Protein tyrosine phosphatases as adhesion receptors.

The intracellular segments of classic adhesion molecules such as N-CAM do not show structural similarity to any known signaling molecules. This suggests that their effects on signaling responses must be exerted indirectly through associated proteins. In contrast, many receptor protein tyrosine phosphatases (RPTPs) possess extracellular segments with homology to cell adhesion molecules linked directly to intracellular segments comprising one or two protein tyrosine phosphatase catalytic domains. Therefore, the RPTPs have the potential for direct modulation of catalytic function through engagement of the extracellular segment, suggesting they could be direct signal transducers of cell contact phenomena. In the past few years, some RPTPs have been shown to effect cell-cell adhesion directly via homophilic binding or indirectly by association with known cell adhesion molecules. In addition, RPTPs have been localized to points of cell-cell or cell-matrix contact, indicating their potential to regulate these structures.

Protein tyrosine phosphatases: from structure to function.

In the past few years, a diverse family of receptor-like and nontransmembrane protein tyrosine phosphatases (PTPases) have been identified and characterized at the level of primary structure. Progress is now being made towards defining physiological processes in which the activity of PTPases is important. One thing seems clear: the PTPases cannot be regarded simply as antagonists of the protein tyrosine kinases (PTKs)--rather, they have the potential to act both positively and negatively in mediating cellular signalling responses.

PTPmu regulates N-cadherin-dependent neurite outgrowth.

Cell adhesion is critical to the establishment of proper connections in the nervous system. Some receptor-type protein tyrosine phosphatases (RPTPs) have adhesion molecule-like extracellular segments with intracellular tyrosine phosphatase domains that may transduce signals in response to adhesion. PTPmu is a RPTP that mediates cell aggregation and is expressed at high levels in the nervous system. In this study, we demonstrate that PTPmu promotes neurite outgrowth of retinal ganglion cells when used as a culture substrate. In addition, PTPmu was found in a complex with N-cadherin in retinal cells. To determine the physiological significance of the association between PTPmu and N-cadherin, the expression level and enzymatic activity of PTPmu were perturbed in retinal explant cultures. Downregulation of PTPmu expression through antisense techniques resulted in a significant decrease in neurite outgrowth on an N-cadherin substrate, whereas there was no effect on laminin or L1-dependent neurite outgrowth. The overexpression of a catalytically inactive form of PTPmu significantly decreased neurite outgrowth on N-cadherin. These data indicate that PTPmu specifically regulates signals required for neurites to extend on an N-cadherin substrate, implicating reversible tyrosine phosphorylation in the control of N-cadherin function. Together, these results suggest that PTPmu plays a dual role in the regulation of neurite outgrowth.

Homophilic binding of PTP mu, a receptor-type protein tyrosine phosphatase, can mediate cell-cell aggregation.

The receptor-like protein tyrosine phosphatase, PTPmu, displays structural similarity to cell-cell adhesion molecules of the immunoglobulin superfamily. We have investigated the ability of human PTPmu to function in such a capacity. Expression of PTPmu, with or without the PTPase domains, by recombinant baculovirus infection of Sf9 cells induced their aggregation. However, neither a chimeric form of PTPmu, containing the extracellular and transmembrane segments of the EGF receptor and the intracellular segment of PTPmu, nor the intracellular segment of PTPmu expressed as a soluble protein induced aggregation. PTPmu mediates aggregation via a homophilic mechanism, as judged by lack of incorporation of uninfected Sf9 cells into aggregates of PTPmu-expressing cells. Homophilic binding has been demonstrated between PTPmu-coated fluorescent beads (Covaspheres) and endogenously expressed PTPmu on MvLu cells. Additionally the PTPmu-coated beads specifically bound to a bacterially expressed glutathione-S-transferase fusion protein containing the extracellular segment of PTPmu (GST/PTPmu) adsorbed to petri dishes. Covaspheres coated with the GST/PTPmu fusion protein aggregated in vitro and also bound to PTPmu expressed endogenously on MvLu cells. These results suggest that the ligand for this transmembrane PTPase is another PTPmu molecule on an adjacent cell. Thus homophilic binding interactions may be an important component of the function of PTPmu in vivo.

Receptor protein tyrosine phosphatase PTPmu associates with cadherins and catenins in vivo.

The extracellular segment of the receptor-type type protein tyrosine phosphatase PTPmu, possesses an MAM domain, an immunoglobulin domain, and four fibronectin type-III repeats. It binds homophilically, i.e., PTPmu on the surface of one cell binds to PTPmu on an apposing cell, and the binding site lies within the immunoglobulin domain. The intracellular segment of PTPmu has two PTP domains and a juxtamembrane segment that is homologous to the conserved intracellular domain of the cadherins. In cadherins, this segment interacts with proteins termed catenins to mediate association with the actin cytoskeleton. In this article, we demonstrate that PTPmu associates with a complex containing cadherins, alpha- and beta-catenin in mink lung (MvLu) cells, and in rat heart, lung, and brain tissues. Greater than 80% of the cadherin in the cell is cleared from Triton X-100 lysates of MvLu cells after immunoprecipitation with antibodies to PTPmu; however, the complex is dissociated when lysates are prepared in more stringent, SDS-containing RIPA buffer. In vitro binding studies demonstrated that the intracellular segment of PTPmu binds directly to the intracellular domain of E-cadherin, but not to alpha- or beta-catenin. Consistent with their ability to interact in vivo, PTPmu, cadherins, and catenins all localized to points of cell-cell contact in MvLu cells, as assessed by immunocytochemical staining. After pervanadate treatment of MvLu cells, which inhibits cellular tyrosine phosphatase activity including PTPmu, the cadherins associated with PTPmu are now found in a tyrosine-phosphorylated form, indicating that the cadherins may be an endogenous substrate for PTPmu. These data suggest that PTPmu may be one of the enzymes that regulates the dynamic tyrosine phosphorylation, and thus function, of the cadherin/catenin complex in vivo.

Dynamic interaction of PTPmu with multiple cadherins in vivo.

There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPmu associates with the cadherin-catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S. M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977- 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPmu interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPmu and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPmu and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPmu from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPmu in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513-1517) have asserted that the association we observed between PTPmu and the cadherin-catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPmu, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPmu obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPmu antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPmu and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.

One-pot synthesis of nanochain particles for targeting brain tumors.

To synthesize multi-component nanochains, we developed a simple 'one-pot' synthesis, which exhibited high yield and consistency. The nanochains particles consist of parent nanospheres chemically linked into a higher-order, chain-like assembly. The one-pot synthesis is based on the addition of two types of parent nanospheres in terms of their surface chemical functionality (e.g., decorated with PEG-NH2 or PEG-COOH). By reacting the two types of parent nanospheres at a specific ratio (∼2 : 1) for a short period of time (∼30 min) under rigorous stirring, nanochains were formed. For example, we show the synthesis of iron oxide nanochains with lengths of about 125 nm consisting of 3-5 constituting nanospheres. The chain-like shaped nanoparticle possessed a unique ability to target and rapidly deposit on the endothelium of glioma sites via vascular targeting. To target and image invasive brain tumors, we used iron oxide nanochains with the targeting ligand being the fibronectin-targeting peptide CREKA. Overexpression of fibronectin is strongly associated with the perivascular regions of glioblastoma multiforme and plays a critical role in migrating and invasive glioma cells. In mice with invasive glioma tumors, 3.7% of the injected CREKA-targeted nanochains was found in gliomas within 1 h. Notably, the intratumoral deposition of the nanochain was ∼2.6-fold higher than its spherical variant. Using MR imaging, the precise targeting of nanochains to gliomas provided images with the exact topology of the disease including their margin of infiltrating edges and distant invasive sites.

Increasing N-CAM-mediated cell-cell adhesion does not reduce invasion of RSV-transformed WC5 rat cerebellar cells.

The WC5 rat cerebellar cell line, infected with a Rous sarcoma virus (RSV) that is temperature-sensitive for pp60v-src transformation, expresses high levels of the neural cell adhesion molecule, N-CAM, when grown at the non-permissive temperature for pp60v-src activity. At the permissive temperature, N-CAM expression is 4- to 10-fold reduced and the cells aggregate poorly. To evaluate the effects of variations in N-CAM expression, we compared the invasive ability of transformed WC5 cells that express low levels of N-CAM with transformed cells in which N-CAM-mediated adhesion was restored. WC5 cells were transfected with expression vectors containing cDNAs encoding the 120 or 180 kDa forms of chicken N-CAM linked to constitutive promoters. Several permanently transfected lines that expressed chicken N-CAM at the cell surface were isolated. These cell lines showed enhanced aggregation at the permissive temperature relative to untransfected WC5 cells or cells transfected with control constructs. By comparing the ability of control and transfected WC5 cells to invade reconstituted extracellular matrix, we tested the effect of variations in N-CAM-mediated adhesion on invasion. Clones that expressed high levels of N-CAM showed invasion rates that were similar to control cells, indicating that increasing N-CAM-mediated adhesion does not inhibit the invasiveness of RSV-transformed WC5 cells.

Identification of the homophilic binding site of the receptor protein tyrosine phosphatase PTP mu.

The receptor-type protein tyrosine phosphatase PTP mu comprises an extracellular segment containing a MAM domain, an immunoglobulin domain and four fibronectin type III repeats, a transmembrane segment, and two intracellular PTP domains. We have previously shown that PTP mu binds homophilically, i.e. PTP mu on the surface of one cell binds to PTP mu on an apposing cell, and that the extracellular segment alone is sufficient for homophilic binding. In this study we report that in MvLu cells PTP mu is proteolytically processed into two noncovalently associated fragments, one comprising most of the extracellular segment (approximately 100 kDa) and the other containing predominantly the transmembrane and intracellular portions (approximately 100 kDa). We have also identified the homophilic binding site within the extracellular segment. We have generated, expressed, and purified various fragments of the extracellular segment of PTP mu and have used fluorescent beads (Covaspheres) coated with these fragments in three binding assays: (i) measurement of bead aggregation, (ii) binding of beads to surfaces of dishes coated with purified PTP mu, or (iii) binding to MvLu cells. Only beads coated with recombinant fragments that contained the immunoglobulin domain underwent aggregation or bound to surfaces displaying PTP mu, suggesting that neither the MAM domain nor the fibronectin type III repeats bound homophilically in these assays. The fragment containing the Ig domain alone bound as well as any other Ig domain-containing fragment, suggesting that the Ig domain is both necessary and sufficient for homophilic binding under these conditions.

Purification and characterization of the human protein tyrosine phosphatase, PTP mu, from a baculovirus expression system.

The receptor like PTPase, PTP mu, displays structural similarity in its extracellular segment to members of the immunoglobulin superfamily of cell adhesion molecules. The full length form of PTP mu (200 kD) and a construct expressing only the intracellular PTPase domain-containing segment (80 kD) were expressed in the baculovirus/Sf9 cell system, purified and characterized. Full length PTP mu was membrane associated while the truncated form was recovered in the soluble fraction. PTP mu preferentially dephosphorylated a reduced carboxamidomethylated and maleylated derivative of lysozyme (RCML) over other tyrosine phosphorylated substrates such as myelin basic protein (MBP) or the synthetic peptide EDNDYINASL. The enzymatic properties of the soluble, truncated form of the enzyme were examined in detail. The pH optimum was 7.5. It dephosphorylated RCML with a Km of 400 nM and a Vmax of 725 nmol/min/mg. This form of the enzyme was 2 fold more active than full length PTP mu. Trypsinization of the full length form inhibited activity. Vanadate and molybdate, potent tyrosine phosphatase inhibitors, abolished activity of the enzyme. Zn++ and Mn++ ions, polylysine, poly-glu/tyr, and spermine were also inhibitory.

Invasion of Rous sarcoma virus-transformed retinal cells: role of cell motility.

Transformation of retinal neuro-epithelial cells by Rous sarcoma virus (RSV) leads to many alterations in cell phenotype, including changes in cell movement, cell-cell adhesion and protease secretion. To define and quantitate the alterations in cell movement, we analyzed video recordings of cultured cells using the computer-assisted Dynamic Morphology System (DMS). Control neuro-epithelial cells showed very low levels of translocation and membrane activity. After transformation, neuro-epithelial cells exhibited increased membrane activity, although directed cell translocation remained low. Developing retinas also contain a small proportion of Müller glial cells, which were purified by repeated passaging of control cultures. In contrast to neuro-epithelial cells, both control and RSV-transformed glial cells showed high levels of translocation and membrane activity. To analyze how different kinds of cell movement affect invasive behavior, we compared the ability of control and RSV-transformed cells to invade the chorio-allantoic membrane of developing chicken embryos. Control neuro-epithelial cells were not invasive. RSV-transformed neuro-epithelial cells, which showed low levels of translocation as revealed by DMS, were invasive. Similarly, RSV-transformed glial cells were invasive while control glial cells, which translocated, were not invasive. These results suggest that high levels of cell translocation are not necessary for invasion. In addition, the results suggest that elevated membrane activity in neuro-epithelial cells may be important for their invasion.

Invasion by WC5 rat cerebellar cells is independent of RSV-induced changes in growth and adhesion.

The WC5 rat cerebellar cell line, which is infected with a Rous sarcoma virus that is temperature-sensitive for pp60src transformation, shows temperature-dependent expression of the neural-cell-adhesion molecule (N-CAM) and glial fibrillary acidic protein (GFAP). We found that WC5 cells maintained at the non-permissive temperature in both monolayer cultures and spheroids are subject to density-dependent inhibition of growth, whereas cells maintained at the permissive temperature continued to grow. The movement of isolated WC5 cells at both temperatures was similar, while the migration of WC5 cells out of 3-dimensional aggregates was faster at the non-permissive temperature. We tested whether the RSV-induced changes affect the invasion of the WC5 cells in 2 in vitro assays: the chorio-allantoic-membrane assay and the chick-heart-fragment assay. In both assays, WC5 cells grown at either temperature were invasive. These results indicate that growth rate is unrelated to invasion and that loss of N-CAM-mediated cell-cell adhesion is not necessary for invasion.

The PTPmu protein-tyrosine phosphatase binds and recruits the scaffolding protein RACK1 to cell-cell contacts.

PTPmu, an Ig superfamily receptor protein-tyrosine phosphatase, promotes cell-cell adhesion and interacts with the cadherin-catenin complex. The signaling pathway downstream of PTPmu is unknown; therefore, we used a yeast two-hybrid screen to identify additional PTPmu interacting proteins. The membrane-proximal catalytic domain of PTPmu was used as bait. Sequencing of two positive clones identified the scaffolding protein RACK1 (receptor for activated protein C kinase) as a PTPmu interacting protein. We demonstrate that RACK1 interacts with PTPmu when co-expressed in a recombinant baculovirus expression system. RACK1 is known to bind to the src protein-tyrosine kinase. This study demonstrates that PTPmu association with RACK1 is disrupted by the presence of constituitively active src. RACK1 is thought to be a scaffolding protein that recruits proteins to the plasma membrane via an unknown mechanism. We have shown that the association of endogenous PTPmu and RACK1 in a lung cell line is increased at high cell density. We also demonstrate that the recruitment of RACK1 to both the plasma membrane and cell-cell contact sites is dependent upon the presence of the PTP mu protein in these cells. Therefore, PTPmu may be one of the proteins that recruits RACK1 to points of cell-cell contact, which may be important for PTPmu-dependent signaling in response to cell-cell adhesion.

Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway.

The Slit protein guides neuronal and leukocyte migration through the transmembrane receptor Roundabout (Robo). We report here that the intracellular domain of Robo interacts with a novel family of Rho GTPase activating proteins (GAPs). Two of the Slit-Robo GAPs (srGAPs) are expressed in regions responsive to Slit. Slit increased srGAP1-Robo1 interaction and inactivated Cdc42. A dominant negative srGAP1 blocked Slit inactivation of Cdc42 and Slit repulsion of migratory cells from the anterior subventricular zone (SVZa) of the forebrain. A constitutively active Cdc42 blocked the repulsive effect of Slit. These results have demonstrated important roles for GAPs and Cdc42 in neuronal migration. We propose a signal transduction pathway from the extracellular guidance cue to intracellular actin polymerization.