scholarly journals The Plasminogen Receptor, , and Macrophage Function

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Lindsey A. Miles ◽  
Shahrzad Lighvani ◽  
Nagyung Baik ◽  
Nicholas M. Andronicos ◽  
Emily I. Chen ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Cell Surface ◽  
Protein Identification ◽  
Transmembrane Protein ◽  
Membrane Topology ◽  
Plasminogen Activation ◽  
Macrophage Recruitment ◽  
Multidimensional Protein Identification Technology ◽  
C Terminus ◽  
Proteomics Approach

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, . 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.

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

Blood ◽  
2010 ◽  
Vol 115 (7) ◽  
pp. 1319-1330 ◽  
Author(s):  
Nicholas M. Andronicos ◽  
Emily I. Chen ◽  
Nagyung Baik ◽  
Hongdong Bai ◽  
Caitlin M. Parmer ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Identification ◽  
Control Point ◽  
Cell Types ◽  
Plasminogen Activation ◽  
Cell Surfaces ◽  
Developmentally Regulated ◽  
Carboxypeptidase B ◽  
Multidimensional Protein Identification Technology ◽  
Pathologic Processes

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-RKT (C9orf46 homolog). Plg-RKT was highly colocalized on the cell surface with the urokinase receptor, uPAR. Our data suggest that Plg-RKT also interacts directly with tissue plasminogen activator. Furthermore, Plg-RKT markedly promoted cell surface plasminogen activation. Database searching revealed that Plg-RKT 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.

scholarly journals Regulation of macrophage migration by a novel plasminogen receptor Plg-RKT

Blood ◽  
2011 ◽  
Vol 118 (20) ◽  
pp. 5622-5630 ◽  
Author(s):  
Shahrzad Lighvani ◽  
Nagyung Baik ◽  
Jenna E. Diggs ◽  
Sophia Khaldoyanidi ◽  
Robert J. Parmer ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Human Peripheral Blood ◽  
Plasminogen Activation ◽  
Blood Monocytes ◽  
Chemotactic Migration ◽  
Peripheral Blood Monocytes ◽  
Macrophage Recruitment ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract 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-RKT, which exposes a C-terminal lysine on the cell surface. In the present study, we investigated the role of Plg-RKT in macrophage invasion, chemotactic migration, and recruitment. Plg-RKT 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-RKT mAb. Treatment of monocytes with anti–Plg-RKT 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-RKT mAb (by 64%). In a mouse model of thioglycollate-induced peritonitis, anti–Plg-RKT mAb markedly inhibited macrophage recruitment (by 58%), concomitant with a reduction in pro–MMP-9 activation in the inflamed peritoneum. Treatment with anti–Plg-RKT mAb did not further reduce the low level of macrophage recruitment in plasminogen-null mice. We conclude that Plg-RKT plays a key role in the plasminogen-dependent regulation of macrophage invasion, chemotactic migration, and recruitment in the inflammatory response.

scholarly journals Exploring the membrane topology of prohormone convertase 1 in AtT20 Cells: in situ analysis by immunofluorescence microscopy

F1000Research ◽  
2012 ◽  
Vol 1 ◽  
pp. 9
Author(s):  
Niamh X Cawley ◽  
Meera Sridhar ◽  
Hong Hong ◽  
Peng Loh
Keyword(s):  
Endocrine Cell ◽  
Transmembrane Protein ◽  
Membrane Topology ◽  
Amino Terminus ◽  
Prohormone Convertase ◽  
C Terminus ◽  
Prohormone Convertase 1 ◽  
Extraction Properties

Prohormone convertase 1 (PC1) was previously characterized as a partially transmembrane protein in purified chromaffin granules of bovine adrenal medulla1. This was challenged with experiments on transfected PC1 in COS1 cells, a non-endocrine cell line2. To address this issue, we undertook to analyze its extraction properties in vitro and its immunocytochemical localization in situ in AtT20 cells, an endocrine cell line that expresses PC1. Most of the 87 kDa form of PC1 was resistant to carbonate extraction suggesting that it had properties of a transmembrane protein. Under semi-permeabilized conditions whereby only the plasma membrane was permeabilized, the carboxy-terminus of PC1 was specifically immunostained whereas the amino-terminus was not. These results indicate that the amino-terminus of PC1 was within the lumen of the Golgi and granules, and some of the C-terminus was exposed to the cytosol. Thus, endogenous PC1 can assume a transmembrane orientation in situ in AtT20 cells.

scholarly journals Effect of Extension of the Cytoplasmic Domain of Human Immunodeficiency Type 1 Virus Transmembrane Protein gp41 on Virus Replication

2004 ◽  
Vol 78 (10) ◽  
pp. 5157-5169 ◽  
Author(s):  
Woan-Eng Chan ◽  
Ya-Lin Wang ◽  
Hui-Hua Lin ◽  
Steve S.-L. Chen
Keyword(s):  
Cell Surface ◽  
Virus Replication ◽  
Transmembrane Protein ◽  
Cytoplasmic Tail ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Virus Infectivity ◽  
Gag Protein ◽  
C Terminus ◽  
Protein Gp41

ABSTRACT The biological significance of the presence of a long cytoplasmic domain in the envelope (Env) transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1) is still not fully understood. Here we examined the effects of cytoplasmic tail elongation on virus replication and characterized the role of the C-terminal cytoplasmic tail in interactions with the Gag protein. Extensions with six and nine His residues but not with fewer than six His residues were found to severely inhibit virus replication through decreased Env electrophoretic mobility and reduced Env incorporation compared to the wild-type virus. These two mutants also exhibited distinct N glycosylation and reduced cell surface expression. An extension of six other residues had no deleterious effect on infectivity, even though some mutants showed reduced Env incorporation into the virus and/or decreased cell surface expression. We further show that these elongated cytoplasmic tails in a format of the glutathione S-transferase fusion protein still interacted effectively with the Gag protein. In addition, the immediate C terminus of the cytoplasmic tail was not directly involved in interactions with Gag, but the region containing the last 13 to 43 residues from the C terminus was critical for Env-Gag interactions. Taken together, our results demonstrate that HIV-1 Env can tolerate extension at its C terminus to a certain degree without loss of virus infectivity and Env-Gag interactions. However, extended elongation in the cytoplasmic tail may impair virus infectivity, Env cell surface expression, and Env incorporation into the virus.

scholarly journals Sequence Requirements for Trafficking of the CRAM Transmembrane Protein to the Flagellar Pocket of African Trypanosomes

2000 ◽  
Vol 20 (14) ◽  
pp. 5149-5163 ◽  
Author(s):  
Hong Yang ◽  
David G. Russell ◽  
Baijing Zheng ◽  
Manami Eiki ◽  
Mary Gwo-Shu Lee
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Surface Glycoprotein ◽  
Cell Surface Expression ◽  
Flagellar Pocket ◽  
African Trypanosomes ◽  
Cytoplasmic Extension ◽  
C Terminus

ABSTRACT CRAM is a cysteine-rich acidic transmembrane protein, highly expressed in the procyclic form of Trypanosoma brucei. Cell surface expression of CRAM is restricted to the flagellar pocket of trypanosomes, the only place where receptor mediated endocytosis takes place in the parasite. CRAM can function as a receptor and was hypothesized to be a lipoprotein receptor of trypanosomes. We study mechanisms involved in the presentation and routing of CRAM to the flagellar pocket of insect- and bloodstream-form trypanosomes. By deletional mutagenesis, we found that deleting up to four amino acids from the C terminus of CRAM did not affect the localization of CRAM at the flagellar pocket. Shortening the CRAM protein by 8 and 19 amino acids from the C terminus resulted in the distribution of the CRAM protein in the endoplasmic reticulum (ER) (the CRAM protein is no longer uniquely sequestered at the flagellar pocket). This result indicates that the truncation of the CRAM C terminus affected the transport efficiency of CRAM from the ER to the flagellar pocket. However, when CRAM was truncated between 29 and 40 amino acids from the C terminus, CRAM was not only distributed in the ER but also located to the flagellar pocket and spread to the cell surface and the flagellum. Replacing the CRAM transmembrane domain with the invariant surface glycoprotein 65-derived transmembrane region did not affect the flagellar pocket location of CRAM. These results indicate that the CRAM cytoplasmic extension may exhibit two functional domains: one domain near the C terminus is important for efficient export of CRAM from the ER, while the second domain is of importance for confining CRAM to the flagellar pocket membrane.

scholarly journals The N Terminus of the Transmembrane Protein BP180 Interacts with the N-terminal Domain of BP230, Thereby Mediating Keratin Cytoskeleton Anchorage to the Cell Surface at the Site of the Hemidesmosome

2000 ◽  
Vol 11 (1) ◽  
pp. 277-286 ◽  
Author(s):  
Susan B. Hopkinson ◽  
Jonathan C. R. Jones
Keyword(s):  
Cell Surface ◽  
Bullous Pemphigoid ◽  
Transmembrane Protein ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus ◽  
Α6β4 Integrin ◽  
Two Hybrid ◽  
Insight Into

In epidermal cells, the keratin cytoskeleton interacts with the elements in the basement membrane via a multimolecular junction called the hemidesmosome. A major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid autoantigen (BP230/BPAG1), which connects directly to the keratin-containing intermediate filaments of the cytoskeleton via its C terminus. A second bullous pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component of the hemidesmosome. Using yeast two-hybrid technology and recombinant proteins, we show that an N-terminal fragment of BP230 can bind directly to an N-terminal fragment of BP180. We have also explored the consequences of expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in vitro. Unexpectedly, this fragment disrupts the distribution of BP180 in transfected cells but has no apparent impact on the organization of endogenous BP230 and α6β4 integrin. We propose that the BP230 N terminus competes with endogenous BP230 protein for BP180 binding and inhibits incorporation of BP180 into the cell surface at the site of the hemidesmosome. These data provide new insight into those interactions of the molecules of the hemidesmosome that are necessary for its function in integrating epithelial and connective tissue types.

Mechanisms of Signaling through Urokinase Receptor and the Cellular Response

1999 ◽  
Vol 82 (08) ◽  
pp. 305-311 ◽  
Author(s):  
Yuri Koshelnick ◽  
Monika Ehart ◽  
Hannes Stockinger ◽  
Bernd Binder
Keyword(s):  
Cell Surface ◽  
Proteolytic Activity ◽  
Tumor Invasion ◽  
Cellular Response ◽  
Transmembrane Protein ◽  
Urokinase Receptor ◽  
Biological Processes ◽  
In Vivo Models ◽  
Proteolytic Activation ◽  
Core Proteins

IntroductionThe urokinase-urokinase receptor (u-PA-u-PAR) system seems to play a crucial role in a number of biological processes, including local fibrinolysis, tumor invasion, angiogenesis, neointima and atherosclerotic plaque formation, inflammation, and matrix remodeling during wound healing and development.1-6 Binding of urokinase to its specific receptor provides cells with a localized proteolytic potential. It stimulates conversion of cell surface-bound plasminogen into active plasmin, which, in turn, is required for proteolytic degradation of basement membrane components, including fibronectin, collagen, laminin, and proteoglycan core proteins.7 Moreover, plasmin activates other matrix-degrading enzymes, such as matrix metalloproteinases.8 Overexpression of u-PA/u-PAR correlates with tumor invasion and metastasis formation,9-13 while reduction of cell-surface bound u-PA and inhibition of u-PAR expression leads to a significant decrease of invasive and metastatic activity.14 Specific antagonists that suppress binding of u-PA to u-PAR have been shown to inhibit cell-surface plasminogen activation, tumor growth, and angiogenesis both in vitro and in vivo models.15,16 Independently of its proteolytic activity, u-PA is implicated in many biological processes that seem to require u-PAR-mediated intracellular signal transduction, such as proliferation, chemotactic movement and adhesion, migration, and differentiation.17 Data obtained in the late 1980s indicated that u-PA not only provides cells with local proteolytic activity, but might also be capable of transducing signals to the cell.18-22 At that time, however, the u-PAR has just been isolated, cloned, and identified as a glycosylphosphatidylinositol (GPI)-linked protein and not a transmembrane protein. Signaling via the u-PAR was, therefore, regarded as being unlikely, and the effects of u-PA on cell proliferation18-22 were thought to be mediated by proteolytic activation of latent growth factors. The assumption of direct signaling via u-PAR was, in fact, considered controversial, until about 10 years later when a physical association between u-PAR and signaling proteins was found.23 From this report on, several proteins associated with u-PAR have been identified. Now, u-PAR seems to be part of a large “signalosome” associated and interacting with several proteins on both the outside and inside of the cell.

scholarly journals Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Andreia Peixoto ◽  
Dylan Ferreira ◽  
Rita Azevedo ◽  
Rui Freitas ◽  
Elisabete Fernandes ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cell Surface ◽  
Protein Identification ◽  
Glucose Deprivation ◽  
Invasive Bladder Cancer ◽  
Nutrient Deprivation ◽  
Molecular Heterogeneity ◽  
Cell Models ◽  
Membrane Expression ◽  
Bladder Tumours

Abstract Background Muscle invasive bladder cancer (MIBC) remains amongst the deadliest genitourinary malignancies due to treatment failure and extensive molecular heterogeneity, delaying effective targeted therapeutics. Hypoxia and nutrient deprivation, oversialylation and O-glycans shortening are salient features of aggressive tumours, creating cell surface glycoproteome fingerprints with theranostics potential. Methods A glycomics guided glycoproteomics workflow was employed to identify potentially targetable biomarkers using invasive bladder cancer cell models. The 5637 and T24 cells O-glycome was characterized by mass spectrometry (MS), and the obtained information was used to guide glycoproteomics experiments, combining sialidase, lectin affinity and bottom-up protein identification by nanoLC-ESI-MS/MS. Data was curated by a bioinformatics approach developed in-house, sorting clinically relevant molecular signatures based on Human Protein Atlas insights. Top-ranked targets and glycoforms were validated in cell models, bladder tumours and metastases by MS and immunoassays. Cells grown under hypoxia and glucose deprivation disclosed the contribution of tumour microenvironment to the expression of relevant biomarkers. Cancer-specificity was validated in healthy tissues by immunohistochemistry and MS in 20 types of tissues/cells of different individuals. Results Sialylated T (ST) antigens were found to be the most abundant glycans in cell lines and over 900 glycoproteins were identified potentially carrying these glycans. HOMER3, typically a cytosolic protein, emerged as a top-ranked targetable glycoprotein at the cell surface carrying short-chain O-glycans. Plasma membrane HOMER3 was observed in more aggressive primary tumours and distant metastases, being an independent predictor of worst prognosis. This phenotype was triggered by nutrient deprivation and concomitant to increased cellular invasion. T24 HOMER3 knockdown significantly decreased proliferation and, to some extent, invasion in normoxia and hypoxia; whereas HOMER3 knock-in increased its membrane expression, which was more pronounced under glucose deprivation. HOMER3 overexpression was associated with increased cell proliferation in normoxia and potentiated invasion under hypoxia. Finally, the mapping of HOMER3-glycosites by EThcD-MS/MS in bladder tumours revealed potentially targetable domains not detected in healthy tissues. Conclusion HOMER3-glycoforms allow the identification of patients’ subsets facing worst prognosis, holding potential to address more aggressive hypoxic cells with limited off-target effects. The molecular rationale for identifying novel bladder cancer molecular targets has been established. Graphical abstract

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