RESUMEN
The inappropriate expression of the c-MET cell surface receptor in many human solid tumors necessitates the development of companion diagnostics to identify those patients who could benefit from c-MET targeted therapies. Tumor tissues are formalin fixed and paraffin embedded (FFPE) for histopathologic evaluation, making the development of an antibody against c-MET that accurately and reproducibly detects the protein in FFPE samples an urgent need. We have developed a monoclonal antibody (mAb), designated MET4, from a panel of MET-avid mAbs, based on its specific staining pattern in FFPE preparations. The accuracy of MET4 immunohistochemistry (MET4-IHC) was assessed by comparing MET4-IHC in FFPE cell pellets with immunoblotting analysis. The technical reproducibility of MET4-IHC possessed a percentage coefficient of variability of 6.25% in intra-assay and interassay testing. Comparison with other commercial c-MET antibody detection reagents demonstrated equal specificity and increased sensitivity for c-MET detection in prostate tissues. In cohorts of ovarian cancers and gliomas, MET4 reacted with ovarian cancers of all histologic subtypes (strong staining in 25%) and with 63% of gliomas. In addition, MET4 bound c-MET on the surfaces of cultured human cancer cells and tumor xenografts. In summary, the MET4 mAb accurately and reproducibly measures c-MET expression by IHC in FFPE tissues and can be used for molecular imaging in vivo. These properties encourage further development of MET4 as a multipurpose molecular diagnostics reagent to help to guide appropriate selection of patients being considered for treatment with c-MET-antagonistic drugs.
Asunto(s)
Anticuerpos Monoclonales , Biomarcadores de Tumor/análisis , Neoplasias/patología , Proteínas Proto-Oncogénicas c-met/análisis , Biomarcadores de Tumor/normas , Femenino , Formaldehído , Glioma , Humanos , Inmunohistoquímica , Masculino , Neoplasias/química , Neoplasias/diagnóstico , Neoplasias Ováricas , Adhesión en Parafina , Fijación del TejidoRESUMEN
Hepatocyte growth factor (HGF) activates the Met receptor tyrosine kinase by binding and promoting receptor dimerization. Here we describe a mechanistic basis for designing Met antagonists based on NK1, a natural variant of HGF containing the N-terminal and the first kringle domain. Through detailed biochemical and structural analyses, we demonstrate that both mouse and human NK1 induce Met dimerization via a conserved NK1 dimer interface. Mutations designed to alter the NK1 dimer interface abolish its ability to promote Met dimerization but retain full Met-binding activity. Importantly, these NK1 mutants act as Met antagonists by inhibiting HGF-mediated cell scattering, proliferation, branching, and invasion. The ability to separate the Met-binding activity of NK1 from its Met dimerization activity thus provides a rational basis for designing Met antagonists. This strategy of antagonist design may be applicable for other growth factor receptors by selectively abolishing the receptor activation ability but not the receptor binding of the growth factors.
Asunto(s)
Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Neuroquinina-1/metabolismo , Animales , Línea Celular , Dimerización , Disulfuros/química , Perros , Escherichia coli/genética , Heparina/farmacología , Factor de Crecimiento de Hepatocito/agonistas , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Histidina/química , Humanos , Concentración 50 Inhibidora , Riñón/citología , Kringles , Luz , Ratones , Modelos Biológicos , Mutación , Oligopéptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Neuroquinina-1/genética , Proteínas Recombinantes de Fusión/metabolismo , Dispersión de Radiación , Difracción de Rayos XRESUMEN
Mineralocorticoid receptor (MR) controls sodium homeostasis and blood pressure through hormone binding and coactivator recruitment. Here, we report a 1.95 A crystal structure of the MR ligand binding domain containing a single C808S mutation bound to corticosterone and the fourth LXXLL motif of steroid receptor coactivator-1 (SRC1-4). Through a combination of biochemical and structural analyses, we demonstrate that SRC1-4 is the most potent MR binding motif and mutations that disrupt the MR/SRC1-4 interactions abolish the ability of the full-length SRC1 to coactivate MR. The structure also reveals a compact steroid binding pocket with a unique topology that is primarily defined by key residues of helices 6 and 7. Mutations swapping a single residue at position 848 from helix H7 between MR and glucocorticoid receptor (GR) switch their hormone specificity. Together, these findings provide critical insights into the molecular basis of hormone binding and coactivator recognition by MR and related steroid receptors.
Asunto(s)
Hormonas/metabolismo , Receptores de Mineralocorticoides/química , Factores de Transcripción/química , Aldosterona/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células COS , Chlorocebus aethiops , Corticosterona/metabolismo , Cristalografía por Rayos X , Genes Reporteros/genética , Histona Acetiltransferasas , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Coactivador 1 de Receptor Nuclear , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The functional interaction between the orphan nuclear receptors small heterodimer partner (SHP) and liver receptor homolog 1 (LRH-1), where SHP binds to LRH-1 and represses its constitutive transcriptional activity, is crucial for regulating genes involved in cholesterol homeostasis. Here, we report structural and biochemical analyses of the LRH-1/SHP interaction. The crystal structure and modeling studies of the LRH-1 ligand-binding domain bound to either of the two LXXLL-related motifs of SHP show that the receptor undergoes conformational changes to accommodate the SHP docking and reveal key residues that determine the potency and selectivity of SHP binding. Through a combination of mutagenesis and binding studies, we demonstrate that only the second SHP LXXLL motif is required for repressing LRH-1, and this motif displays a strong preference for binding to LRH-1 over the closely related receptor steroidogeneic factor 1 (SF-1). Structural comparisons indicate that this binding selectivity is determined by residues flanking the core LXXLL motifs. These results establish a structural model for understanding how SHP interacts with LRH-1 to regulate cholesterol homeostasis and provide new insights into how nuclear receptor/coregulator selectivity is achieved.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Homeostasis/fisiología , Modelos Moleculares , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencias de Aminoácidos/genética , Animales , Colesterol/metabolismo , Cristalografía , Ratones , Mutagénesis , Plásmidos/genética , Unión Proteica , Receptores Citoplasmáticos y Nucleares/genética , TransfecciónRESUMEN
The orphan nuclear receptor steroidogenic factor 1 (SF-1) regulates the differentiation and function of endocrine glands. Although SF-1 is constitutively active in cell-based assays, it is not known whether this transcriptional activity is modulated by ligands. Here, we describe the 1.5 angstroms crystal structure of the SF-1 ligand binding domain in complex with an LXXLL motif from a coregulator protein. The structure reveals the presence of a phospholipid ligand in a surprisingly large pocket (approximately 1600 angstroms3), with the receptor adopting the canonical active conformation. The bound phospholipid is readily exchanged and modulates SF-1 interactions with coactivators. Mutations designed to reduce the size of the SF-1 pocket or to disrupt hydrogen bonds with the phospholipid abolish SF-1/coactivator interactions and significantly reduce SF-1 transcriptional activity. These findings provide evidence that SF-1 is regulated by endogenous ligands and suggest an unexpected relationship between phospholipids and endocrine development and function.