RESUMEN
Previously, we identified a human ST3Gal-V mRNA variant peculiarly characterized by the presence of a translational start codon localized up-stream and in-frame with the one that is usually considered as unique translation initiation site in the human gene. In this study we demonstrate, by cDNA transfection experiments, mutational analyses, enzyme activity assays, and endoglycosidase-H treatments, that the in vivo expression of this transcript gives rise to two human ST3Gal-V isoforms with distinct characteristics. Produced by a leaky scanning mechanism, they carry different N-glycan structures and exhibit differences in their GM(3) synthase activity that might be relevant for the modulation of GM(3) cellular content.
Asunto(s)
Variación Genética , Nitrógeno/metabolismo , Placenta/metabolismo , Sialiltransferasas/biosíntesis , Sialiltransferasas/química , Western Blotting , Endonucleasas/metabolismo , Femenino , Gangliósido G(M3)/metabolismo , Glicosilación , Humanos , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Embarazo , ARN Mensajero/genética , Sialiltransferasas/metabolismo , Especificidad por SustratoRESUMEN
Gangliosides are well-known regulators of cell differentiation through specific interactions with growth factor receptors. Previously, our group provided the first evidence about stable association of ganglioside GM(3) to EGFR/ErbB2 heterodimers in mammary epithelial cells. Goals of the present study were to better define the role of gangliosides in EGFR/ErbB2 heterodimerization and receptor phosphorylation events and to analyze their involvement in mammary cell differentiation. Experiments have been conducted using the ceramide analogue (+/-)-treo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ceramide glucosyltransferase resulting in the endogenous ganglioside depletion, and the lactogenic hormone mix DIP (dexamethasone, insulin, prolactin), which induces cell differentiation and beta-casein mRNA synthesis. In addition, treatments of ganglioside-depleted cells with exogenous GM(3) have been carried out to ascertain the specific involvement of this ganglioside. Results from co-immunoprecipitation and Western blot experiments have shown that the endogenous ganglioside depletion resulted in the disappearance of SDS-stable EGFR/ErbB2 heterodimers and in the appearance of tyrosine-phosphorylated EGFR also in the absence of EGF stimulation; exogenous GM(3) added in combination with [D]-PDMP reversed both these effects. In contrast, the tyrosine phosphorylation of ErbB2 in ganglioside-depleted cells occurred only after EGF stimulation. Moreover, when ganglioside-depleted cells were treated with DIP in absence of EGF, beta-casein gene expression appeared strongly down-regulated, and beta-casein mRNA levels were partially restored by exogenous GM(3) treatment. Altogether, although the involvement of other ganglioside species cannot be excluded, these findings sustain the ganglioside GM(3) as an essential molecule for EGFR/ErbB2 heterodimer stability and important regulator of EGFR tyrosine phosphorylation, but it is not crucial for tyrosine phosphorylation of the heterodimerization partner ErbB2. Moreover, modulation of EGFR phosphorylation may explain how gangliosides contribute to regulate the lactogenic hormone-induced mammary cell differentiation.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Gangliósidos/farmacología , Receptor ErbB-2/metabolismo , Secuencia de Bases , División Celular , Línea Celular , Cartilla de ADN , Dimerización , Humanos , FosforilaciónRESUMEN
Gangliosides are known to modulate the activation of receptor tyrosine-kinases (RTKs). Recently, we demonstrated the functional relationship between ErbB2 and ganglioside GM(3) in HC11 epithelial cell line. In the present study we investigated, in the same cells, the ErbB2 activation state and its tendency to form stable molecular complexes with the epidermal growth factor receptor (EGFR) and with ganglioside GM(3) upon EGF stimulation. Results from co-immunoprecipitation experiments and western blot analyses indicate that tyrosine-phosphorylated ErbB2 and EGFR monomers and stable ErbB2/EGFR high molecular complexes (heterodimers) are formed following EGF stimulation, even if the receptors co-immunoprecipitates also in the absence of the ligand; these data suggest the existence of pre-dimerization inactive receptor clusters on the cell surface. High performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of the ganglioside fractions extracted from the immunoprecipitates demonstrate that GM(3), but not other gangliosides, is tightly associated to the tyrosine-phosphorylated receptors. Furthermore, we show that GM(3) is preferentially and in a SDS-resistant manner associated to the activated ErbB2/EGFR complexes and EGFR monomer, but not to ErbB2. Altogether our data support the hypothesis that the modulating effects produced by GM(3) on ErbB2 activation are mediated by EGFR.
Asunto(s)
Receptores ErbB/metabolismo , Gangliósido G(M3)/metabolismo , Fosfotirosina/metabolismo , Receptor ErbB-2/metabolismo , Animales , Western Blotting , Dimerización , Factor de Crecimiento Epidérmico/farmacología , Inmunoprecipitación , RatonesRESUMEN
All human GM3 synthase mRNA variants until now identified predict a protein of 362 amino acids having substrate activity highly restricted to lactosylceramide. In this report we describe the identification of a new GM3 synthase transcript containing an additional translation start codon, located upstream and in-frame with that up to now considered unique translation initiation site in the human GM3 synthase gene. In vitro expression studies showed that the new transcript produces a longer form of human GM3 synthase, that is efficiently translocated into the microsomal lumen and glycosylated. Moreover, stable cDNA transfection into mammalian cells gives rise to a threefold increase of GM3 synthase activity, associated to a broader substrate specificity. Although this transcript has been initially identified in the human placenta, RT-PCR analyses verified the expression of an identical mRNA also in undifferentiated HL60 cells, but not in the monocytic lineage. Altogether, these results are the first demonstration of the existence of a new isoform of human GM3 synthase, which could play an important role during HL60 cell differentiation. The functional relevance of the existence of two isoforms of GM3 synthase is also discussed.
Asunto(s)
Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Diferenciación Celular/genética , Codón Iniciador , ADN Complementario/genética , Células HL-60 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Placenta/enzimología , ARN Mensajero/metabolismo , Especificidad por SustratoRESUMEN
We analyzed the role of gangliosides in the association of the ErbB2 receptor tyrosine-kinase (RTK) with lipid rafts in mammary epithelial HC11 cells. Scanning confocal microscopy experiments revealed a strict ErbB2-GM3 colocalization in wild-type cells. In addition, analysis of membrane fractions obtained using a linear sucrose gradient showed that ErbB2, epidermal growth factor receptor (EGFR) and Shc-p66 (proteins correlated with the ErbB2 signal transduction pathway) were preferentially enriched in lipid rafts together with gangliosides. Blocking of endogenous ganglioside synthesis by (+/-)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP) induced a drastic cell-surface redistribution of ErbB2, EGFR and Shc-p66, within the Triton-soluble fractions, as revealed by linear sucrose-gradient analysis. This redistribution was partially reverted when exogenous GM3 was added to ganglioside-depleted HC11 cells. The results point out the key role of ganglioside GM3 in retaining ErbB2 and signal-transduction-correlated proteins in lipid rafts.
Asunto(s)
Células Epiteliales/metabolismo , Gangliósido G(M3)/fisiología , Glándulas Mamarias Animales/citología , Microdominios de Membrana/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Membrana Celular/metabolismo , Células Cultivadas , Colesterol/metabolismo , Cromatografía Líquida de Alta Presión , Células Epiteliales/efectos de los fármacos , Receptores ErbB/metabolismo , Immunoblotting , Inmunoprecipitación , Ratones , Microscopía Confocal , Morfolinas/farmacología , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
The functional relationship between ganglioside GM(3) and two tyrosine-kinase receptors, the normal protein p185(c-neu) and the mutant oncogenic protein p185(neu), was examined in HC11 cells and in MG1361 cells, respectively. In the former, p185(c-neu) expression and activation are controlled by EGF addition to the culture medium and by epidermal growth factor receptor (EGFR) activity, whereas the latter express unchangingly high levels of constitutively activated p185(neu). Studies were carried out using (+/-)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ganglioside biosynthesis resulting in ganglioside depletion, and addition of exogenous GM(3) to the culture medium. In HC11 cells treated with only [D]-PDMP, p185(c-neu) levels remain similar to control cells, whereas levels of tyrosine-phosphorylated p185(c-neu) increase after treatment with [D]-PDMP in combination with EGF. When exogenous GM(3) is added in combination with [D]-PDMP and EGF, the enhanced phosphorylated-p185(c-neu) returns to control levels. Interestingly, EGFR levels also vary and, analogously to phosphorylated-p185(c-neu), the increase of EGFR content consequent to the [D]-PDMP and EGF addition is reversed by exogenous GM(3). In contrast, the addition of neither [D]-PDMP nor exogenous GM(3) modifies expression and tyrosine-phosphorylation levels of p185(neu) in MG1361 cells. These findings indicate that changes in GM(3) content modulate the tyrosine-phosphorylated p185(c-neu) levels in a reversible manner, but this is not specific for p185(c-neu) because EGFR levels are also modified. Furthermore, these data suggest that GM(3) may play a functional role by affecting the internalisation pathway of p185(c-neu)/EGFR heterodimers, but not of p185(neu) homodimers.