6B4 Proteoglycan/Phosphacan, an Extracellular Variant of Receptor-like Protein-tyrosine Phosphatase ζ/RPTPβ, Binds Pleiotrophin/Heparin-binding Growth-associated Molecule (HB-GAM)

Abstract

A major chondroitin sulfate proteoglycan in the brain, 6B4 proteoglycan/phosphacan, corresponds to the extracellular region of a receptor-like protein-tyrosine phosphatase, PTPzeta/RPTPbeta. Here, we purified and characterized 6B4 proteoglycan-binding proteins from rat brain. From the CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) extract of brain microsomal fractions, 18-, 28-, and 40-kDa proteins were specifically isolated using 6B4 proteoglycan-Sepharose. N-terminal amino acid sequencing identified the 18-kDa protein as pleiotrophin/heparin-binding growth-associated molecule (HB-GAM). Scatchard analysis of 6B4 proteoglycan-pleiotrophin binding revealed low (Kd = 3 nM) and high (Kd = 0.25 nM) affinity binding sites. Chondroitinase ABC digestion of the proteoglycan decreased the binding affinities to a single value (Kd = 13 nM) without changing the number of binding sites. This suggested the presence of two subpopulations of the proteoglycan with different chondroitin sulfate structures. Heparin potently inhibited binding of 6B4 proteoglycan to pleiotrophin (IC50 = 3.5 ng/ml). Heparan sulfate and chondroitin sulfate C inhibited moderately (IC50 = 150 and 400 ng/ml, respectively), but, in contrast, chondroitin sulfate A and keratan sulfate were poor inhibitors (IC50 > 100 microg/ml). Immunofluorescence and immunoblotting analyses indicated that both 6B4 proteoglycan and PTPzeta are located on cortical neurons. Anti-6B4 proteoglycan antibody added to the culture medium suppressed pleiotrophin-induced neurite outgrowth of cortical neurons. These results suggested that interaction between 6B4 proteoglycan and pleiotrophin is required for the action of pleiotrophin, and chondroitin sulfate chains on 6B4 proteoglycan play regulatory roles in its binding.

Increased trabecular bone formation in mice lacking the growth factor midkine

“…Although we cannot rule that Mdk simply acts through modulating the well-known effects of heparin on bone cells, there are several in vitro studies demonstrating binding of Mdk and Ptn to specific membrane proteins, such as syndecans, integrins, low-density lipoprotein receptor-related proteins, the receptor tyrosine kinase ALK, or the receptor tyrosine phosphatase Rptpz. (4,(56)(57)(58)(59)(60)(61)(62)(63)(64)(65) Interestingly, we have observed previously that the expressions of both syndecan-3 and Rptpz increase in the course of osteoblast differentiation and that mice lacking Rptpz display a skeletal phenotype. (27,29) However, since the deficiency of Rptpz resulted in low bone mass, and since Rptpz-deficient calvarial osteoblasts, unlike Mdk-deficient cultures, displayed a marked increase in their proliferation rate, we would assume that Mdk regulates bone formation by other mechanisms.…”

Translocations involving anaplastic lymphoma kinase (ALK)

“…It seems surprising that the profound and multiple e ects of pleiotrophin might be mediated solely by the ALK receptor. Indeed, other cellular receptors for pleiotrophin have been identi®ed, protein tyrosine phosphatase zeta/RPTP beta and syndecan-3, that may mediate some of these e ects (Maeda et al, 1996;Raulo et al, 1994). In addition, further studies are required to establish pleiotrophin as the sole ligand for the ALK receptor, and the physiological relevance of this receptor/ligand pair.…”

Expression of VEGF and pleiotrophin in deer antler

“…Receptors associated with pleiotrophin and its neurotrophic effects are N-syndecan/syndecan-3 and RPTPb/PTPf (Raulo et al, 1994;Maeda et al, 1996).…”