N-syndecan (syndecan-3) was previously isolated as a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and suggested to mediate the neurite growth-promoting signal from cell matrixbound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. In the present study, we have transfected N18 neuroblastoma cells with a rat N-syndecan cDNA and show that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinaseactive fractions that bind to the cytosolic moiety of Nsyndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. We suggest that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway. HB-GAM1 was initially isolated from neonatal rat brain as a neurite outgrowth-promoting protein, the expression of which in brain corresponds to the stage of rapid axonal growth (1). Molecular cloning of full-length cDNA identified a novel secretory sequence (2). The same cDNA sequence was reported for pleiotrophin, a protein suggested to be mitogen for fibroblastic cells (3, 4). The HB-GAM/pleiotrophin sequence shares approximately 50% homology with the midkine protein involved in retinoic acid-induced cell differentiation (5-7).The expression of HB-GAM in the axon pathways of the brain and in the basement membranes outside of brain (8 -9) and the neurite outgrowth-promoting property of HB-GAM in vitro (see Refs. 1 and 4) have suggested interaction of matrixassociated HB-GAM with a cell surface receptor. Furthermore, HB-GAM is expressed at the surface of developing muscle cells and is suggested to play a role in the development of nerve/ muscle contacts (10 -12). N-syndecan (syndecan-3) has recently been isolated from detergent extracts of perinatal rat brain as a receptor or coreceptor for HB-GAM using recombinant HB-GAM as an affinity matrix (13). N-syndecan is localized at the surface of neurites and their growth cones in rat primary neurons growing on HB-GAM-coated matrix in vitro (13). Furthermore, HB-GAM and N-syndecan are spatiotemporally co-expressed in developing rat brain (14).The cell surface N-syndecan interacts with HB-GAM through its heparan sulfate chains (15). This interaction is enhanced by assembly of the heparan sulfat...

Heparin-binding growth-associated molecule (HB-GAM) is an extracellular matrix-associated protein implicated in the development and plasticity of neuronal connections of brain. Binding to cell surface heparan sulfate is indispensable for the biological activity of HB-GAM. In the present paper we have studied the structure of recombinant HB-GAM using heteronuclear NMR. These studies show that HB-GAM contains two ␤-sheet domains connected by a flexible linker. Both of these domains contain three antiparallel ␤-strands. In addition to this domain structure, HB-GAM contains the Nand C-terminal lysine-rich sequences that lack a detectable structure and appear to form random coils. Studies using CD and NMR spectroscopy suggest that HB-GAM undergoes a conformational change upon binding to heparin, and that the binding occurs primarily to the ␤-sheet domains of the protein. Search of sequence data bases shows that the ␤-sheet domains of HB-GAM are homologous to the thrombospondin type I repeat (TSR). Sequence comparisions show that the ␤-sheet structures found previously in midkine, a protein homologous with HB-GAM, also correspond to the TSR motif. We suggest that the TSR sequence motif found in various extracellular proteins defines a ␤-sheet structure similar to that found in HB-GAM and midkine. In addition to the apparent structural similarity, a similarity in biological functions is suggested by the occurrence of the TSR sequence motif in a wide variety of proteins that mediate cell-to-extracellular matrix and cell-to-cell interactions, in which the TSR domain mediates specific cell surface binding.Heparin-binding growth-associated molecule (HB-GAM) 1 (p18) was originally isolated from rat brain as an 18-kDa neurite outgrowth-promoting protein, the expression of which in brain tissue peaks during the perinatal stage of rapid axon growth and synapse formation (1). HB-GAM is highly homologous with the midkine (MK) sequence (2-4), and these proteins thus form a two-member family of small extracellular proteins that are conserved in vertebrates.In developing tissues HB-GAM associates with extracellular matrix of axonal tracts and of synapses (5, 6). It is also clearly expressed in developing basement membranes outside of brain (7) and in the cartilage matrix (8). N-syndecan (syndecan-3) acts as a receptor of HB-GAM in brain neurons in vitro (9) and localizes in many anatomical areas to the same developing fiber tracts as HB-GAM (10, 11). The heparan sulfate structure of brain N-syndecan is exceptionally heparin-like, especially in its high content of 2-0-sulfo-iduronic acid residues, which is of importance in the HB-GAM binding carbohydrate epitope, the minimal size of which appears to be 10 monosaccharide residues (12). The neurite outgrowth-promoting effect, based on HB-GAM/N-syndecan interaction, was very recently shown to be mediated by the cortactin/src-kinase signaling pathway to the cytoskeleton of neurites (13). These findings have led to the concept that N-syndecan mediates HB-GAM-induced neurite growth (for r...

Bone has an enormous capacity for growth, regeneration, and remodeling. This capacity is largely due to induction of osteoblasts that are recruited to the site of bone formation. The recruitment of osteoblasts has not been fully elucidated, though the immediate environment of the cells is likely to play a role via cell– matrix interactions. We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix–associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation. Intriguingly, N-syndecan, which acts as a receptor for HB-GAM, is expressed by osteoblasts/osteoblast precursors, whose ultrastructural phenotypes suggest active cell motility. The hypothesis that HB-GAM/N-syndecan interaction mediates osteoblast recruitment, as inferred from developmental studies, was tested using osteoblast-type cells that express N-syndecan abundantly. These cells migrate rapidly to HB-GAM in a haptotactic transfilter assay and in a migration assay where HB-GAM patterns were created on culture wells. The mechanism of migration is similar to that previously described for the HB-GAM–induced migratory response of neurons. Our hypothesis that HB-GAM/N-syndecan interaction participates in regulation of osteoblast recruitment was tested using two different in vivo models: an adjuvant-induced arthritic model and a transgenic model. In the adjuvant-induced injury model, the expression of HB-GAM and of N-syndecan is strongly upregulated in the periosteum accompanying the regenerative response of bone. In the transgenic model, the HB-GAM expression is maintained in mesenchymal tissues with the highest expression in the periosteum. The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness. HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.

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