Pleiotrophin is an 18-kDa heparin-binding growth factor, which uses chondroitin sulfate (CS) proteoglycan, PTP as a receptor. It has been suggested that the D-type structure (GlcA(2S)␤1-3GalNAc(6S)) in CS contributes to the high affinity binding between PTP and pleiotrophin. Here, we analyzed the interaction of shark cartilage CS-D with pleiotrophin using a surface plasmon resonance biosensor to reveal the importance of D-type structure. CS-D was partially digested with chondroitinase ABC, and fractionated using a Superdex 75pg column. The >18-mer CS fractions showed significant binding to pleiotrophin, and the longer fractions had stronger affinity for pleiotrophin than the shorter ones. The ϳ46-mer CS fraction bound to densely immobilized pleiotrophin with high affinity (K D ‫؍‬ ϳ30 nM), and the binding reactions fitted the bivalent analyte model. However, when the density of the immobilized pleiotrophin was lowered, the strength of affinity remarkably decreased (K D ‫؍‬ ϳ2.5 M), and the reactions no longer fitted the model and were considered to be monovalent binding. The 20ϳ24-mer fractions showed low affinity binding to densely immobilized pleiotrophin (K D ‫؍‬ 3ϳ20 M), which seemed to be monovalent. When ϳ22-mer CS oligosaccharides were fractionated by strong anion exchange HPLC, each fraction differed in affinity for pleiotrophin (K D ‫؍‬ 0.36 ϳ >10 M), and the affinity correlated with the amounts of D-and E-(GlcA␤1-3GalNAc(4S,6S)) type oversulfated structures. These results suggest that the binding of pleiotrophin to CS is regulated by multivalency with CS ϳ20 mer as a unit and by the amounts of oversulfated structures.Pleiotrophin, also known as heparin-binding growth-associated molecule (HB-GAM) 2 or heparin affin regulatory peptide (HARP), is an 18-kDa growth factor that shows 45% amino acid sequence identity with midkine (1, 2). Pleiotrophin and midkine share many biological activities such as the promotion of neurite outgrowth and migration of embryonic neurons and osteoblasts (1, 2). Furthermore, it has been revealed that both use a common signal-transducing receptor, receptortype protein-tyrosine phosphatase (PTP) (3-7). PTP is synthesized as a membrane-bound chondroitin sulfate (CS) proteoglycan, and its extracellular variant, which is generated by alternative splicing, is secreted as a major soluble CS proteoglycan in the brain, phosphacan (8, 9).The binding of phosphacan to pleiotrophin and midkine depends on the CS portion of this proteoglycan, and the removal of CS resulted in a remarkable decrease in the binding affinity (9, 10). It was revealed that pleiotrophin inactivated the tyrosine phosphatase activity of this receptor leading to an increase in the tyrosine phosphorylation levels of specific substrates such as cat-1 and ␤-catenin (4, 11). Several researchers suggested that pleiotrophin induces the dimerization of PTP, which results in the inactivation of its enzymatic activity (4, 11).On the other hand, midkine and pleiotrophin easily formed noncovalently bound multimers, and it has be...

PTPzeta and lectican family members are major chondroitin sulfate proteoglycans (CS-PGs) in the brain, which bind with many proteins via core protein and CS portions. Recent studies revealed that the oversulfated structures in CS constitute high affinity binding sites for various growth factors and axon guidance molecules, and play important roles in the proliferation of neural progenitor cells, neurite extension and neuronal migration. PTPzeta uses pleiotrophin as a ligand. The CS portion of PTPzeta constitutes a part of the pleiotrophin-binding site, and oversulfated D unit increases the binding affinity. Pleiotrophin-PTPzeta signaling regulates the morphogenesis of Purkinje cell by controlling the tyrosine phosphorylation of a Notch-related transmembrane protein, DNER. In the brain of adult animals, a subset of neurons are surrounded by CS-PG-rich extracellular matrix called perineuronal net, in which lecticans form complexes with hyaluronic acid and tenascin-R. CS-PGs in the perineuronal net regulate ocular dominance plasticity in the visual cortex by enhancing the uptake of Otx2 homeoprotein by parvalbumin-positive interneurons in a CS-dependent manner. These studies revealed unexpectedly complex mechanisms of CS-PG functions.

Protein tyrosine phosphatase (PTP) is a receptor type protein tyrosine phosphatase that uses pleiotrophin as a ligand. Pleiotrophin inactivates the phosphatase activity of PTP, resulting in the increase of tyrosine phosphorylation levels of its substrates. We studied the functional interaction between PTP and DNER, a Notch-related transmembrane protein highly expressed in cerebellar Purkinje cells. PTP and DNER displayed patchy colocalization in the dendrites of Purkinje cells, and immunoprecipitation experiments indicated that these proteins formed complexes. Several tyrosine residues in and adjacent to the tyrosine-based and the second C-terminal sorting motifs of DNER were phosphorylated and were dephosphorylated by PTP, and phosphorylation of these tyrosine residues resulted in the accumulation of DNER on the plasma membrane. DNER mutants lacking sorting motifs accumulated on the plasma membrane of Purkinje cells and Neuro-2A cells and induced their process extension. While normal DNER was actively endocytosed and inhibited the retinoic-acid-induced neurite outgrowth of Neuro-2A cells, pleiotrophin stimulation increased the tyrosine phosphorylation level of DNER and suppressed the endocytosis of this protein, which led to the reversal of this inhibition, thus allowing neurite extension. These observations suggest that pleiotrophin-PTP signaling controls subcellular localization of DNER and thereby regulates neuritogenesis.Protein tyrosine phosphatase (PTP), also known as RPTP/␤, is a receptor type protein tyrosine phosphatase that is synthesized as a chondroitin sulfate proteoglycan (12,16,17,21,28,32). There are three major splice variants of this molecule, the full-length form (PTP-A), the short receptor form (PTP-B), and the secreted form (phosphacan) (Fig.

With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.

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