Ring finger protein 34 (RNF34) interacts with and promotes Î³-aminobutyric acid type-A receptor degradation via ubiquitination of the Î³2 subunit.

Jin, Hongbing; Chiou, Tzu-Ting; Serwanski, David R; Miralles, Celia P; Pinal, Noelia; De Blas, Angel L

Jin, Hongbing; Chiou, Tzu-Ting; Serwanski, David R; Miralles, Celia P; Pinal, Noelia; De Blas, Angel L.

Afiliación

Jin H; From the Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269.
Chiou TT; From the Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269.
Serwanski DR; From the Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269.
Miralles CP; From the Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269.
Pinal N; From the Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269.
De Blas AL; From the Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269 angel.deblas@uconn.edu.

J Biol Chem ; 289(42): 29420-36, 2014 Oct 17.

Article en En | MEDLINE | ID: mdl-25193658

RESUMEN

We have found that the large intracellular loop of the Î³2 GABAA receptor (R) subunit (Î³2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABAARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the Î³2 GABAAR subunit by increasing the ratio of ubiquitinated/nonubiquitinated Î³2. Mutating several lysines of the Î³2IL into arginines makes the Î³2 subunit resistant to RNF34-induced degradation. RNF34 also reduces the expression of the Î³2 subunit when α1 and ß3 subunits are co-assembled with Î³2. This effect is partially reversed by leupeptin or MG132, indicating that both the lysosomal and proteasomal degradation pathways are involved. Immunofluorescence of cultured hippocampal neurons shows that RNF34 forms clusters and that a subset of these clusters is associated with GABAergic synapses. This association is also observed in the intact rat brain by electron microscopy immunocytochemistry. RNF34 is not expressed until the 2nd postnatal week of rat brain development, being highly expressed in some interneurons. Overexpression of RNF34 in hippocampal neurons decreases the density of Î³2 GABAAR clusters and the number of GABAergic contacts that these neurons receive. Knocking down endogenous RNF34 with shRNA leads to increased Î³2 GABAAR cluster density and GABAergic innervation. The results indicate that RNF34 regulates postsynaptic Î³2-GABAAR clustering and GABAergic synaptic innervation by interacting with and ubiquitinating the Î³2-GABAAR subunit promoting GABAAR degradation.

Asunto(s)

Proteínas Portadoras/metabolismo; Receptores de GABA-B/metabolismo; Animales; Encéfalo/embriología; Regulación de la Expresión Génica; Cobayas; Células HEK293; Hipocampo/embriología; Hipocampo/metabolismo; Humanos; Lisosomas/metabolismo; Neuronas/metabolismo; Complejo de la Endopetidasa Proteasomal/metabolismo; Unión Proteica; Transporte de Proteínas; Ratas; Sinapsis/metabolismo; Técnicas del Sistema de Dos Híbridos; Ubiquitina/metabolismo; Ubiquitinación

Palabras clave

E3 Ubiquitin Ligase; GABAA Receptors; Interaction; Lysosome; Proteasome; RNF34; Synapse; Ubiquitination; Yeast Two-hybrid

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Portadoras / Receptores de GABA-B Límite: Animals / Humans Idioma: En Revista: J Biol Chem Año: 2014 Tipo del documento: Article Pais de publicación: Estados Unidos

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