RESUMO
Among the members of the fibroblast growth factor receptor family the FGFR4 has demonstrated strong dependence on heparin-like material for its activation by fibroblast growth factors. We have produced and characterized a recombinant human FGFR4 extracellular domain (FGFR4ed), in order to study its biochemical properties in isolated conditions. The FGFR4ed was expressed in an insect cell system and purified from the culture medium by Ni(2+)-affinity and gel filtration chromatography. Pure FGFR4ed was tested for FGF- and heparin-binding by covalent crosslinking experiments and by biosensor analysis. In solution, FGFR4ed formed complexes with acidic FGF (FGF-1) and basic FGF (FGF-2), both in the presence and absence of heparin. Immobilized FGFR4 also bound FGF-8 besides FGF-1 and FGF-2. Furthermore, heparin alone induced receptor oligomerization on the surface of the receptor coupled chip. Thus, the recombinant FGFR4ed revealed properties described for the cellular form of this receptor and can be used for interaction studies.
Assuntos
Histidina , Engenharia de Proteínas/métodos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Técnicas Biossensoriais , Células Cultivadas , Cromatografia de Afinidade , Dimerização , Fator 1 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 8 de Crescimento de Fibroblasto , Fatores de Crescimento de Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Insetos/citologia , Peptídeos/genética , Peptídeos/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Amplification and enhanced expression of the erbB2/HER-2/neu gene has been associated with an increased growth rate and poor prognosis of human breast cancer. We have studied the relationship between erbB2 expression and the regulation of cell growth by estrogen and anti-estrogens in the human breast cancer cell line ZR-75-1 in vitro and in athymic nude mice, pS2 being used as a marker gene for estrogen-stimulated gene expression. Only low amounts of erbB2 mRNA were seen in the cells grown in vitro in the presence of estrogen which stimulated the cells to proliferate rapidly and induced the expression of pS2 mRNA. Upon hormone withdrawal, erbB2 mRNA and protein increased, while pS2 mRNA declined to an undetectable level and cell proliferation slowed down. Opposite but more rapid changes were observed upon estrogen addition. The anti-estrogens toremifene and tamoxifen inhibited estrogen induction of pS2 expression, down-regulation of erbB2 expression and proliferation of the ZR-75-I cells in a concentration-dependent manner. Similar results were obtained in nude mice. ZR-75-I cells formed tumors only in mice carrying estrogen pellets. In these tumors little erbB2 mRNA was seen. Concomitant administration of toremifene or tamoxifen increased erbB2 mRNA and abolished pS2 mRNA. Our results show that enhanced expression of erbB2 is associated with hormone deprivation and growth arrest of the estrogen-dependent breast cancer cell line ZR-75-I. Thus, in mammary epithelial cells, erbB2 may have important estrogen-regulated functions which are not related to cell proliferation.