activity were found between NC1 domains and endostatins and are related to the oligomerization state. Endostatin effects are not restricted to endothelial cells, but also control renal epithelial cells and neuronal guidance in C. elegans. Cellular receptors are still insufficiently characterized and include for endostatin-XVIII heparan sulfate proteoglycans. Receptor engagement elicits various downstream effects including tyrosine kinase and gene activation. Much remains to be learned, however, about details of the signal transduction cascades and how they interfere with pro-angiogenic factors under physiological conditions and during therapeutic treatment.

Animals↗