Proteomics-based discovery of a novel, structurally unique, and developmentally regulated plasminogen receptor, Plg-RKT, a major regulator of cell surface plasminogen activation

Abstract

Activation of plasminogen, the zymogen of the primary thrombolytic enzyme, plasmin, is markedly promoted when plasminogen is bound to cell surfaces, arming cells with the broad spectrum proteolytic activity of plasmin. In addition to its role in thrombolysis, cell surface plasmin facilitates a wide array of physiologic and pathologic processes. Carboxypeptidase B-sensitive plasminogen binding sites promote plasminogen activation on eukaryotic cells. However, no integral membrane plasminogen receptors exposing carboxyl terminal basic residues on cell surfaces have been identified. Here we use the exquisite sensitivity of multidimensional protein identification technology and an inducible progenitor cell line to identify a novel differentiation-induced integral membrane plasminogen receptor that exposes a C-terminal lysine on the cell surface, Plg-R KT (C9orf46 homolog). Plg-R KT was highly colocalized on the cell surface with the urokinase receptor, uPAR.Our data suggest that Plg-R KT also interacts directly with tissue plasminogen activator. Furthermore, Plg-R KT IntroductionLocalization of plasminogen on cell surfaces is a crucial control point for positive regulation of cell surface plasmin proteolytic activity that facilitates both physiologic and pathologic processes, 1,2 including macrophage recruitment during the inflammatory response, [3][4][5][6] tissue remodeling, 7 wound healing, 8,9 tumor cell invasion and metastasis, 10-12 skeletal myogenesis, 13 neuroendocrine prohormone processing, 14,15 and neurite outgrowth. 16,17 Cell surface plasminogen binding sites promote plasminogen activation by reducing the Km (11-to 60-fold) for plasminogen activation. [18][19][20][21][22][23][24] Active plasmin also associates with the cell surface, where its activity is protected from inhibitors. 25,26 Plasminogen binding sites are very broadly distributed on both eukaryotic and prokaryotic cells. 27 Of the many eukaryotic cells examined to date, only erythrocytes do not bind plasminogen. 28 The interactions of plasminogen with eukaryotic cells are mediated by lysine binding sites within the disulfide-bonded kringle domains of plasminogen. 18,29 Therefore, plasminogen binding to eukaryotic cells is blocked in the presence of lysine and lysine analogs, including ⑀-aminocaproic acid (EACA). 27 Because most cell types have a very high capacity for plasminogen, no single molecule can account for the entire plasminogen binding capacity of a given cell type. 27 However, a subset of plasminogen binding proteins exposing C-terminal basic residues on cell surfaces are predominantly responsible for the ability of eukaryotic cells to enhance plasminogen activation because carboxypeptidase B (CpB) treatment abrogates cell surface-dependent plasminogen activation. 24 Correspondingly, plasminogen-dependent macrophage recruitment in vivo is mediated by CpB-sensitive plasminogen receptors, and plasminogen binding to recruited macrophages is increased, compared with peripheral blood monocytes. 6,30 Therefore, we probed the monocyte ...

Monoclonal antibodies detect receptor-induced binding sites in Glu-plasminogen

“…We have designated these Abs as anti-Pg-RIBS mAbs, according to the terminology of Zamarron and colleagues. 9 To further address the relationships between changes in Glu-Pg conformation and cellular binding sites, we found that Glu-Pg binding to the C-terminal peptide of the recently described Pg receptor, Plg-R KT , 18 was detected with the anti-Pg RIBS mAbs and the binding was not inhibited in the presence of soluble Glu-Pg. Therefore, binding of Glu-Pg to the C-terminus of Plg-R KT mimicked the interaction of Glu-Pg with the cell surface, with respect to conformational changes detected by the anti-Pg RIBS mAbs.…”

“…Thus, in the presence of EACA, soluble Glu-Pg exposes an epitope that is also exposed when Glu-Pg is associated with cells, consistent with binding of Glu-Pg to cellsurface proteins exposing C-terminal lysines, as exemplified by Plg-R KT . 18,38 These anti-Pg RIBS mAbs have many potential uses for future investigations of structure/function relationships in the field of plasminogen activation. For example, they could be of great use in imaging studies of cell-or fibrin-associated Pg.…”

“…9 Reaction of anti-Pg RIBS mAbs with Glu-Pg bound to substrates and regulatory molecules Glu-Pg interacts with the C-terminus of the Pg receptor, Plg-R KT . 15,18 Therefore, we examined whether its interaction with the Plg-R KT C-terminus would mimic the effect of the interaction of Glu-Pg with cells with respect to induction of neoepitope expression. Glu-Pg binding to the C-terminal peptide of Plg-R KT , was detected with representative anti-Pg RIBS mAbs from each epitope group and the binding was not inhibited in the presence of soluble Glu-Pg ( Figure 3A).…”

Urinary Plasmin Activates Collecting Duct ENaC Current in Preeclampsia

“…An integral membrane protein plasminogen receptor, Plg-R KT , has been identified. 32 Plg-R KT promotes activation of plasminogen and colocalizes with urokinase-type plasminogen activator receptor (uPAR). 32,33 Urokinase-type plasminogen activator is abundant in urine and requires activation by uPAR.…”

“…32 Plg-R KT promotes activation of plasminogen and colocalizes with urokinase-type plasminogen activator receptor (uPAR). 32,33 Urokinase-type plasminogen activator is abundant in urine and requires activation by uPAR. The present data show urinary excretion of soluble uPAR at similar levels between PE patients and healthy pregnant women.…”