RESUMEN
Members of the epidermal growth factor (EGF) family of tyrosine kinase receptors are involved in the regulation of cell growth and differentiation, and are found to be expressed in many types of cancers. Activation of these receptors can be elicited by multiple ligands, resulting in the formation of a spectrum of heterodimer complexes and a number of biological outcomes. A clear demonstration of biological activation by a single complex has been difficult to address because of the endogenous expression of HERs (human EGF-like receptors) in many cell lines. We have generated a collection of cell lines expressing all HERs alone or in all pairwise combinations in a clone of NIH 3T3 cells (3T3-7d) devoid of detectable EGF receptor family members. Transformation, as measured by growth in soft agar, only occurred in cells expressing two different HER family members. Transformation with activated Neu and the rate of in vivo tumour formation were also correlated with the expression of multiple HERs in the same cell. To further our understanding of the role of heterodimer signalling, we demonstrated that, within a breast carcinoma cell line, activation of HER-3 results in cellular differentiation, prolonged activation of extracellular-signal-related kinase 1 (ERK1) activity and an increase in p21CIP1/WAF1 nuclear staining. In contrast, activation of HER-4 is mitogenic, induces transient activation of ERK1 activity and decreases the nuclear staining of p21CIP1/WAF1. These differences in biochemical and biological responses are correlated with the contrasting abilities of HER-3 and HER-4 to be down-regulated from the cell surface. The cell-surface localization of HER-3 does not change in response to ligand, whereas activation of HER-4 results in a loss of cell-surface staining followed by accumulation into a perinuclear compartment.
Asunto(s)
Neoplasias de la Mama/patología , Diferenciación Celular , División Celular , Receptores ErbB/fisiología , Células 3T3 , Animales , Receptores ErbB/genética , Humanos , Ratones , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-2/genética , Receptor ErbB-3 , Receptor ErbB-4 , Transfección , Células Tumorales CultivadasRESUMEN
A collection of cell lines expressing each human epidermal growth factor receptor (HER) family member alone or in all pairwise combinations in a clone of NIH3T3 cells (3T3-7d) devoid of detectable epidermal growth factor receptor family members has been generated. Transformation, as measured by growth in soft agar, occurred only in the presence of appropriate ligand and only in cells expressing two different HER family members. Transfection of oncogenic neu (Tneu), conferred ligand-independent transformation only in cells which co-expressed HER1, HER3, or HER4, but not when expressed alone or with HER2. Cell lines were also tested for their ability to form tumors in animals. None of the cell lines expressing single HER family members was able to form tumors in animals with the exception of HER1, which was weakly tumorigenic. Although unable to form tumors when expressed alone, HER2 was tumorigenic when expressed with HER1 or HER3, but not HER4. Of all complexes analyzed, cells expressing HER1 + HER2 were the most aggressive. The relationship between HER1 activation, intracellular calcium fluxes, and phospholipase Cgamma1 activation is well established. We found that activation of HER1 was required for the induction of a calcium flux and the phosphorylation of phospholipase Cgamma1. These activities were independent of, and unaffected by, the co-expression of any other family member. Further, heregulin stimulation of all cell lines including those containing HER1 did not demonstrate any effect on intracellular calcium levels or phospholipase Cgamma1 phosphorylation. This demonstrates that heregulin induced cellular transformation by activating HER3- and HER4-containing complexes does not require the activation of either phospholipase Cgamma1 or the mobilization of intracellular calcium.
Asunto(s)
Transformación Celular Neoplásica , Receptores ErbB/fisiología , Neurregulina-1 , Proteínas Proto-Oncogénicas/fisiología , Receptor ErbB-2/fisiología , Células 3T3 , Animales , Calcio/metabolismo , Proteínas Portadoras/fisiología , Factor de Crecimiento Epidérmico/fisiología , Glicoproteínas/fisiología , Humanos , Isoenzimas/metabolismo , Ratones , Fosfolipasa C gamma , Fosfotirosina/metabolismo , Receptor ErbB-3 , Receptor ErbB-4 , Transducción de Señal , Fosfolipasas de Tipo C/metabolismoRESUMEN
The EGF receptor family of tyrosine kinase growth factor receptors is expressed in a variety of cell types and has been implicated in the progression of certain human adenocarcinomas. The most recent addition to this family of receptors, HER4, was expressed in NIH 3T3 cells to determine its biological and biochemical characteristics. Cells expressing HER4 were responsive to heregulin beta2 as demonstrated by an increase in HER4 tyrosine phosphorylation and ability to form foci on a cell monolayer. HER4 exhibited in vitro kinase activity and was able to phosphorylate the regulatory subunit of phosphatidylinositol 3-kinase and SHC. Peptide competition studies identified tyrosine 1056 of HER4 as the phosphatidylinositol 3-kinase binding site and tyrosines 1188 and 1242 as two potential SHC binding sites. Interestingly, transfection of HER4 into NIH 3T3 cells conferred responsiveness to EGF with respect to colony formation in soft agar. It was also found that in response to heregulin beta2, endogenous murine HER1 or transfected human HER1 became phosphorylated when HER4 was present. This demonstrates that HER1 and HER4 can exist in a heterodimer complex and likely activate each other by transphosphorylation.
Asunto(s)
Receptores ErbB/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Sitios de Unión , División Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/biosíntesis , Receptores ErbB/química , Humanos , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptor ErbB-4 , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Transfección , TirosinaRESUMEN
This report describes the isolation and recombinant expression of a cDNA clone encoding HER4, the fourth member of the human epidermal growth factor receptor (EGFR) family. The HER4/erbB4 gene encodes a 180-kDa transmembrane tyrosine kinase (HER4/p180erbB4) whose extracellular domain is most similar to the orphan receptor HER3/p160erbB3, whereas its cytoplasmic kinase domain exhibits 79% and 77% identity with EGFR and HER2/p185erbB2, respectively. HER4 is most predominantly expressed in several breast carcinoma cell lines, and in normal skeletal muscle, heart, pituitary, brain, and cerebellum. In addition, we describe the partial purification of a heparin-binding HER4-stimulatory factor from HepG2 cells. This protein was found to specifically stimulate the intrinsic tyrosine kinase activity of HER4/p180erbB4 while having no direct effect on the phosphorylation of EGFR, HER2, or HER3. Furthermore, this heparin-binding protein induces phenotypic differentiation, and tyrosine phosphorylation, of a human mammary tumor cell line that overexpresses both HER4 and HER2. These findings suggest that this ligand-receptor interaction may play a role in the growth and differentiation of some normal and transformed cells.
Asunto(s)
Receptores ErbB/genética , Receptores ErbB/fisiología , Proteínas Tirosina Quinasas/genética , Receptores de Superficie Celular/genética , Secuencia de Bases , Células , Clonación Molecular , Receptores ErbB/metabolismo , Expresión Génica , Ligandos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , Receptor ErbB-4 , Receptores de Superficie Celular/fisiología , Alineación de Secuencia , Transducción de Señal , Distribución TisularRESUMEN
The present study reports that smooth muscle cells can be derived from the adult rat aorta without being exposed to serum containing platelet releasate, these cells can also be grown and passaged in medium lacking detectable quantities of platelet-derived growth factor (PDGF). As reported earlier, smooth muscle cells grown from the newborn or injured vessels have a distinctive epithelioid appearance. The platelet factor independent smooth muscle cells have a similar appearance. Unlike the cells from newborn animals or from injured vessels, smooth muscle cells derived in this way from the adult animal do not produce PDGF, although they do display PDGF receptors. Studies in serum-free medium show that the PDGF-independent cells can respond to PDGF when all other components of serum are absent; however, growth of these smooth muscle cells proceeds equally well in low concentrations of serum, independent of the presence of PDGF or other growth factors detectable by growth-promoting assays using either 3T3 cells or smooth muscle cells whose growth is dependent on factors present in whole blood serum. Taken together with recent observations in vivo, these data suggest that growth of at least some smooth muscle cells may depend on mechanisms independent of release of growth factors by platelets.