RESUMO
Protein tagging is widely used in approaches ranging from affinity purification to fluorescence-based detection in live cells. However, an intrinsic limitation of tagging is that the native function of the protein may be compromised or even abolished by the presence of the tag. Here we describe and characterize a set of small, innocuous protein tags (inntags) that we anticipate will find application in a variety of biological techniques.
Assuntos
Epitopos/análise , Epitopos/química , Imunofluorescência/métodos , Imunoprecipitação/métodos , Proteínas/análise , Proteínas/imunologia , Animais , Anticorpos Monoclonais , Epitopos/genética , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The nerve growth factor receptor TrkA (tropomyosin-related kinase receptor) participates in the survival and differentiation of several neuronal populations. The C-terminal tail of TrkA contains a PPXY motif, the binding site of the E3 ubiquitin-ligase Nedd4-2 (neural precursor cell expressed, developmentally down-regulated 4-2). In order to analyze the role of Nedd4-2 ubiquitination on TrkA function, we generated three TrkA mutants, by introducing point mutations on conserved hydrophobic amino acids - Leu784 and Val790 switched to Ala. TrkA mutants co-localized and co-immunoprecipitated more efficiently with Nedd4-2 and consequently a strong increase in the basal multimonoubiquitination of the mutant receptors was observed. In addition, we found a decrease in TrkA abundance because of the preferential sorting of mutant receptors towards the late endosome/lysosome pathway instead of recycling back to the plasma membrane. Despite the reduction in the amount of membrane receptor caused by the C-terminal changes, TrkA mutants were able to activate signaling cascades and were even more efficient in promoting neurite outgrowth than the wild-type receptor. Our results demonstrate that the C-terminal tail hydrophobicity of TrkA regulates Nedd4-2 binding and activity and therefore controls receptor turnover. In addition, TrkA multimonoubiquitination does not interfere with the activation of signaling cascades, but rather potentiates receptor signaling leading to differentiation.
Assuntos
Cisteína Endopeptidases/metabolismo , Lisossomos/metabolismo , Receptor trkA/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologia , Animais , Biotina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Cisteína Endopeptidases/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Proteínas de Fluorescência Verde/genética , Imunoprecipitação/métodos , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Mutação/genética , Fator de Crescimento Neural/farmacologia , Células PC12 , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Transporte Proteico/efeitos dos fármacos , Ratos , Receptor trkA/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção/métodos , Ubiquitinação/efeitos dos fármacos , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Tropomyosin-related kinase A (TrkA) receptor mediates the effects exerted by nerve growth factor on several subpopulations of neuronal cells. Ligand binding to TrkA induces receptor autophosphorylation on several tyrosine residues and the activation of signaling cascades. In this study, we describe a new site relevant for TrkA regulation, the tyrosine 701 (Y701), which is important for receptor trafficking and activation. Y701 replacement by aspartate or phenylalanine reduces receptor internalization rate and decreases the colocalization and association of TrkA with clathrin heavy chain, demonstrating that Y701 constitutes a YxxPhi (YRKF701-704) trafficking motif relevant for the regulation of receptor endocytosis. In accordance with this hypothesis, the colocalization of Y701 mutant receptors with a lysosomal marker is also reduced giving support to the involvement of the YRKF701-704 motif in the lysosomal targeting of TrkA receptors. Contrary to what was expected, substitution of Y701 for an Asp in order to mimic phosphorylation, impairs TrkA ability to mediate nerve growth factor-induced differentiation, although the mutant receptor retains its in vitro kinase activity. This is the first evidence that a Tyr residue can simultaneously regulate TrkA receptor trafficking and activity.
Assuntos
Receptor trkA/metabolismo , Tirosina/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Animais , Ácido Aspártico/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Cadeias Pesadas de Clatrina/metabolismo , Imunoprecipitação/métodos , Proteínas de Membrana Lisossomal/metabolismo , Mutação/fisiologia , Células PC12 , Transporte Proteico/fisiologia , Ratos , Transfecção/métodosRESUMO
Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs.