RESUMO
PURPOSE: To evaluate the plasma concentration of the soluble form of the urokinase-type plasminogen activator receptor ((s)uPAR), an established biomarker of chronic inflammation, in patients affected by neovascular age-related macular degeneration. METHODS: Forty consecutive patients affected by age-related macular degeneration and 52 subjects with no history of the disease were included in this case-control study. The two groups of individuals considered for the study were matched for age, sex, and class of medications taken. Plasma concentration of suPAR was measured using a specific ELISA assay (suPARnostic, Birkeroed, Denmark). RESULTS: The case and control groups were similar for age, gender distribution, weight, height, and systolic and diastolic blood pressure, as well as for dyslipidemia and high blood pressure medication (P > 0.28). The plasma concentrations of suPAR were significantly increased in patients with neovascular age-related macular degeneration when compared to controls (6.19 ± 2.2 ng/ml, vs 5.21 ± 1.5, respectively, mean ± SD P = 0.01). CONCLUSIONS: Patients with neovascular age-related macular degeneration display increased plasma levels of suPAR, suggesting that chronic inflammation may be involved in the pathogenesis of the disease.
Assuntos
Inflamação/sangue , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Degeneração Macular Exsudativa/sangue , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Angiofluoresceinografia , Fundo de Olho , Humanos , Inflamação/diagnóstico , Macula Lutea/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Tomografia de Coerência Óptica , Degeneração Macular Exsudativa/diagnósticoRESUMO
The urokinase-type plasminogen activator receptor (uPAR) is a non-integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure-function analyses of uPAR, VN and integrins, we document that uPAR-mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin-matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non-canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non-integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch.
Assuntos
Integrinas/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais/fisiologia , Vitronectina/metabolismo , Adesão Celular/fisiologia , Células HEK293 , Humanos , Integrinas/genética , Mutação , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Vitronectina/genéticaRESUMO
Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.
Assuntos
Motivos de Aminoácidos , Adesão Celular , Plasminogênio/metabolismo , Domínios e Motivos de Interação entre Proteínas , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vitronectina/química , Vitronectina/metabolismo , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Fibrinolisina/metabolismo , Fibronectinas/metabolismo , Expressão Gênica , Humanos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ligação Proteica , Proteólise , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Extensive evidence implicates the urokinase plasminogen activator receptor (uPAR) in tumor growth, invasion, and metastasis. Recent studies have substantiated the importance of the interaction between uPAR and the extracellular matrix protein vitronectin (VN) for the signaling activity of the receptor in vitro, however, the possible relevance of this interaction for the activity of uPAR in tumor growth and metastasis has not been assessed. We generated a panel of HEK293 cell lines expressing mouse uPAR (muPAR(WT)), an uPAR mutant specifically deficient in VN binding (muPAR(W32A)), and a truncation variant (muPAR(ΔD1)) deficient in both VN and uPA binding. In vitro cells expressing muPAR(WT) display increased cell adhesion, spreading, migration, and proliferation associated with increased p130Cas and MAPK signaling. Disruption of VN binding or ablation of both VN and uPA binding specifically abrogates these activities of uPAR. When xenografted into SCID (severe combined immunodeficiency) mice, the expression of muPAR(WT), but not muPAR(W32A) or muPAR(ΔD1), accelerates tumor development, demonstrating that VN binding is responsible for the tumor-promoting activity of uPAR in vivo. In an orthotopic xenograft model using MDA-MB-231 cells in RAG1(-/-)/VN(-/-) mice, we document that host deficiency in VN strongly impairs tumor formation. These 2 lines of in vivo experimentation independently demonstrate an important role for VN in tumor growth even if the uPAR dependence of the effect in the MDA-MB-231 model remains to be ascertained.
Assuntos
Proliferação de Células , Neoplasias/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vitronectina/metabolismo , Vitronectina/fisiologia , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Transplante de Neoplasias , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Transplante Heterólogo , Carga Tumoral/genética , Vitronectina/genéticaRESUMO
The urokinase plasminogen activator receptor (uPAR) is a cell surface receptor involved in a multitude of physiologic and pathologic processes. uPAR regulates simultaneously a branch of the plasminogen activator system and modulates cell adhesion and intracellular signaling by interacting with extracellular matrix components and signaling receptors. The multiple uPAR functions are deeply interconnected, and their integration determines the effects that uPAR expression triggers in different contexts. The proteolytic function of uPAR affects both the signaling and the adhesive functions of the receptor, whereas these latter two are closely interconnected. This review focuses on the molecular mechanisms that connect and mutually regulate the different uPAR functions.
Assuntos
Adesão Celular/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Animais , Humanos , Proteólise , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de SinaisRESUMO
We studied the molecular forms of the GPI-anchored urokinase plasminogen activator receptor (uPAR-mEGFP) in the human embryo kidney (HEK293) cell membrane and demonstrated that the binding of the amino-terminal fragment (ATF) of urokinase plasminogen activator is sufficient to induce the dimerization of the receptor. We followed the association kinetics and determined precisely the dimeric stoichiometry of uPAR-mEGFP complexes by applying number and brightness (N&B) image analysis. N&B is a novel fluctuation-based approach for measuring the molecular brightness of fluorophores in an image time sequence in live cells. Because N&B is very sensitive to long-term temporal fluctuations and photobleaching, we have introduced a filtering protocol that corrects for these important sources of error. Critical experimental parameters in N&B analysis are illustrated and analyzed by simulation studies. Control experiments are based on mEGFP-GPI, mEGFP-mEGFP-GPI, and mCherry-GPI, expressed in HEK293. This work provides a first direct demonstration of the dimerization of uPAR in live cells. We also provide the first methodological guide on N&B to discern minor changes in molecular composition such as those due to dimerization events, which are involved in fundamental cell signaling mechanisms.
Assuntos
Membrana Celular/metabolismo , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência/métodos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Algoritmos , Simulação por Computador , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Immunoblotting , Microscopia de Fluorescência/instrumentação , Multimerização Proteica , Reprodutibilidade dos Testes , Vitronectina/metabolismoRESUMO
Expression of the membrane receptor uPAR induces profound changes in cell morphology and migration, and its expression correlates with the malignant phenotype of cancers. To identify the molecular interactions essential for uPAR function in these processes, we carried out a complete functional alanine scan of uPAR in HEK293 cells. Of the 255 mutant receptors characterized, 34 failed to induce changes in cell morphology. Remarkably, the molecular defect of all of these mutants was a specific reduction in integrin-independent cell binding to vitronectin. A membrane-tethered plasminogen activator inhibitor-1, which has the same binding site in vitronectin as uPAR, replicated uPAR-induced changes. A direct uPAR-vitronectin interaction is thus both required and sufficient to initiate downstream changes in cell morphology, migration, and signal transduction. Collectively these data demonstrate a novel mechanism by which a cell adhesion molecule lacking inherent signaling capability evokes complex cellular responses by modulating the contact between the cell and the matrix without the requirement for direct lateral protein-protein interactions.
Assuntos
Movimento Celular/fisiologia , Receptores de Superfície Celular/fisiologia , Vitronectina/fisiologia , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Adesão Celular/fisiologia , Linhagem Celular , Cricetinae , Cricetulus , Teste de Complementação Genética , Humanos , Mutagênese Sítio-Dirigida , Mapeamento de Interação de Proteínas , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transdução de Sinais , Vitronectina/química , Vitronectina/metabolismoRESUMO
uPAR, the receptor for urokinase plasminogen activator, is a regulator of the uptake by macrophages of apoptotic neutrophils (efferocytosis). Its role and mechanism appear to be complex and possibly controversial.
RESUMO
The urokinase plasminogen activator (uPA) receptor (uPAR) is a GPI-linked cell surface protein that facilitates focused plasmin proteolytic activity at the cell surface. uPAR has been detected in macrophages infiltrating the central nervous system (CNS) and soluble uPAR has been detected in the cerebrospinal fluid during a number of CNS pathologies. However, its expression by resident microglial cells in vivo remains uncertain. In this work, we aimed to elucidate the murine CNS expression of uPAR and uPA as well as that of tissue plasminogen activator and plasminogen activator inhibitor 1 (PAI-1) during insults generating distinct and well-characterized inflammatory responses; acute intracerebral lipopolysaccharide (LPS), acute kainate-induced neurodegeneration, and chronic neurodegeneration induced by prion disease inoculation. All three insults induced marked expression of uPAR at both mRNA and protein level compared to controls (naïve, saline, or control inoculum-injected). uPAR expression was microglial in all cases. Conversely, uPA transcription and activity was only markedly increased during chronic neurodegeneration. Dissociation of uPA and uPAR levels in acute challenges is suggestive of additional proteolysis-independent roles for uPAR. PAI-1 was most highly expressed upon LPS challenge, whereas tissue plasminogen activator mRNA was constitutively present and less responsive to all insults studied. These data are novel and suggest much wider involvement of the uPAR/uPA system in CNS function and pathology than previously supposed.
Assuntos
Encefalite/metabolismo , Hipocampo/metabolismo , Microglia/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Análise de Variância , Animais , Western Blotting , Encefalite/induzido quimicamente , Encefalite/complicações , Feminino , Hipocampo/efeitos dos fármacos , Imuno-Histoquímica , Ácido Caínico/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Doenças Priônicas/complicações , Doenças Priônicas/metabolismo , Príons/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
The urokinase plasminogen activator receptor (uPAR) and its ligand (uPA) play an important role in cell migration and extracellular proteolysis. We previously described uPAR/uPA overexpression in the cerebrospinal fluid (CSF) and brain tissues of patients with human immunodeficiency virus (HIV)-related cerebral diseases. In this study, we examined uPAR/uPA expression by immunohistochemistry (IHC) in brains of HIV patients with opportunistic cerebral lesions and in HIV-positive/negative controls. uPAR was found in macrophages/microglia with the highest levels in cytomegalovirus (CMV) encephalitis, toxoplasmosis, and lymphomas; in cryptococcosis and progressive multifocal leukoencephalopathy (PML) cases, only a few positive cells were found and no positivity was observed in controls. uPA expression was demonstrated only in a few macrophages/microglia and lymphocytes in all the cases and HIV-positive controls without different pattern of distribution; no uPA immunostaining was found in cryptococcosis and HIV-negative controls. The higher expression of uPAR/uPA in most of the opportunistic cerebral lesions supports their role in these diseases, suggesting their contribution to tissue injury.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/metabolismo , Encefalopatias/metabolismo , Encéfalo/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Adulto , Idoso , Encéfalo/patologia , Encefalopatias/microbiologia , Encefalopatias/parasitologia , Feminino , Humanos , Ligantes , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells present in all tissue and organs. Cell-matrix interactions play fundamental roles during embryonic development, morphogenesis, tissue homoeostasis, wound healing, and tumourigenesis. Cell-matrix communication is kept in balance by physical contact and by transmembrane integrin receptors providing the dynamic link between the extracellular and intracellular environments through bi-directional signalling. The urokinase-type plasminogen activator receptor (uPAR) is a plasma membrane receptor overexpressed during inflammation and in almost all human cancers. One of its functions is to endorse ECM remodelling through the activation of plasminogen and downstream proteases, including matrix-metalloproteases (MMPs). Beside its role in ECM degradation, uPAR modulates cell-matrix contact through a direct engagement with the ECM component, vitronectin (Vn), and by regulating the activity state of integrins thus promoting or inhibiting integrin signalling and integrin-mediated cell adhesion to other ECM components, like fibronectin and collagen. In this review we have centred our attention on the non-proteolytic function of uPAR as a mediator of cell adhesion and downstream signalling.
Assuntos
Adesão Celular , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Vitronectina/metabolismo , Animais , Matriz Extracelular/metabolismo , Adesões Focais , HIV-1/metabolismo , Humanos , Integrinas/metabolismo , Modelos Biológicos , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
The oligomerization of glycosylphosphatidylinositol-anchored proteins is thought to regulate their association with membrane microdomains, subcellular sorting, and activity. However, these mechanisms need to be comprehensively explored in living, unperturbed cells, without artificial clustering agents, and using fluorescent protein-tagged chimeras that are fully biologically active. We expressed in human embryo kidnay 293 (HEK293) cells a biologically active chimera of the urokinase plasminogen activator receptor (uPAR), the uPAR-mEGFP-GPI. We also produced HEK293/D2D3-mEGFP-GPI cells expressing the truncated form of the receptor, lacking biological activity. We studied the dynamics and oligomerization of the two proteins, combining fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses, and using subclones with homogenously low expression levels. Overall, the mobile fractions of the two proteins, constituted by monomers and dimers, had comparable diffusion coefficients. However, the diffusion coefficient decreased in monomer-enriched fractions only for the active receptor, suggesting that uPAR monomers might be preferentially engaged in multiprotein transmembrane signaling complexes. Our approach helps in limiting the alteration of the data due to out-of-focus effects and in minimizing the overestimation of the molecular brightness. In addition to a careful design of the cellular model, it gives reliable estimates of diffusion coefficients and oligomerization of GPI-anchored proteins, in steady-state conditions, at low expression levels, and in live, unperturbed cells.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Glicosilfosfatidilinositóis/metabolismo , Rim/metabolismo , Fotometria/métodos , Receptores de Superfície Celular/metabolismo , Algoritmos , Linhagem Celular , Humanos , Cinética , Proteínas de Membrana/metabolismo , Fótons , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Espectrometria de FluorescênciaRESUMO
Urokinase-type plasminogen activator (uPA), an inducer of macrophage adhesion, inhibits HIV-1 expression in PMA-stimulated, chronically infected U1 cells. We investigated whether uPA-dependent cell adhesion played a role in uPA-dependent inhibition of HIV-1 replication in these cells. Monocyte-derived macrophages (MDM) were generated from monocytes of HIV-infected individuals or from cells of seronegative donors infected acutely in vitro. U1 cells were stimulated in the presence or absence of uPA in standard tissue culture (TC) plates, allowing firm cell adhesion or ultra-low adhesion (ULA) plates. Moreover, U1 cells were also maintained in the presence or absence of vitronectin (VN)-containing sera or serum from VN(-/-) mice. Virus production was evaluated by RT activity in culture supernatants, whereas cell adhesion was by crystal violet staining and optical microscopy. uPA inhibited HIV replication in MDM and PMA-stimulated U1 cells in TC plates but not in ULA plates. uPA failed to inhibit HIV expression in U1 cells stimulated with IL-6, which induces virus expression but not cell adhesion in TC plates. VN, known to bind to the uPA/uPA receptor complex, was crucial for these adhesion-dependent, inhibitory effects of uPA on HIV expression, in that they were not observed in TC plates in the presence of VN(-/-) mouse serum. HIV production in control cell cultures was increased significantly in ULA versus TC plates, indicating that macrophage cell adhesion per se curtails HIV replication. In conclusion, uPA inhibits HIV-1 replication in macrophages via up-regulation of cell adhesion to the substrate mediated by VN.
Assuntos
Adesão Celular , Infecções por HIV/terapia , HIV-1/fisiologia , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Replicação Viral , Vitronectina/sangue , Ensaio de Desvio de Mobilidade Eletroforética , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Soropositividade para HIV , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/virologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Ligação Proteica , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Acetato de Tetradecanoilforbol/farmacologia , Células U937RESUMO
The urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein that promotes tissue remodeling, tumor cell adhesion, migration and invasion. uPAR mediates degradation of the extracellular matrix through protease recruitment and enhances cell adhesion, migration and signaling through vitronectin binding and interactions with integrins. Full-length uPAR is released from the cell surface, but the mechanism and significance of uPAR shedding remain obscure. Here we identify transmembrane glycerophosphodiesterase GDE3 as a GPI-specific phospholipase C that cleaves and releases uPAR with consequent loss of function, whereas its homologue GDE2 fails to attack uPAR. GDE3 overexpression depletes uPAR from distinct basolateral membrane domains in breast cancer cells, resulting in a less transformed phenotype, it slows tumor growth in a xenograft model and correlates with prolonged survival in patients. Our results establish GDE3 as a negative regulator of the uPAR signaling network and, furthermore, highlight GPI-anchor hydrolysis as a cell-intrinsic mechanism to alter cell behavior.
Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Diester Fosfórico Hidrolases/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Técnicas de Inativação de Genes/métodos , Células HEK293 , Humanos , Hidrólise , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Modelos Moleculares , Transplante de Neoplasias , Diester Fosfórico Hidrolases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais , Carga Tumoral , Vitronectina/genética , Vitronectina/metabolismoRESUMO
The urokinase plasminogen activator receptor (uPAR) fragments D1 and D2D3 are often found in biological fluids from normal individuals and patients of cancer and other diseases. The D2D3 fragment may possess chemotactic activity depending on its N-terminal sequence. We have developed a sensitive and specific immunoassay for the chemotactic form of D2D3 and show that its level can be measured with high specificity and sensitivity in human serum and urine. Synthetic peptides (residues 84-92) derived from the linker region between domains 1 and 2 of uPAR were used as immunogens to generate mouse monoclonal antibodies. Recombinant soluble uPAR (D1D2D3(1-277)) was used to immunize rabbits to obtain polyclonal antibodies. A sandwich-type immunofluorimetric assay was developed with these antibodies. The assay specifically measures D2D3 containing the 84-88 residues, has a detection limit of 0.25 ng/ml and shows no cross-reactivity with D2D3(93-274). The assay is linear at 0-30 ng/ml, with an intra-assay CV of 10% (n=20), inter-assay CV of 15% (n=9) and a recovery of D2D3(84-274) added to urine samples of between 94% and 105%. A statistically significant difference level of D2D3(84-274) was found in two groups of tumor patients versus healthy volunteers (pAssuntos
Fluorimunoensaio/métodos
, Receptores de Superfície Celular/análise
, Receptores de Superfície Celular/imunologia
, Sequência de Aminoácidos
, Animais
, Anticorpos Monoclonais/biossíntese
, Western Blotting
, Células CHO
, Células COS
, Linhagem Celular
, Fatores Quimiotáticos/análise
, Fatores Quimiotáticos/genética
, Fatores Quimiotáticos/imunologia
, Chlorocebus aethiops
, Cricetinae
, Ensaio de Imunoadsorção Enzimática
, Epitopos/análise
, Epitopos/genética
, Citometria de Fluxo
, Fluorimunoensaio/estatística & dados numéricos
, Humanos
, Imunoprecipitação
, Camundongos
, Fragmentos de Peptídeos/análise
, Fragmentos de Peptídeos/genética
, Fragmentos de Peptídeos/imunologia
, Coelhos
, Receptores de Superfície Celular/genética
, Receptores de Ativador de Plasminogênio Tipo Uroquinase
, Proteínas Recombinantes/genética
, Proteínas Recombinantes/imunologia
, Sensibilidade e Especificidade
RESUMO
The molecular clutch (MC) model proposes that actomyosin-driven force transmission permits integrin-dependent cell migration. To investigate the MC, we introduced diverse talin (TLN) and integrin variants into Flp-In™ T-Rex™ HEK293 cells stably expressing uPAR. Vitronectin variants served as substrate providing uPAR-mediated cell adhesion and optionally integrin binding. This particular system allowed us to selectively analyse key MC proteins and interactions, effectively from the extracellular matrix substrate to intracellular f-actin, and to therewith study mechanobiological aspects of MC engagement also uncoupled from integrin/ligand binding. With this experimental approach, we found that for the initial PIP2-dependent membrane/TLN/f-actin linkage and persistent lamellipodia formation the C-terminal TLN actin binding site (ABS) is dispensable. The establishment of an adequate MC-mediated lamellipodial tension instead depends predominantly on the coupling of this C-terminal TLN ABS to the actomyosin-driven retrograde actin flow force. This lamellipodial tension is crucial for full integrin activation eventually determining integrin-dependent cell migration. In the integrin/ligand-independent condition the frictional membrane resistance participates to these processes. Integrin/ligand binding can also contribute but is not necessarily required.
Assuntos
Movimento Celular/fisiologia , Integrinas/metabolismo , Pseudópodes/metabolismo , Actinas/metabolismo , Imunofluorescência , Adesões Focais/metabolismo , Células HEK293 , Humanos , Ligantes , Microscopia Confocal , Ligação Proteica/fisiologia , Talina/metabolismoRESUMO
The binding of urokinase-type plasminogen activator (uPA) to its glycosyl-phosphatidyl-inositol (GPI) anchored receptor (uPAR) mediates a variety of functions in terms of vascular homeostasis, inflammation and tissue repair. Both uPA and uPAR, as well as their soluble forms detectable in plasma and other body fluids, represent markers of cancer development and metastasis, and they have been recently described as predictors of human immunodeficiency virus (HIV) disease progression, independent of CD4+ T cell counts and viremia. A direct link between the uPA/uPAR system and HIV infection was earlier proposed in terms of cleavage of gp120 envelope by uPA. More recently, a negative regulatory effect on both acutely and chronically infected cells has been linked to the noncatalytic portion of uPA, also referred to as the amino-terminal fragment (ATF). ATF has also been described as a major CD8+ T cell soluble HIV suppressor factor. In chronically infected promonocytic U1 cells this inhibitory effect is exerted at the very late stages of the virus life cycle, involving virion budding and entrapment in intracytoplasmic vacuoles, whereas its mechanism of action in acutely infected cells remains to be defined. Since uPAR is a GPI-anchored receptor it requires association with a signaling-transducing component and different partners, which include CD11b/CD18 integrin and a G-protein coupled receptor homologous to that for the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalanine. Which signaling coreceptor(s) is(are) responsible for uPA-dependent anti-HIV effect remains currently undefined.
Assuntos
Infecções por HIV/fisiopatologia , HIV-1/fisiologia , Receptores de Superfície Celular/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Quimiocinas/imunologia , Progressão da Doença , Infecções por HIV/sangue , Humanos , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Replicação ViralRESUMO
The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored membrane protein, which facilitates extracellular matrix remodeling by focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assembly of its three LU (Ly6/uPAR-like) domains is inherently flexible and binding of uPA drives uPAR into its closed conformation, which presents the higher-affinity state for vitronectin thus providing an allosteric regulatory mechanism. Using a new class of epitope-mapped anti-uPAR monoclonal antibodies (mAbs), we now demonstrate that the reciprocal stabilization is indeed also possible. By surface plasmon resonance studies, we show that these mAbs and vitronectin have overlapping binding sites on uPAR and that they share Arg91 as hotspot residue in their binding interfaces. The crystal structure solved for one of these uPAR·mAb complexes at 3.0Å clearly shows that this mAb preselects the closed uPAR conformation with an empty but correctly assembled large hydrophobic binding cavity for uPA. Accordingly, these mAbs inhibit the uPAR-dependent lamellipodia formation and migration on vitronectin-coated matrices irrespective of the conformational status of uPAR and its occupancy with uPA. This is the first study to the best of our knowledge, showing that the dynamic assembly of the three LU domains in uPARwt can be driven toward the closed form by an external ligand, which is not engaging the hydrophobic uPA binding cavity. As this binding interface is also exploited by the somatomedin B domain of vitronectin, therefore, this relationship should be taken into consideration when exploring uPAR-dependent cell adhesion and migration in vitronectin-rich environments.
Assuntos
Conformação Proteica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Somatomedinas/metabolismo , Vitronectina/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Adesão Celular , Cristalografia por Raios X , Mapeamento de Epitopos , Humanos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Pseudópodes/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Ressonância de Plasmônio de Superfície , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two principal ligands: the serine protease urokinase-type plasminogen activator (uPA) and the provisional matrix protein vitronectin (Vn) (Mertens et al., 2012; Gårdsvoll et al., 2011; Madsen et al., 2007 [2-4]). The data presented here relates to the non-covalent trapping of one of these biologically relevant uPAR-conformations by a novel class of monoclonal antibodies (Zhao et al., 2015 [5]) and to the general mapping of the topographic epitope landscape on uPAR. The methods required to achieve these data include: (1) recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2) developing monoclonal antibodies with unique specificities using this protein as antigen; (3) mapping the functional epitope on uPAR for these mAbs by surface plasmon resonance with a complete library of purified single-site uPAR mutants (Zhao et al., 2015; Gårdsvoll et al., 2006 [5,6]); and finally (4) solving the three-dimensional structures for one of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively].
RESUMO
The urokinase plasminogen activator (uPA) and its receptor (uPAR) play important physiological functions in extracellular proteolysis, as well as cell adhesion and migration. Through dysregulation of these functions, the uPA/uPAR system might be involved in the pathogenesis of AIDS dementia complex (ADC), and, in fact, uPAR has been found to be overexpressed in the cerebrospinal fluid (CSF) and brain tissues of patients with ADC. On the other hand, its ligand uPA has been shown to down-regulate HIV replication in vitro. In this study, we examined uPAR and uPA expression in the brain of HIV-related lesions, as well as CSF levels of soluble uPAR (suPAR), uPA, and complexes between these two molecules (suPAR/uPA) in patients with HIV infection with or without ADC. uPAR was highly expressed by macrophages in both HIV encephalitis (HIV-E) or leukoencephalopathy (HIV-LE), with a distribution exceeding that of HIV p24 antigen. In contrast, uPA was detected only on rare cells in most of the cases. Both uPA and suPAR/uPA complex concentrations were significantly correlated with CSF suPAR levels, and CSF concentrations of both markers were higher in ADC patients than controls. However, uPA levels were substantially lower than corresponding suPAR levels. Although these findings remain correlative, they add support to the hypothesis that uPAR might be an important participant in the events leading to ADC. Additionally, these findings are consistent with a model in which overexpression of uPAR and overproduction of its soluble form may promote HIV replication via binding and removal of uPA from cell surface.