Nagyung Baik

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

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) markedly promoted cell surface plasminogen activation. Database searching revealed that Plg-R(KT) mRNA is broadly expressed by migratory cell types, including leukocytes, and breast cancer, leukemic, and neuronal cells. This structurally unique plasminogen receptor represents a novel control point for regulating cell surface proteolysis.

Regulation of macrophage migration by a novel plasminogen receptor Plg-R KT.

Localization of plasmin on macrophages and activation of pro-MMP-9 play key roles in macrophage recruitment in the inflammatory response. These functions are promoted by plasminogen receptors exposing C-terminal basic residues on the macrophage surface. Recently, we identified a novel transmembrane plasminogen receptor, Plg-R(KT), which exposes a C-terminal lysine on the cell surface. In the present study, we investigated the role of Plg-R(KT) in macrophage invasion, chemotactic migration, and recruitment. Plg-R(KT) was prominently expressed in membranes of human peripheral blood monocytes and monocytoid cells. Plasminogen activation by urokinase-type plasminogen activator (uPA) was markedly inhibited (by 39%) by treatment with anti-Plg-R(KT) mAb. Treatment of monocytes with anti-Plg-R(KT) mAb substantially inhibited invasion through the representative matrix, Matrigel, in response to MCP-1 (by 54% compared with isotype control). Furthermore, chemotactic migration was also inhibited by treatment with anti-Plg-R(KT) mAb (by 64%). In a mouse model of thioglycollate-induced peritonitis, anti-Plg-R(KT) mAb markedly inhibited macrophage recruitment (by 58%), concomitant with a reduction in pro-MMP-9 activation in the inflamed peritoneum. Treatment with anti-Plg-R(KT) mAb did not further reduce the low level of macrophage recruitment in plasminogen-null mice. We conclude that Plg-R(KT) plays a key role in the plasminogen-dependent regulation of macrophage invasion, chemotactic migration, and recruitment in the inflammatory response.

Cell-Surface Actin Binds Plasminogen and Modulates Neurotransmitter Release from Catecholaminergic Cells

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 ∼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 β/γ-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.

The Novel Plasminogen Receptor, Plasminogen ReceptorKT (Plg-RKT), Regulates Catecholamine Release

Neurotransmitter release by catecholaminergic cells is negatively regulated by prohormone cleavage products formed from plasmin-mediated proteolysis. Here, we investigated the expression and subcellular localization of Plg-RKT, a novel plasminogen receptor, and its role in catecholaminergic cell plasminogen activation and regulation of catecholamine release. Prominent staining with anti-Plg-RKT mAb was observed in adrenal medullary chromaffin cells in murine and human tissue. In Western blotting, Plg-RKT was highly expressed in bovine adrenomedullary chromaffin cells, human pheochromocytoma tissue, PC12 pheochromocytoma cells, and murine hippocampus. Expression of Plg-RKT fused in-frame to GFP resulted in targeting of the GFP signal to the cell membrane. Phase partitioning, co-immunoprecipitation with urokinase-type plasminogen activator receptor (uPAR), and FACS analysis with antibody directed against the C terminus of Plg-RKT were consistent with Plg-RKT being an integral plasma membrane protein on the surface of catecholaminergic cells. Cells stably overexpressing Plg-RKT exhibited substantial enhancement of plasminogen activation, and antibody blockade of non-transfected PC12 cells suppressed plasminogen activation. In functional secretion assays, nicotine-evoked [3H]norepinephrine release from cells overexpressing Plg-RKT was markedly decreased (by 51 ± 2%, p < 0.001) when compared with control transfected cells, and antibody blockade increased [3H]norepinephrine release from non-transfected PC12 cells. In summary, Plg-RKT is present on the surface of catecholaminergic cells and functions to stimulate plasminogen activation and modulate catecholamine release. Plg-RKT thus represents a new mechanism and novel control point for regulating the interface between plasminogen activation and neurosecretory cell function.

The Plasminogen Receptor, Plg-R KT , and Macrophage Function

When plasminogen binds to cells its activation to plasmin is markedly enhanced compared to the reaction in solution. Thus, cells become armed with the broad spectrum proteolytic activity of plasmin. Cell-surface plasmin plays a key role in macrophage recruitment during the inflammatory response. Proteins exposing basic residues on the cell surface promote plasminogen activation on eukaryotic cells. We have used a proteomics approach combining targeted proteolysis with carboxypeptidase B and multidimensional protein identification technology, MudPIT, and a monocyte progenitor cell line to identify a novel transmembrane protein, the plasminogen receptor, Plg-RKT. Plg-RKT exposes a C-terminal lysine on the cell surface in an orientation to bind plasminogen and promote plasminogen activation. Here we review the characteristics of this new protein, with regard to membrane topology, conservation of sequence across species, the role of its C-terminus in plasminogen binding, its function in plasminogen activation, cell migration, and its role in macrophage recruitment in the inflammatory response.

New Insights into the Role of Plg-RKT in Macrophage Recruitment

Plasminogen (PLG) is the zymogen of plasmin, the major enzyme that degrades fibrin clots. In addition to its binding and activation on fibrin clots, PLG also specifically interacts with cell surfaces where it is more efficiently activated by PLG activators, compared with the reaction in solution. This results in association of the broad-spectrum proteolytic activity of plasmin with cell surfaces that functions to promote cell migration. Here, we review emerging data establishing a role for PLG, plasminogen receptors and the newly discovered plasminogen receptor, Plg-RKT, in macrophage recruitment in the inflammatory response, and we address mechanisms by which the interplay between PLG and its receptors regulates inflammation.

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

When Glu-plasminogen binds to cells, its activation to plasmin is markedly enhanced compared with the reaction in solution, suggesting that Glu-plasminogen on cell surfaces adopts a conformation distinct from that in solution. However, direct evidence for such conformational changes has not been obtained. Therefore, we developed anti-plasminogen mAbs to test the hypothesis that Glu-plasminogen undergoes conformational changes on its interaction with cells. Six anti-plasminogen mAbs (recognizing 3 distinct epitopes) that preferentially recognized receptor-induced binding sites (RIBS) in Glu-plasminogen were obtained. The mAbs also preferentially recognized Glu-plasminogen bound to the C-terminal peptide of the plasminogen receptor, Plg-R(KT), and to fibrin, plasmin-treated fibrinogen, and Matrigel. We used trypsin proteolysis, immunoaffinity chromatography, and tandem mass spectrometry and identified Glu-plasminogen sequences containing epitopes recognized by the anti-plasminogen-RIBS mAbs: a linear epitope within a domain linking kringles 1 and 2; a nonlinear epitope contained within the kringle 5 domain and the latent protease domain; and a nonlinear epitope contained within the N-terminal peptide of Glu-plasminogen and the latent protease domain. Our results identify neoepitopes latent in soluble Glu-plasminogen that become available when Glu-plasminogen binds to cells and demonstrate that binding of Glu-plasminogen to cells induces a conformational change in Glu-plasminogen distinct from that of Lys-Pg.

Deficiency of Plasminogen Receptor, Plg‐RKT, Causes Defects in Plasminogen Binding and Inflammatory Macrophage Recruitment in vivo

Essentials Plg‐RKT is a novel integral membrane plasminogen receptor. The functions of Plg‐RKT in vivo are not known. Plg‐RKT is a key player in macrophage recruitment in the inflammatory response in vivo. Plg‐RKT deficiency is not compatible with survival of the species.

The plasminogen receptor, Plg-RKT , is essential for mammary lobuloalveolar development and lactation

Essentials Plg‐RKT‐/− female mice give birth, but no offspring of Plg‐RKT−/− female mice survive to weaning. Causal mechanisms of potential lactational failure in Plg‐RKT−/− mice are unknown. Plg‐RKT regulates extracellular matrix remodeling, cell proliferation, apoptosis, fibrin surveillance. Plg‐RKT is essential for lactogenesis and mammary lobuloalveolar development.