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1.
Genes Dev ; 20(8): 927-32, 2006 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-16618804

RESUMEN

Integrins are heterodimeric adhesion receptors associated with bidirectional signaling. In vitro studies support a role for the binding of evolutionarily conserved tyrosine motifs (NPxY) in the beta integrin cytoplasmic tail to phosphotyrosine-binding (PTB) domain-containing proteins, an interaction proposed to be dynamically regulated by tyrosine phosphorylation. Here we show that replacement of both beta1 integrin cytoplasmic tyrosines with alanines, resulting in the loss of all PTB domain interaction, causes complete loss of beta1 integrin function in vivo. In contrast, replacement of beta1 integrin cytoplasmic tyrosines with phenylalanines, a mutation that prevents tyrosine phosphorylation, conserves in vivo integrin function. These results have important implications for the molecular mechanism and regulation of integrin function.


Asunto(s)
Citoplasma/metabolismo , Integrina beta1/fisiología , Tirosina/fisiología , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Integrina beta1/química , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación
2.
Blood ; 106(4): 1268-77, 2005 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-15886326

RESUMEN

The roles of the 2 major platelet-collagen receptors, glycoprotein VI (GPVI) and integrin alpha2beta1, have been intensely investigated using a variety of methods over the past decade. In the present study, we have used pharmacologic and genetic approaches to study human and mouse platelet adhesion to collagen under flow conditions. Our studies demonstrate that both GPVI and integrin alpha2beta1 play significant roles for platelet adhesion to collagen under flow and that the loss of both receptors completely ablates this response. Intracellular signaling mediated by the cytoplasmic adaptor Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) but not by the transmembrane adaptor linker for activation of T cells (LAT) is critical for platelet adhesion to collagen under flow. In addition, reduced GPVI receptor density results in severe defects in platelet adhesion to collagen under flow. Defective adhesion to collagen under flow is associated with prolonged tail-bleeding times in mice lacking one or both collagen receptors. These studies establish platelet-collagen responses under physiologic flow as the consequence of a close partnership between 2 structurally distinct receptors and suggest that both receptors play significant hemostatic roles in vivo.


Asunto(s)
Colágeno/farmacología , Integrina alfa2beta1/fisiología , Adhesividad Plaquetaria , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Coagulación Sanguínea , Humanos , Integrina alfa2beta1/deficiencia , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos , Ratones Noqueados , Perfusión , Fosfoproteínas/fisiología , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/deficiencia , Transducción de Señal
3.
Ann Biomed Eng ; 32(7): 970-6, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15298434

RESUMEN

Glycoprotein VI (GPVI) is a platelet receptor that directly binds collagen. It has been shown by expressing GPVI in rat basophilic leukemia (RBL-2H3) cells that GPVI mediates adhesion to type I collagen under static conditions. However, the ability of GPVI to secure adhesion to collagen type I under flow has not been measured. We studied the interaction of GPVI and type I collagen under hydrodynamic flow using RBL-2H3 cells transfected with the GPVI receptor. We found that GPVI-expressing RBL-2H3 cells adhere to collagen under flow, significantly more so than non-GPVI-expressing RBL-2H3 cells. Inhibition of GPVI by the 11A12 anti-GPVI antibody significantly blocks adhesion to collagen, indicating that GPVI specifically interacts with collagen. Probing the role of signaling in GPVI binding to collagen, we used mutants of GPVI and observed that signal transduction did not inhibit adhesion. To test the correlation between receptor expression and adhesion, we tested three GPVI-expressing RBL-2H3 cell lines (A, B, and C) with different levels of receptor expression. At a single shear rate, the level of adhesion increases monotonically with surface expression. The results, using this model cell line, indicate that GPVI is capable of mediating adhesion to collagen under shear, in a density-dependent fashion that is independent of GPVI signaling.


Asunto(s)
Colágeno Tipo I/metabolismo , Leucemia Basofílica Aguda/metabolismo , Leucemia Basofílica Aguda/patología , Mecanotransducción Celular , Glicoproteínas de Membrana Plaquetaria/metabolismo , Animales , Velocidad del Flujo Sanguíneo , Adhesión Celular , Línea Celular Tumoral , Leucemia Basofílica Aguda/genética , Mutagénesis Sitio-Dirigida , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismo , Estrés Mecánico
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