RESUMO
c-Myc drives uncontrolled cell proliferation in various human cancers. However, in mouse embryo fibroblasts (MEFs), c-Myc also induces apoptosis by activating the p19Arf tumor suppressor pathway. Tbx2, a transcriptional repressor of p19Arf, can collaborate with c-Myc by suppressing apoptosis. MEFs overexpressing c-Myc and Tbx2 are immortal but not transformed. We have performed an unbiased genetic screen, which identified 12 oncogenes that collaborate with c-Myc and Tbx2 to transform MEFs in vitro. One of them encodes the LPA2 receptor for the lipid growth factor lysophosphatidic acid (LPA). We find that LPA1 and LPA4, but not LPA3, can reproduce the transforming effect of LPA2. Using pharmacological inhibitors, we show that the in vitro cell transformation induced by LPA receptors is dependent on the Gi-linked ERK and PI3K signaling pathways. The transforming ability of LPA1, LPA2 and LPA4 was confirmed by tumor formation assays in vivo and correlated with prolonged ERK1/2 activation in response to LPA. Our results reveal a direct role for LPA receptor signaling in cell transformation and tumorigenesis in conjunction with c-Myc and reduced p19Arf expression.
Assuntos
Transformação Celular Neoplásica , Genes myc , Lisofosfolipídeos/fisiologia , Receptores de Ácidos Lisofosfatídicos/fisiologia , Animais , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Embrião de Mamíferos/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/fisiologia , Regulação Neoplásica da Expressão Gênica , Testes Genéticos/métodos , Humanos , Camundongos , Neoplasias/patologiaRESUMO
With the help of 16 chimaeras between human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGF alpha), a detailed analysis was performed on the epitope recognized by two polyclonal antibodies raised against hEGF, and one polyclonal antibody raised against hTGF alpha. All three antibodies recognized essentially the same antigenic site, a non-linear and conformation-dependent sequence that is located near the second and fourth disulphide-bonded cysteines and that includes the start of the B-loop beta-sheet. The epitope recognized by the anti-hEGF antibodies was further characterized using 8 chimaeras between hEGF and an EGF-repeat from Drosophila Notch and was found to include Met(21), Ala(30) and Asn(32). All three polyclonal antibodies were able to neutralize the biological activity of the respective growth factor when tested on 32D murine haematopoietic progenitor cells transfected with ErbB-1, indicating that the receptor binding domain is shielded upon binding of the antibody.
Assuntos
Antígenos/química , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/imunologia , Fator de Crescimento Transformador alfa/química , Fator de Crescimento Transformador alfa/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Sítios de Ligação , Divisão Celular , Relação Dose-Resposta a Droga , Drosophila , Proteínas de Drosophila , Mapeamento de Epitopos , Epitopos/química , Humanos , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Receptores Notch , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Epidermal growth factor (EGF) has been the prototype growth-stimulating peptide for many years. It has a characteristic structure with three disulfide bridges, which is essential for its activity. However, many other proteins, including both growth factors and proteins with unrelated functions, have similar EGF-like domains. This indicates that besides a characteristic conformation provided by the EGF-like domain, specific amino acids are required to provide specificity in protein functioning. Currently, more than 10 different growth factors with an EGF-like domain have been characterized which all exert their action by binding to the four members of the erbB family of receptors. In this review, studies are described on the structure-function relationship of these EGF-like growth factor molecules in an attempt to analyze the individual amino acids that determine their binding specificity to the individual members of the erbB family.
Assuntos
Fator de Crescimento Epidérmico/química , Estrutura Terciária de Proteína/fisiologia , Receptor ErbB-2/fisiologia , Receptor ErbB-3/fisiologia , Sequência de Aminoácidos , Fator de Crescimento Epidérmico/fisiologia , Humanos , Ligantes , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
CD97 is an activation-induced antigen on leukocytes which belongs to a new group of seven-span transmembrane (7-TM) molecules, designated EGF-TM7 family. Family members, including EMR1 and F4/80, are characterized by an extended extracellular region with several N-terminal epidermal growth factor-like (EGF) domains. Alternative splicing of CD97 results in isoforms possessing either three (EGF1, 2, 5), four (EGF1, 2, 3, 5) or five EGF domains (EGF1, 2, 3, 4, 5). We recently identified decay accelerating factor (DAF, CD55), a regulatory protein of the complement cascade, as a cellular ligand of the smallest isoform. Employing mutants of CD97(EGF1, 2, 5) in which the EGF domains have been systematically deleted, we here demonstrate the necessity of at least three tandemly linked EGF domains for the interaction with CD55. Consistent with the involvement of different EGF domains, monoclonal antibodies directed against the first EGF domain as well as the removal of Ca2+, for which binding sites exist in the second and fifth EGF domain, blocked binding to CD55. Compared to CD97(EGF1, 2 ,5) the larger isoforms CD97(EGF1, 2, 3, 5) and CD97(EGF1, 2, 3, 4, 5) have a significantly lower affinity for CD55. Thus, alternative splicing may regulate the ligand specificity of CD97 and probably other members of the EGF-TM7 family.
