RESUMEN
The tie2 receptor tyrosine kinase plays a key role in angiogenesis, and the remodeling and maturation of blood vessels. In this study we have used a factor-dependent cell line (Ba/F3) expressing a chimeric receptor containing the extracellular domain of mouse tie2 and the transmembrane and cytoplasmic domain of the erythropoietin receptor to identify specific binding activity associated with an adipogenic sub-line of 3T3 fibroblasts (3T3-L1). 3T3-L1 fibroblasts are capable of undergoing differentiation to adipocytes under specific culture conditions. When compared to 3T3-L1 cells, the adipocyte differentiated cultures, which contain both pre-adipocytes and adipocytes, exhibited a significantly increased ability to support the growth of Ba/F3 cells expressing the chimeric receptor. Using probes specific for two recently described ligands for tie2, Ang-1 and Ang-2, we have shown that mRNA encoding Ang-1 is upregulated when 3T3-L1 fibroblasts are differentiated to adipocytes. These results suggest that the levels of Ang-1 protein and mRNA in 3T3-L1 cells can be regulated by cellular differentiation in adipose development.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Glicoproteínas de Membrana/genética , 1-Metil-3-Isobutilxantina/farmacología , Células 3T3 , Adipocitos/efectos de los fármacos , Angiopoyetina 1 , Angiopoyetina 2 , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Bioensayo , Diferenciación Celular/efectos de los fármacos , Cartilla de ADN/genética , Dexametasona/farmacología , Femenino , Fibroblastos/efectos de los fármacos , Insulina/farmacología , Ligandos , Glicoproteínas de Membrana/metabolismo , Ratones , Fenotipo , Placenta/metabolismo , Embarazo , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/inmunología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor TIE-2 , Regulación hacia ArribaRESUMEN
Accumulating experimental evidence indicates that endothelial cell growth and blood vessel morphogenesis are processes that are governed by the activity of specifically expressed receptor tyrosine kinases (RTKs). We have used two new rat monoclonal antibodies (mAbs) to study the expression and phosphorylation of one such receptor, mouse Tie2 (tyrosine kinase that contains immunoglobulin-like loops and epidermal-growth-factor-similar domains 2]), in transfected cells, endothelioma cell lines and mouse tissues. The Tie2 receptor was found to be constitutively autophosphorylated when over-expressed in COS7 cells. In contrast, the endogenous Tie2 protein was not phosphorylated in endothelioma cell lines. However, in these cell lines, Tie2 could be induced to become tyrosine phosphorylated, and this activation was found to be independent of Tie1. Studying Tie2 receptor activity during angiogenesis in mouse development, the receptor was only weakly phosphorylated in the early postnatal mouse brain whereas a stronger activation could be detected in mouse embryos at day 10.5 post coitum.
Asunto(s)
Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Anticuerpos Monoclonales , Encéfalo/embriología , Encéfalo/metabolismo , Células COS , Células Cultivadas , Femenino , Expresión Génica , Ratones , Ratones Noqueados , Fosforilación , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptor TIE-1 , Receptor TIE-2 , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores TIE , Distribución Tisular , TransfecciónRESUMEN
The cDNA of a novel mouse cell surface receptor (tie2) has been isolated from a mouse lung library. The predicted amino acid sequence of tie2 encodes a protein of 1122 amino acids, with an extracellular domain and an intracellular tyrosine kinase domain bisected by a transmembrane region. The extracellular domain consists of two Ig-like domains, three cysteine-rich domains and three fibronectin type III repeats whilst the intracellular tyrosine kinase domain has a short insert region of 15 amino acids. In vitro transcription/translation of the tie2 cDNA demonstrates that it encodes a glycoprotein of some 145 kDa. The tie2 protein exhibits a high degree of similarity to the cell surface receptor tie, (Partanen, J. et al., (1992) Mol. Cell. Biol., 12, 1698-1707), and together with this protein defines a new class of cell surface receptor.