Neurotrophin receptors of the Trk family play a vital role in the survival of developing neurons and the process of axonogenesis. The Trk family are receptor protein tyrosine kinases (RTKs) and their signalling in response to neurotrophins is critically dependent upon their ability to transphosphorylate and act as signalling centres for multiple adaptor proteins and distinct, downstream pathways. Such phosphotyrosine signalling also depends upon the appropriate counter-regulation by phosphatases. A large family of receptor-like protein tyrosine phosphatases (RPTPs) are also expressed in developing neurons and in this study we have examined the ability of the phosphatase PTPsigma to interact with and regulate Trk proteins in transfected HEK 293T cells. PTPsigma can bind differentially to Trk proteins, binding stably in complexes with TrkA and TrkC, but not TrkB. The transmembrane domains of PTPsigma and TrkA appear to be sufficient for the direct or indirect interaction between these two receptors. Furthermore, PTPsigma is shown to dephosphorylate all three Trk receptors and suppress their phosphorylation in the presence of neurotrophins. In addition, overexpression of PTPsigma in primary sensory neurons in culture inhibits neurite outgrowth without affecting the short-term survival of these neurons. PTPsigma can thus show differential complex formation with different Trk family members and in neurons can selectively target the neurite-forming signalling pathway driven by TrkA.

Signaling through receptor protein tyrosine phosphatases (RPTPs) can influence diverse processes, including axon development, lymphocyte activation, and cell motility. The molecular regulation of these enzymes, however, is still poorly understood. In particular, it is not known if, or how, the dimerization state of RPTPs is related to the binding of extracellular ligands. Protein tyrosine phosphatase (PTP) is an RPTP with major isoforms that differ in their complements of fibronectin type III domains and in their ligand-binding specificities. In this study, we show that PTP forms homodimers in the cell, interacting at least in part through the transmembrane region. Using this knowledge, we provide the first evidence that PTP ectodomains must be presented as dimers in order to bind heterophilic ligands. We also provide evidence of how alternative use of fibronectin type III domain complements in two major isoforms of PTP can alter the ligand binding specificities of PTP ectodomains. The data suggest that the alternative domains function largely to change the rotational conformations of the amino-terminal ligand binding sites of the ectodomain dimers, thus imparting novel ligand binding properties. These findings have important implications for our understanding of how heterophilic ligands interact with, and potentially regulate, RPTPs.Many cell-signaling events are regulated through reversible tyrosine phosphorylation of proteins. This phosphorylation cycle is controlled by the counterbalanced actions of two enzyme families, protein tyrosine kinases and protein tyrosine phosphatases (PTPs), each of which has cytoplasmic and receptorlike members. While the regulation and actions of many receptor protein tyrosine kinases (RTKs) are well characterized, our related understanding of the receptor-type PTPs (RPTPs) remains far from complete.Twenty-one human RPTPs have been identified, and highly conserved orthologues and homologues exist in vertebrates and invertebrates (3). Several of these RPTPs have particularly well-documented roles in neural development (16,26,40), cell adhesion, and motility (7, 51). RPTPs are type I transmembrane proteins with varied extracellular domain structures, and in many cases, it is still unclear what their ligands are. It is also unclear how, and in most cases if, such ligands directly control RPTP activity. Moreover, although RTKs are obligate dimers during ligand activation and signaling, dimerization has been demonstrated for only a small number of RPTPs, and we have only a few clues as to how this dimerization is linked to signaling control. Furthermore, it is not clear whether ligand binding is influenced by, or influences, the RPTP dimerization state.The RPTPs PTP␣, Sap-1, and CD45 form dimers normally in the cell, and this dimerization can block the catalytic action of these enzymes (23,24,64). PTP␣ and CD45 may be inhibited by steric constraints, possibly through an inhibitory wedge structure (8, 29), although this is still controversial (35). With PTP␣ and Sap-1, there is also e...

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Vertebrate nervous system development relies on a multitude of guidance cues to stimulate axonal extension and stable synaptic contacts with targets such as muscles. Interpretation of these environmental signals by growth cones involves multiple receptor classes such as cell adhesion molecules (CAMs) Reversible tyrosine phosphorylation, catalyzed by receptor tyrosine kinases and receptor tyrosine phosphatases, plays an essential part in cell signaling during axonal development. Receptor protein tyrosine phosphatase-r has been implicated in the growth, guidance and repair of retinal axons. This phosphatase has also been implicated in motor axon growth and innervation. Insect orthologs of receptor protein tyrosine phosphatase-r are also implicated in the recognition of muscle target cells. A potential extracellular ligand for vertebrate receptor protein tyrosine phosphatase-r has been previously localized in developing skeletal muscle. The identity of this muscle ligand is currently unknown, but it appears to be unrelated to the heparan sulfate ligands of receptor protein tyrosine phosphatase-r. In this study, we have used affinity chromatography and tandem MS to identify nucleolin as a binding partner for receptor protein tyrosine phosphatase-r in skeletal muscle tissue. Nucleolin, both from tissue lysates and in purified form, binds to receptor protein tyrosine phosphatase-r ectodomains. Its expression pattern also overlaps with that of the receptor protein tyrosine phosphatase-r-binding partner previously localized in muscle, and nucleolin can also be found in retinal basement membranes. We demonstrate that a significant amount of muscle-associated nucleolin is present on the cell surface of developing myotubes, and that two nucleolin-binding components, lactoferrin and the HB-19 peptide, can block the interaction of receptor protein tyrosine phosphatase-r ectodomains with muscle and retinal basement membranes in tissue sections. These data suggest that muscle cell surface-associated nucleolin represents at least part of the muscle binding site for axonal receptor protein tyrosine phosphatase-r and that nucleolin may also be a necessary component of basement membrane binding sites of receptor protein tyrosine phosphatase-r.Abbreviations AP, alkaline phosphatase; CAM, cell adhesion molecule; FGF, fibroblast growth factor; FITC, fluorescein isothiocyanate; HB-19, 5[Kw(CH 2 N)PR]-TASP; HSPG, heparin sulfate proteoglycan; PLAP, placental alkaline phosphatase; PTP, protein tyrosine phosphatase; RAP, receptor affinity probe; RPTP, receptor protein tyrosine phosphatase; RTK, receptor protein tyrosine kinase.