RESUMO
Grb2 is an adaptor protein that links receptor and cytoplasmic tyrosine kinases to the Ras signalling pathway by binding the Ras-specific guanine nucleotide exchange factor, Sos1, through its SH3 domains. The Grb2-SH3 domain binding has been localized to the carboxy-terminal two hundred amino acids of Sos1 (Sos1-c). By using real time biospecific interaction analysis (BIAcore), we studied the kinetic parameters and binding affinity of the Grb2-Sos1-c interaction. The binding of Grb2 to Sos1-c is a high affinity interaction with a moderate association rate (9.45 x 10(4) per M per s), a slow dissociation rate (13.8 x 10(-5) s), and an affinity constant of 1.48 nM. BIAcore measurements on isolated N-terminal and C-terminal SH3 domains (NSH3 and CSH3) further indicate that the high affinity Grb2-Sos1-c interaction is primarily mediated through the NSH3 domain (Kd = 1.68 nM). The CSH3 domain shows substantially reduced binding to Sos1-c in these measurements. Inhibition studies with BIAcore using proline rich peptides derived from the C-terminus of Sos1 show that there is a single major binding site for Grb2 in Sos1. This binding site is contained within the peptide N20, which corresponds to amino acids 1143-1162 of Sos1. This peptide completely blocks the Grb2-Sos1-c and NSH3-Sos1-c interactions with IC50 values of 8 microM and 4 microM respectively. The discrete interaction between the NSH3 domain and the N20 peptide may be amenable for drug discovery through screening or peptidomimetic approaches.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Fúngicas/metabolismo , Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Domínios de Homologia de src , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína Adaptadora GRB2 , Camundongos , Dados de Sequência Molecular , Proteínas/química , Coelhos , Proteína SOS1RESUMO
The recombinant oncotoxin OLX-209 [e23(Fv)PE38KDEL] has been developed to target cancers with erbB-2 expression and is nearing a clinical trial. Important in clinical planning is the selection of patients on the basis of tumor expression of erbB-2. ErbB-2 gene amplification occurs in cancers of the breast, stomach, and ovary. Patients with these diseases and evident overexpression are candidates for OLX-209 therapy. In lung cancer, overexpression of erbB-2 is also frequent, but in most cases, it is not caused by gene amplification. This study demonstrates that OLX-209 has activity on lung cancer cells with varying levels of erbB-2 expression in the presence and absence of gene amplification. In vitro sensitivity of cell lines to OLX-209 is related to erbB-2 expression level. Normal bronchial epithelial cells were not sensitive. Effective treatment of lung cancer cell lines growing as xenografts in nude mice was shown with Calu-3 (a lung adenocarcinoma line with high levels of p185(erbB-2) caused by gene amplification) and three other lung adenocarcinomas (A549, NCI-H1466, and 201T) with lower levels of p185(erbB-2) and no gene amplification. The 201T cell line was isolated recently from a lung tumor with erbB-2 expression in the original tumor. The results of this study indicate that patients with erbB-2-positive, non-small cell lung cancer should be included in clinical trials of OLX-209.