RESUMEN
RhoA organizes actin stress fibres and is necessary for cell transformation by oncogenes such as src and ras. Moreover, RhoA is implicated in cadherin clustering during the formation of adherens junctions. The catenin p120 has also been implicated in cadherin clustering through an unknown mechanism. Here we show that p120 selectively inhibits RhoA activity in vitro and in vivo. RhoA inhibition and the interaction of p120 with cadherins are mutually exclusive, suggesting a mechanism for regulating the recruitment and exchange of RhoA at nascent cell-cell contacts. By affecting RhoA activation, p120 could modulate cadherin functions, including suppression of invasion, neurite extension and junction formation.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Fosfoproteínas/metabolismo , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Células 3T3 , Animales , Cadherinas/metabolismo , Cateninas , Moléculas de Adhesión Celular/genética , Guanosina Difosfato/metabolismo , Humanos , Lisofosfolípidos/farmacología , Ratones , Fenotipo , Fosfoproteínas/genética , Células Tumorales Cultivadas , Proteína de Unión al GTP cdc42/biosíntesis , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP rac1/biosíntesis , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo , Catenina deltaRESUMEN
p120(ctn) is a catenin whose direct binding to the juxtamembrane domain of classical cadherins suggests a role in regulating cell-cell adhesion. The juxtamembrane domain has been implicated in a variety of roles including cadherin clustering, cell motility, and neuronal outgrowth, raising the possibility that p120 mediates these activities. We have generated minimal mutations in this region that uncouple the E-cadherin-p120 interaction, but do not affect interactions with other catenins. By stable transfection into E-cadherin-deficient cell lines, we show that cadherins are both necessary and sufficient for recruitment of p120 to junctions. Detergent-free subcellular fractionation studies indicated that, in contrast to previous reports, the stoichiometry of the interaction is extremely high. Unlike alpha- and beta-catenins, p120 was metabolically stable in cadherin-deficient cells, and was present at high levels in the cytoplasm. Analysis of cells expressing E-cadherin mutant constructs indicated that p120 is required for the E-cadherin-mediated transition from weak to strong adhesion. In aggregation assays, cells expressing p120-uncoupled E-cadherin formed only weak cell aggregates, which immediately dispersed into single cells upon pipetting. As an apparent consequence, the actin cytoskeleton failed to insert properly into peripheral E-cadherin plaques, resulting in the inability to form a continuous circumferential ring around cell colonies. Our data suggest that p120 directly or indirectly regulates the E-cadherin-mediated transition to tight cell-cell adhesion, possibly blocking subsequent events necessary for reorganization of the actin cytoskeleton and compaction.
Asunto(s)
Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Adhesión Celular , Fosfoproteínas/metabolismo , Animales , Sitios de Unión , Células CHO , Cadherinas/genética , Cateninas , Adhesión Celular/fisiología , Cricetinae , Citoplasma/metabolismo , Expresión Génica , Humanos , Uniones Intercelulares , Células L , Ratones , Fosforilación , Catenina deltaRESUMEN
We have generated the first monoclonal antibodies (MAbs) to Armadillo repeat gene deleted in velo-cardiofacial syndrome (ARVCF), a recently identified Armadillo repeat-containing protein closely related to the catenin p120ctn. Six ARVCF-specific MAbs were characterized for isotype, species cross-reactivity, and utility in assays including immunofluorescence, immunoprecipitation, and Western blotting. All six antibodies were isotyped as IgG1 and several cross-reacted with ARVCF from a variety of species including human, rat, dog, and monkey, but not mouse. Importantly, none of the ARVCF MAbs cross-reacted with p120ctn, despite the high homology between these proteins. MAbs 3B2 and 4B1 were consistently the best in all applications and will provide valuable tools for further study of the role of ARVCF in cells.
Asunto(s)
Anomalías Múltiples/genética , Anomalías Múltiples/inmunología , Anticuerpos Monoclonales/inmunología , Armadillos/genética , Armadillos/inmunología , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Formación de Anticuerpos , Sitios de Unión , Western Blotting , Cadherinas/metabolismo , Cateninas , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Línea Celular/química , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/inmunología , Síndrome de DiGeorge/genética , Síndrome de DiGeorge/inmunología , Perros , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Haplorrinos , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/inmunología , Humanos , Hibridomas/inmunología , Uniones Intercelulares/química , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , Ratas , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Secuencias Repetitivas de Ácidos Nucleicos/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Insuficiencia Velofaríngea/genética , Insuficiencia Velofaríngea/inmunología , Catenina deltaRESUMEN
This report describes the generation and characterization of a panel of monoclonal antibodies (MAb) to the catenin p120ctn. p120ctn (formerly p120cas) is a cadherin-binding protein with structural similarity to the classical catenins beta-catenin and plakoglobin. It was originally identified as a prominent Src substrate and subsequently as a substrate for the Platelet Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF), and Colony Stimulating Factor-1 (CSF-1) receptor tyrosine kinases. To facilitate further study of p120 function, we have generated novel MAbs to both the N- and C-terminal ends of p120 and compared them to previously described antibodies to these regions.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Moléculas de Adhesión Celular/inmunología , Fosfoproteínas/inmunología , Animales , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos , Western Blotting , Cateninas , Línea Celular , Reacciones Cruzadas , Perros , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Hibridomas/inmunología , Ratones , Pruebas de Precipitina , Ratas , Especificidad de la Especie , Catenina deltaRESUMEN
The norepinephrine (NE) transporter (NET), a target of many clinically prescribed antidepressants, regulates noradrenergic neurotransmission by efficiently clearing NE from synaptic spaces after release. To advance our understanding of NET gene structure, regulation, and potential associations with complex behavioral trait loci, we amplified a mouse norepinephrine transporter (mNET) cDNA from placenta total RNA and utilized mNET probes to isolate and characterize the mNET gene. Inferred translation of the major open reading frame of the mNET cDNA predicts a 617-amino acid protein with 12 putative membrane-spanning regions and 94% identity to human NET. The coding exons of the mNET cDNA were found to be spread across >36 kb of 129/Svj genomic DNA, with exon-intron boundaries bearing consensus gt/ag splice sites. Sequence upstream (202 bp) of the inferred translation initiation site matched the sequence of 5' rapid amplification of cDNA ends products from brain mRNA with no evidence for intervening introns and is preceded by a TATA box and canonical transcriptional regulatory elements that may play a role in mNET expression in vivo. Probes derived from mNET cDNA identified species-specific MspI restriction fragment length variations within the mNET gene that were utilized to position the gene (Slc6a5) to murine chromosome 8, one recombinant distal to D8Mit15. This site is within a recently defined quantitative trait locus defined for ethanol sensitivity in LSXSS recombinant inbred mice, Lore4. The status of Slc6a5 as a candidate gene for alcohol sensitivity is discussed with respect to studies noting ethanol-induced alterations in brain NE receptors, NE receptor-linked adenylate cyclase, and NE transport.