Heparan sulfate proteoglycans are ligands for receptor protein tyrosine phosphatase sigma

The complex pattern of neural connectivity established during nervous system development relies on the ability of the axon's motile tip, the growth cone, to receive, transduce, and integrate multiple environmental signals. Protein phosphorylation on tyrosine residues plays a key role in these processes (21, 29). Two major families of enzymes, the protein tyrosine kinases and the protein tyrosine phosphatases (PTPs), control cellular phosphotyrosine levels. These enzymes are found in both cytoplasmic and transmembrane (receptor-like) forms, and the biochemical interactions between them lead to a diversity of cellular behaviors (25, 76). Receptor protein tyrosine phosphatases (RPTPs) have recently joined the list of molecules involved in neural development and in particular in axon growth and guidance (reviewed in references 6, 68, 72, and 79). Type 2 RPTPs, containing cell adhesion molecule (CAM)-like extracellular regions, may be particularly well equipped to trigger signals involving cell-cell or cell-extracellular matrix contacts (68). Recent experiments with Drosophila have demonstrated the involvement of the RPTPs DLAR and DPTP69D in motor (19, 20, 50), retinal (27, 57), and midline (73) axon guidance. In leech, a LAR gene-related RPTP (HmLAR2) is implicated in Comb cell behavior, specifically in process outgrowth and mutual avoidance by sibling growth cones (2, 28). Several vertebrate RPTPs have been shown to promote neurite outgrowth in cell culture, including cPTPσ (51), RPTPκ (23), RPTPμ (10), and RPTPδ (82). Moreover, it has recently been shown that RPTPδ also has a potential guidance function, at least in vitro (74). In mice, gene deficiencies in type 2 RPTPs lead to various abnormalities. LAR deficiency leads to a reduction in size of basal forebrain cholinergic neurons, diminished hippocampal innervation, and defects in other tissues, such as the mammary gland (63, 78, 86). RPTPδ deficiency leads to impaired learning and enhanced hippocampal long-term potentiation (77). The most extreme defects are seen in RPTPσ-deficient mice, which show poor fecundity, hypomyelination of peripheral nerves, ataxias, and abnormalities in development of the hypothalamus and pituitary (24, 80). Although the developmental mechanism of these defects is not known, it is of interest that the avian orthologue of RPTPσ, cPTPσ (69, 85), regulates axon outgrowth of embryonic neurons (51). Despite this accumulation of functional data, much less is known about the extracellular cues that trigger signal transduction though RPTPs. Several RPTPs interact homophilically in trans, including RPTPμ (8), RPTPκ (62), and RPTPδ (82), but the effects of such homophilic interactions on enzyme function are as yet unclear. The laminin-nidogen complex has been shown to be a heterotypic ligand for a nonneural isoform of LAR (59), while type 5 RPTPζ can bind to several molecules, including contactin and tenascin and the cytokines midkine and pleiotrophin (60). Significantly, pleiotrophin has also recently been shown to suppress the catalytic activity of RPTPζ (53). cPTPσ, originally described as CRYPα (69), is a type 2 RPTP expressed as two major isoforms: cPTPσ1 (CRYPα1) has three immunoglobulin-like (Ig) domains and four fibronectin type III (FNIII) domains in its extracellular region, while cPTPσ2 (CRYPα2) has four extra FNIII domains. Both isoforms are strongly expressed in the chicken embryo nervous system, in particular within retinal and tectal axons and on their growth cones (70, 71). Moreover cPTPσ1 promotes intraretinal axon growth in vitro and controls growth cone morphology via the maintenance of lamellipodia (51). One or more ligands for cPTPσ are localized in the retinal and tectal basal lamina (BL) and on the glial endfeet of these tissues (33, 51), but the identity of these ligands has remained elusive. To understand how cPTPσ may function at the molecular and cellular levels, we sought to identify the molecular nature of these ligands. Here we show that cPTPσ is a heparin binding protein and that extracellular matrix heparan sulfate proteoglycans (HSPGs), in particular agrin and collagen XVIII, are binding partners for cPTPσ in vitro. Receptor affinity probe assays on tissue sections reveal that the binding of cPTPσ ectodomains to HSPGs is absolutely dependent on the presence of heparan sulfate (HS) side chains. Site-directed mutagenesis in a putative heparin and HS (heparin/HS) binding site in cPTPσ completely abolished this interaction. These data, together with the overlapping expression patterns of cPTPσ, agrin, and collagen XVIII in the developing chick retina, suggest that the reported interactions are physiologically relevant and that HSPGs could be a major ligand class for cPTPσ.