Carlos Arregui

Activation of the repulsive receptor Roundabout inhibits N-cadherin-mediated cell adhesion

The formation of axon trajectories requires integration of local adhesive interactions with directional information from attractive and repulsive cues. Here, we show that these two types of information are functionally integrated; activation of the transmembrane receptor Roundabout (Robo) by its ligand, the secreted repulsive guidance cue Slit, inactivates N-cadherin-mediated adhesion. Loss of N-cadherin-mediated adhesion is accompanied by tyrosine phosphorylation of β-catenin and its loss from the N-cadherin complex, concomitant with the formation of a supramolecular complex containing Robo, Abelson (Abl) kinase and N-cadherin. Local formation of such a receptor complex is an ideal mechanism to steer the growth cone while still allowing adhesion and growth in other directions.

Turn‐off, drop‐out: Functional state switching of cadherins

The classic cadherins are a group of calcium dependent, homophilic cell–cell adhesion molecules that drive morphogenetic rearrangements and maintain the integrity of cell groups through the formation of adherens junctions. The formation and maintenance of cadherin‐mediated adhesions is a multistep process and mechanisms have evolved to regulate each step. This suggests that functional state switching plays an important role in development. Among the many challenges ahead is to determine the developmental role that functional state switching plays in tissue morphogenesis and to define the roles of each of the several regulatory interactions that participate in switching. One correlate of the loss of cadherin‐mediated adhesion, the “turn‐off” of cadherin function, is the exit, or “drop‐out” of cells from neural and epithelial layers and their conversion to a motile phenotype. We suggest that epithelial mesenchymal conversions may be initiated by signaling pathways that result in the loss of cadherin function. Tyrosine phosphorylation of β‐catenin is one such mechanism. Enhanced phosphorylation of tyrosine residues on β‐catenin is almost invariably associated with loss of the cadherin‐actin connection concomitant with loss of adhesive function. There are several tyrosine kinases and phosphatases that have been shown to have the potential to alter the phosphorylation state of β‐catenin and thus the function of cadherins. Our laboratory has focused on the role of the nonreceptor tyrosine phosphatase PTP1B in regulating the phosphorylation of β‐catenin on tyrosine residues. Our data suggest that PTP1B is crucial for maintenance of N‐cadherin‐mediated adhesions in embryonic neural retina cells. By using an L‐cell model system constitutively expressing N‐cadherin, we have worked out many of the molecular interactions essential for this regulatory interaction. Extracellular cues that bias this critical regulatory interaction toward increased phosphorylation of β‐catenin may be a critical component of many developmental events.

PTP1B Modulates the Association of β-Catenin with N-cadherin through Binding to an Adjacent and Partially Overlapping Target Site

The nonreceptor tyrosine phosphatase PTP1B associates with the cytoplasmic domain of N-cadherin and may regulate cadherin function through dephosphorylation of β-catenin. We have now identified the domain on N-cadherin to which PTP1B binds and characterized the effect of perturbing this domain on cadherin function. Deletion constructs lacking amino acids 872–891 fail to bind PTP1B. This domain partially overlaps with the β-catenin binding domain. To further define the relationship of these two sites, we used peptides to compete in vitro binding. A peptide representing the most NH2-terminal 8 amino acids of the PTP1B binding site, the region of overlap with the β-catenin target, effectively competes for binding of β-catenin but is much less effective in competing PTP1B, whereas two peptides representing the remaining 12 amino acids have no effect on β-catenin binding but effectively compete for PTP1B binding. Introduction into embryonic chick retina cells of a cell-permeable peptide mimicking the 8 most COOH-terminal amino acids in the PTP1B target domain, the region most distant from the β-catenin target site, prevents binding of PTP1B, increases the pool of free, tyrosine-phosphorylated β-catenin, and results in loss of N-cadherin function. N-cadherin lacking this same region of the PTP1B target site does not associate with PTP1B or β-catenin and is not efficiently expressed at the cell surface of transfected L cells. Thus, interaction of PTP1B with N-cadherin is essential for its association with β-catenin, stable expression at the cell surface, and consequently, cadherin function.

PTP1B regulates neurite extension mediated by cell-cell and cell-matrix adhesion molecules

N‐cadherin and β1‐integrin adhesion and signaling play important roles in growth cone adhesion and guidance. Each of these adhesion receptor systems is composed of multiprotein complexes, and both adhesion and downstream signaling events are regulated through the interaction of protein tyrosine kinases and phosphatases with many of the proteins that make up these complex systems. Work from our laboratory reported that the nonreceptor protein tyrosine phosphatase PTP1B is localized to adherens junctions and focal adhesion complexes and regulates both N‐cadherin‐ and β1‐integrin‐mediated adhesion. PTP1B appears to modulate integrin‐mediated adhesion through regulation of src activation and cadherin‐mediated adhesion through dephosphorylation of β‐catenin. We have continued these studies and report that PTP1B is localized to the tips of growing neurites and that introduction of a noncatalytic mutant of PTP1B into PC12 cells results in inhibition of N‐cadherin‐ and β1‐integrin‐mediated neurite outgrowth but is without effect on neurite outgrowth on poly‐L‐lysine. Moreover, suppressing the level of PTP1B in primary embryonic chick neural retina cells using antisense oligonucleotides also inhibits N‐cadherin‐ and β1‐integrin‐mediated neurite outgrowth. Neither of these techniques reduces the levels of expression of either adhesion receptor. We conclude that PTP1B is a regulatory component of the molecular complex required for both N‐cadherin and β1‐integrin‐mediated axon growth. J. Neurosci. Res. 63:143–150, 2001.

The juxtamembrane domain of cadherin regulates integrin-mediated adhesion and neurite outgrowth

Axons are guided along their trajectories during development by many different systems of adhesion, attraction, and repulsion. Thus, many distinct, and potentially competing, receptor systems respond to environmental cues, and the information must be coordinated inside the growth cone to ensure that extension follows the appropriate path. In this brief review we bring together two studies, each of which has defined different aspects of a pathway through which N‐cadherin regulates β1‐integrin function allowing for coordinated responses to environmental cues during neurite extension. First we review progress in defining the binding to cells and the subsequent effects on adhesion and neurite outgrowth of the chondroitin sulfate proteoglycan, neurocan. Neurocan binds to a cell surface glycosyltransferase associated with N‐cadherin (but not integrin), initiating a signal which results in loss of cadherin and integrin‐function—suggesting that these two adhesion receptor systems engage in cross‐talk, allowing coordinate regulation. Second, we review the use of “Trojan” peptides, peptides which mimic specific sequences in the cytoplasmic domain of N‐cadherin attached to a cell permeation sequence, to reveal protein–protein interactions critical to cadherin–integrin cross‐talk. One peptide mimicking a 20 amino acid sequence in the juxtamembrane region of N‐cadherin has the same effect as neurocan, blocking both cadherin‐ and integrin‐mediated adhesion and neurite outgrowth. Both neurocan and the peptide cause the release of the non‐receptor tyrosine kinase Fer from the cadherin complex and its binding to the integrin complex. These data define an epigenetic pathway through which environmental cues are capable of coordinately regulating the activity of two developmentally important adhesion systems. J. Neurosci. Res. 58:727–734, 1999.

Dendritic arbors of developing retinal ganglion cells are stabilized by β1-integrins

The architecture of dendritic arbors is a defining characteristic of neurons and is established through a sequential but overlapping series of events involving process outgrowth and branching, stabilization of the global pattern, and synapse formation. To investigate the roles of cadherins and beta1-integrins in maintaining the global architecture of the arbor, we used membrane permeable peptides and transfection with dominant-negative constructs to disrupt adhesion molecule function in intact chick neural retina at a stage when the architecture of the ganglion cell (RGC) arbor is established but synapse formation is just beginning. Inactivation of beta1-integrins induces rapid dendrite retraction, with loss of dynamic terminal filopodia followed by resorption of major branches. Disruption of N-cadherin-beta-catenin interactions has no effect; however, dendrites do retract following perturbation of the juxtamembrane region of N-cadherin, which disrupts N-cadherin-mediated adhesion and initiates a beta1-integrin inactivating signal. Thus, developing RGC dendritic arbors are stabilized by beta1-integrin-dependent processes.

Regulation of signaling by protein-tyrosine phosphatases: potential roles in the nervous system.

During neuronal development, cells respond to a variety of environmental cues through cell surface receptors that are coupled to a signaling transduction machinery based on protein tyrosine phosphorylation and dephosphorylation. Receptor and non-receptor tyrosine kinases have received a great deal of attention; however, in the last few years, receptor (plasma membrane associated) and non-receptor protein-tyrosine phosphatases (PTPs) have also been shown to play important roles in development of the nervous system. In many cases PTPs have provocative distribution patterns or have been shown to be associated with specific cell adhesion and growth factor receptors. Additionally, altering PTP expression levels or activity impairs neuronal behavior. In this review we outline what is currently known about the role of PTPs in development, differentiation and neuronal physiology.

Cloning of Rat Olfactory Bulb Tubulin Tyrosine Ligase cDNA: A Dominant Negative Mutant and an Antisense cDNA Increase the Proliferation Rate of Cells in Culture*

In this paper we describe the cloning of rat olfactory bulb tubulin tyrosine ligase (TTL) cDNA, and investigate the physiological role of TTL in cultured CHO-K1 cells. Comparison of the deduced amino acid sequence of rat TTL cDNA with those of bovine and pig showed approximately 90% of identity. Transient transfection of CHO-K1 cells with a dominant negative mutant of TTL that contains the binding site to the substrate (tubulin) but not the catalytic domain, significantly decreased the endogenous TTL activity as determined in vitro. Similar results were obtained using a construction encoding for the antisense sequence of TTL. The reduction in TTL activity is not accompanied by a decrease in the tyrosination levels of microtubules, as judged by immunofluorescence analysis. Strikingly, the number of cells in the plates transfected with the mutant TTL or the antisense TTL cDNA was, after 72 h of culture, two and three times higher, respectively, than the number of cells in the control plates. These results support the hypothesis that TTL may play a role in the regulation of the cell cycle in living cells.