RESUMO
Receptors for PDGF play an important role in cell proliferation and migration and have been implicated in certain cancers. The 44-amino acid E5 protein of bovine papillomavirus binds to and activates the PDGFß receptor (PDGFßR), resulting in oncogenic transformation of cultured fibroblasts. Previously, we isolated an artificial 36-amino acid transmembrane protein, pTM36-4, which transforms cells because of its ability to activate the PDGFßR despite limited sequence similarity to E5. Here, we demonstrated complex formation between the PDGFßR and three pTM36-4 mutants: T21E, T21Q, and T21N. T21Q retained wild type transforming activity and activated the PDGFßR in a ligand-independent manner as a consequence of binding to the transmembrane domain of the PDGFßR, but T21E and T21N were severely defective. In fact, T21N substantially inhibited E5-induced PDGFßR activation and transformation in both mouse and human fibroblasts. T21N did not prevent E5 from binding to the receptor, and genetic evidence suggested that T21N and E5 bind to nonidentical sites in the transmembrane domain of the receptor. T21N also inhibited transformation and PDGFßR activation induced by v-Sis, a viral homologue of PDGF-BB, as well as PDGF-induced mitogenesis and signaling by preventing phosphorylation of the PDGFßR at particular tyrosine residues. These results demonstrated that T21N acts as a novel inhibitor of the PDGFßR and validated a new strategy for designing highly specific short transmembrane protein inhibitors of growth factor receptors and possibly other transmembrane proteins.
Assuntos
Ativadores de Enzimas/metabolismo , Fibroblastos/metabolismo , Mutação de Sentido Incorreto , Proteínas Oncogênicas v-sis/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Substituição de Aminoácidos , Animais , Papillomavirus Bovino 1/genética , Papillomavirus Bovino 1/metabolismo , Bovinos , Linhagem Celular , Transformação Celular Viral/genética , Fibroblastos/patologia , Humanos , Masculino , Camundongos , Proteínas Oncogênicas v-sis/genética , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Fosforilação/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/agonistas , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/genéticaRESUMO
PDGF is one of the most potent serum mitogens, and the signalling mechanism by way of its receptor tyrosine-kinase has been extensively studied since its first purification in 1979. The identification of homology between the simian sarcoma virus oncogene, v-sis, and the B-chain of PDGF, as well as the frequent over-expression of both the ligands and receptors in various tumours and stroma led to the proposal of the PDGF-mediated autocrine and paracrine hypothesis. Consistent with the important roles of PDGF in the growth and survival of cells, the expression and activity of PDGF receptors are tightly controlled by both positive and negative feedback mechanisms at different levels. The deregulation of the control system can result in serious pathological conditions such as chronic inflammation and tumours. Understanding the molecular mechanisms for the regulatory system and the signalling pathway of PDGF is essential in order to find effective therapies in the diseases where PDGF is involved.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteínas Oncogênicas v-sis/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Dimerização , Humanos , Fosforilação , Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/fisiologiaRESUMO
Specific oncogenes that contribute to the pathogenesis of canine osteosarcoma (OS) have not been identified. In the process of characterizing four OS cell lines, we have found one cell line, CO8, that overexpresses the sis oncogene, which encodes the platelet-derived growth factor (PDGF)-beta. The expression of an important downstream transcriptional target of the PDGF signaling pathway, c-myc, is also elevated fourfold. Conditioned medium from CO8 alone specifically induces tyrosine phosphorylation and therefore the activation of the PDGF-alpha and PDGF-beta receptors on murine 3T3 cells. All of the canine OS lines tested contain PDGF receptors and therefore are capable of responding to PDGE Given the importance of PDGF in promoting cell proliferation, migration, and cell survival, the activation of the sis oncogene and the resultant growth factor autocrine loop potentially contribute to the pathogenesis of a subset of canine osteosarcomas.
