RESUMO
Grb7 is a member of a family of molecular adapters which are able to contribute positively but also negatively to signal transduction and whose precise roles remain obscure. Rnd1 is a member of the Rho family, but, as opposed to usual GTPases, it is constitutively bound to GTP. We show here that Rnd1 and Grb7 interact, in two-hybrid assays, in vitro, and in pull-down experiments performed with SK-BR3, a breast cancer cell line that overexpresses Grb7. This interaction involves switch II loop of Rnd1, a region crucial for guanine nucleotide exchange in all GTPases, and a Grb7 SH2 domain, a region crucial for Grb7 interaction with several activated receptors. The contribution of the interaction between Rnd1 and Grb7 to their respective functions and properties is discussed.
Assuntos
Proteínas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Anticorpos/imunologia , Sítios de Ligação , Escherichia coli/genética , Proteína Adaptadora GRB7 , Células HeLa , Humanos , Peso Molecular , Fosforilação , Fosfotirosina/imunologia , Fosfotirosina/metabolismo , Testes de Precipitina , Ligação Proteica , Proteínas/química , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência/genética , Homologia de Sequência de Aminoácidos , Transfecção , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/isolamento & purificação , Domínios de Homologia de srcRESUMO
Some of the most potent antiinflammatory and immunosuppressive agents are synthetic glucocorticoids. However, major side effects severely limit their therapeutic use. The development of improved glucocorticoid-based drugs will require the separation of beneficial from deleterious effects. One possibility toward this goal is to try to dissociate two main activities of glucocorticoids, i.e. transactivation and transrepression. Screening of a library of compounds using transactivation and AP-1 transrepression models in transiently transfected cells identified dissociated glucocorticoids, which exert strong AP-1 inhibition but little or no transactivation. Importantly, despite high ligand binding affinity, the prototypic dissociated compound, RU24858, acted as a weak agonist and did not efficiently antagonize dexamethasone-induced transcription in transfected cells. Similar results were obtained in hepatic HTC cells for the transactivation of the endogenous tyrosine amino transferase gene (TAT), which encodes one of the enzymes involved in the glucocorticoid-dependent stimulation of neoglucogenesis. To investigate whether dissociated glucocorticoids retained the antiinflammatory and immunosuppressive potential of classic glucocorticoids, several in vitro and in vivo models were used. Indeed, secretion of the proinflammatory lymphokine interleukin-1beta was severely inhibited by dissociated glucocorticoids in human monocytic THP 1 cells. Moreover, in two in vivo models, these compounds exerted an antiinflammatory and immunosuppressive activity as potent as that of the classic glucocorticoid prednisolone. These results may lead to an improvement of antiinflammatory and immunosuppressive therapies and provide a novel concept for drug discovery.
Assuntos
Anti-Inflamatórios/farmacologia , Glucocorticoides/farmacologia , Hidroxicorticosteroides , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Colagenases/efeitos dos fármacos , Colagenases/genética , Desoximetasona/análogos & derivados , Relação Dose-Resposta a Droga , Genes Reporter , Células HeLa/efeitos dos fármacos , Humanos , Imunossupressores/farmacologia , Interleucina-1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Regiões Promotoras Genéticas , Ratos , Receptores de Glucocorticoides/efeitos dos fármacos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Timo/efeitos dos fármacos , Fator de Transcrição AP-1/efeitos dos fármacos , Fator de Transcrição AP-1/genética , Transfecção , Tirosina Transaminase/efeitos dos fármacos , Tirosina Transaminase/metabolismoRESUMO
Src homology 3 (SH3) domains are conserved modules which participate in protein interaction by recognizing proline-rich motifs on target molecules. To identify new SH3-containing proteins, we performed a two-hybrid screen with a proline-rich region of human SOS-1. One of the specific SOS-1 interacting clones that were isolated from a mouse brain cDNA library defines a new protein that was named amphiphysin 2 because of its homology to the previously reported amphiphysin. Amphiphysin 2 is expressed in a number of mouse tissues through multiple RNA transcripts. Here, we report the amino acid sequence of a brain form of amphiphysin 2 (BRAMP2) encoded by a 2. 5-kilobase mRNA. BRAMP2 associates in vitro with elements of the endocytosis machinery such as alpha-adaptin and dynamin. On a biosensor surface, the BRAMP2/dynamin interaction appeared to be direct and partly dependent on a proline-rich sequence of dynamin. Association with dynamin was also observed in PC12 cells after cell stimulation with nerve growth factor, suggesting that amphiphysin 2 may be connected to receptor-dependent signaling pathways. This hypothesis is strengthened by the ability of BRAMP2 to interact with the p21(ras) exchange factor SOS, in vitro, as a possible point of interconnection between the endocytic and signaling pathways.