Miles Lindsey A, Andronicos Nicholas M, Baik Nagyung, Parmer Robert J
Department of Cell Biology, Division of Vascular Biology, The Scripps Research Institute, La Jolla, California, 92037, USA.
J Neurosci. 2006 Dec 13;26(50):13017-24. doi: 10.1523/JNEUROSCI.2070-06.2006.
An emerging area of research has documented a novel role for the plasminogen activation system in the regulation of neurotransmitter release. Prohormones, secreted by cells within the sympathoadrenal system, are processed by plasmin to bioactive peptides that feed back to inhibit secretagogue-stimulated release. Catecholaminergic cells of the sympathoadrenal system are prototypic prohormone-secreting cells. Processing of prohormones by plasmin is enhanced in the presence of catecholaminergic cells, and the enhancement requires binding of plasmin(ogen) to cellular receptors. Consequently, modulation of the local cellular fibrinolytic system of catecholaminergic cells results in substantial changes in catecholamine release. However, mechanisms for enhancing prohormone processing and cell-surface molecules mediating the enhancement on catecholaminergic cells have not been investigated. Here we show that plasminogen activation was enhanced >6.5-fold on catecholaminergic cells. Carboxypeptidase B treatment decreased cell-dependent plasminogen activation by approximately 90%, suggesting that the binding of plasminogen to proteins exposing C-terminal lysines on the cell surface is required to promote plasminogen activation. We identified catecholaminergic plasminogen receptors required for enhancing plasminogen activation, using a novel strategy combining targeted specific proteolysis using carboxypeptidase B with a proteomics approach using two-dimensional gel electrophoresis, radioligand blotting, and tandem mass spectrometry. Two major plasminogen-binding proteins that exposed C-terminal lysines on the cell surface contained amino acid sequences corresponding to beta/gamma-actin. An anti-actin monoclonal antibody inhibited cell-dependent plasminogen activation and also enhanced nicotine-dependent catecholamine release. Our results suggest that cell-surface-expressed forms of actin bind plasminogen, thereby promoting plasminogen activation and increased prohormone processing leading to inhibition of neurotransmitter release.
一个新兴的研究领域已证明纤溶酶原激活系统在调节神经递质释放方面具有新作用。交感肾上腺系统内的细胞分泌的激素原，经纤溶酶加工成生物活性肽，这些肽会反馈抑制促分泌剂刺激的释放。交感肾上腺系统的儿茶酚胺能细胞是典型的分泌激素原的细胞。在儿茶酚胺能细胞存在的情况下，纤溶酶对激素原的加工会增强，且这种增强需要纤溶酶（原）与细胞受体结合。因此，调节儿茶酚胺能细胞的局部细胞纤溶系统会导致儿茶酚胺释放发生显著变化。然而，尚未研究增强激素原加工的机制以及介导儿茶酚胺能细胞上这种增强作用的细胞表面分子。在此我们表明，儿茶酚胺能细胞上的纤溶酶原激活增强了6.5倍以上。羧肽酶B处理使细胞依赖性纤溶酶原激活降低了约90%，这表明纤溶酶原与细胞表面暴露C末端赖氨酸的蛋白质结合是促进纤溶酶原激活所必需的。我们采用一种新颖的策略，将使用羧肽酶B的靶向特异性蛋白水解与使用二维凝胶电泳、放射性配体印迹和串联质谱的蛋白质组学方法相结合，确定了增强纤溶酶原激活所需的儿茶酚胺能纤溶酶原受体。两种在细胞表面暴露C末端赖氨酸的主要纤溶酶原结合蛋白含有与β/γ-肌动蛋白相对应的氨基酸序列。一种抗肌动蛋白单克隆抗体抑制细胞依赖性纤溶酶原激活，还增强了尼古丁依赖性儿茶酚胺释放。我们的结果表明，细胞表面表达的肌动蛋白形式结合纤溶酶原，从而促进纤溶酶原激活并增加激素原加工，导致神经递质释放受到抑制。
