Isolation of a neuronal cell surface receptor of heparin binding growth-associated molecule (HB-GAM). Identification as N-syndecan (syndecan-3).
HB-GAM (heparin binding growth-associated molecule; pleiotrophin) is a secretory, extracellular matrix-associated protein that is strongly expressed in developing nervous tissues and belongs to a novel family of differentiation/growth factors. It promotes axonal growth from perinatal rat brain neurons and is suggested to be mitogenic for some cell types and to display cell-transforming activity. Since the receptors of HB-GAM in cells are unknown, we have started isolation of putative cell surface receptors from brain neurons and from perinatal rat brain. For this purpose, recombinant HB-GAM was produced with the aid of a baculovirus vector and used as an affinity matrix in receptor isolation. A detergent-solubilized component from cultured brain neurons and from brain was identified that binds specifically to HB-GAM and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad smear with an apparent molecular mass of about 200 kDa. This cell surface component was found to contain heparan sulfate chains, which are bound to a core protein with an apparent molecular mass of 120 kDa. Gel electrophoretic characteristics, immunochemical analysis, and partial peptide sequencing revealed that the cell surface component isolated as an HB-GAM receptor is N-syndecan (syndecan-3). In a solid phase binding assay, N-syndecan was found to bind to HB-GAM in a similar manner as to basic fibroblast growth factor (KD = 0.6 nM). Immunofluorescence microscopy indicated that in brain neurons, N-syndecan occurs at the surface of the cell soma and of the neurites that grow along HB-GAM-coated substrates. Anti-N-syndecan antibodies added to culture media had an inhibitory effect on HB-GAM-induced neurite outgrowth. We suggest that N-syndecan mediates the neurite outgrowth-promoting signal from HB-GAM to the cytoskeleton of growing neurites.
Raulo
E
E
Laboratory of Molecular Neurobiology, University of Helsinki, Finland.
Chernousov
M A
MA
Carey
D J
DJ
Nolo
R
R
Rauvala
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D008562:Membrane Glycoproteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016501:Neurites; D009474:Neurons / Q000378:metabolism*; D011509:Proteoglycans / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D053670:Syndecan-3
D000906:Antibodies; D002352:Carrier Proteins; D016207:Cytokines; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011509:Proteoglycans; C508303:Sdc3 protein, rat; D053670:Syndecan-3; C066845:pleiotrophin