RESUMO
A fibroblast growth factor receptor 1 variant missing 37 amino acids from the carboxy-terminal tyrosine kinase catalytic domain was discovered in human lung fibroblasts and several other human cell lines. The receptor variant binds specifically to acidic fibroblast growth factor but has no tyrosine kinase activity. It was found that cellular transfectants expressing the fibroblast growth factor receptor 1 variant are mitogenically inactive and ligand binding to the receptor causes neither receptor autophosphorylation nor phospholipase C-gamma transphosphorylation. The fibroblast growth factor receptor 1 variant therefore represents an inactive receptor for acidic fibroblast growth factor. Since both kinase and kinase-deficient receptor forms are expressed in cells, it is conceivable that the kinase-deficient receptor plays an important role in regulating cellular responses elicited by acidic fibroblast growth factor stimulation.
Assuntos
Fator 1 de Crescimento de Fibroblastos/farmacologia , Variação Genética , Receptores Proteína Tirosina Quinases , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Primers do DNA , DNA Complementar , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fibroblastos , Humanos , Pulmão/enzimologia , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Sondas RNA , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleases , Deleção de Sequência , Timidina/metabolismo , TransfecçãoRESUMO
Tyrosine phosphorylation is a key step in the regulation of many cellular events including signal transduction of stimulated growth factor receptor tyrosine kinases. Upon ligand activation, these proteins undergo dimerization and subsequent auto- and transphosphorylation events on specific tyrosine residues, which enables them to interact with several cellular signaling proteins. We have used synthetic peptides encompassing all the tyrosine residues of a tyrosine kinase and employed them as substrates in in vitro kinase reactions. Using this assay we have shown that short tyrosine-containing peptides derived from the cytoplasmic domain of the mouse platelet-derived growth factor beta receptor (PDGF-R) can serve as specific phosphorylation targets for the kinase. These peptides include 7 out of 8 tyrosines that are known auto- or transphosphorylation sites in vivo, as previously determined by peptide mapping and mutational analyses. We have also identified 10 additional tyrosine-containing peptides that are phosphorylated and represent possible novel auto- or transphosphorylation sites of PDGF-R. The presented method greatly simplifies the mapping of auto- or transphosphorylation sites in tyrosine kinases and provides a valuable tool in the analysis of signaling mechanisms involving these proteins.
