Ns (neuroserpin) is a member of the serpin (serine protease inhibitor) gene family that is primarily expressed within the central nervous system. Its principal target protease is tPA (tissue plasminogen activator), which is thought to contribute to synaptic plasticity and to be secreted in a stimulus-dependent manner. In the present study, we demonstrate in primary neuronal cultures that Ns co-localizes in LDCVs (large dense core vesicles) with the regulated secretory protein chromogranin B. We also show that Ns secretion is regulated and can be specifically induced 4-fold by secretagogue treatment. A novel 13-amino-acid sorting signal located at the C-terminus of Ns is identified that is both necessary and sufficient to target Ns to the regulated secretion pathway. Its deletion renders Ns no longer responsive to secretagogue stimulation, whereas PAI-Ns [Ns (neuroserpin)-PAI-1 (plasminogen activator inhibitor-1) chimaera appending the last 13 residues of Ns sequence to the C-terminus of PAI-1] shifts PAI-1 secretion into a regulated secretory pathway.
The serine proteinase inhibitor, plasminogen activator inhibitor type-1 (PAI-1), binds to the adhesion protein vitronectin with high affinity at a site that is located directly adjacent to the vitronectin RGD integrin binding sequence. The binding of PAI-1 to vitronectin sterically blocks integrin access to this site and completely inhibits the binding of purified integrins to vitronectin; however, its inhibition of endothelial and smooth muscle cell adhesion to vitronectin is at most 50 -75%. Because PAI-1 binds vitronectin with ϳ10 -100-fold higher affinity than purified integrins, we have analyzed the mechanism whereby these cells are able to overcome this obstacle. Our studies exclude proteolytic removal of PAI-1 from vitronectin as the mechanism, and show instead that cell adhesion in the presence of PAI-1 is dependent on integrin-cytoskeleton engagement. Disrupting endothelial or smooth muscle cell actin polymerization and/or focal adhesion assembly reduces cell adhesion to vitronectin in the presence of PAI-1 to levels similar to that observed for the binding of purified integrins to vitronectin. Furthermore, endothelial cell, but not smooth muscle cell adhesion to vitronectin in the presence of PAI-1 requires both polymerized microtubules and actin, further demonstrating the importance of the cytoskeleton for integrin-mediated adhesion. Finally, we show that cell adhesion in the presence of PAI-1 leads to colocalization of PAI-1 with the integrins ␣v3 and ␣v5 at the cell-matrix interface.Cell adhesion receptors play important roles in maintaining cell anchorage and polarity in addition to promoting migration and differentiation. Many of the known adhesion receptors belong to the integrin family of noncovalent heterodimeric transmembrane proteins that are expressed by most cell types. The integrin family consists of at least 18 ␣-and 8 -subunits that combine to form as many as 24 different ␣ dimers (1). The extracellular domains of both integrin subunits are required for binding to adhesion proteins. In addition, integrins need physiological concentrations of divalent cations such as calcium or magnesium. Integrins have relatively short cytoplasmic tails that interact with an intracellular protein complex, termed focal contacts, which include adaptor proteins and kinases. The focal contacts control the linkage between the cytoskeleton and integrins and mediate intracellular signaling. Integrins direct forces generated by the cytoskeleton onto the extracellular matrix (ECM) 2 to produce the traction necessary for cell adhesion and migration (2). Understanding the mechanism and control of integrin-mediated cell adhesion and migration is of special interest because of their importance in processes such as wound healing and angiogenesis as well as pathologies associated with these processes.In general, integrins recognize short linear peptide sequences on adhesion proteins, the most prevalent being arginine-glycine-aspartic acid (RGD), which is found in many ECM proteins such as fibronectin, collagens, ...
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