RESUMO
Controlling the number of functional gamma-aminobutyric acid A (GABA(A)) receptors in neuronal membranes is a crucial factor for the efficacy of inhibitory neurotransmission. Here we describe the direct interaction of GABA(A) receptors with the ubiquitin-like protein Plic-1. Furthermore, Plic-1 is enriched at inhibitory synapses and is associated with subsynaptic membranes. Functionally, Plic-1 facilitates GABA(A) receptor cell surface expression without affecting the rate of receptor internalization. Plic-1 also enhances the stability of intracellular GABA(A) receptor subunits, increasing the number of receptors available for insertion into the plasma membrane. Our study identifies a previously unknown role for Plic-1, a modulation of GABA(A) receptor cell surface number, which suggests that Plic-1 facilitates accumulation of these receptors in dendritic membranes.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Receptores de GABA-A/metabolismo , Ubiquitinas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Relacionadas à Autofagia , Membrana Celular/metabolismo , Estabilidade de Medicamentos , Isoformas de Proteínas/metabolismo , Ratos , Frações Subcelulares/metabolismo , Distribuição Tecidual , Ubiquitinas/metabolismoRESUMO
Two GABA(B) receptors, GABA(B)R1 and GABA(B)R2, have been cloned recently. Unlike other G protein-coupled receptors, the formation of a heterodimer between GABA(B)R1 and GABA(B)R2 is required for functional expression. We have used the yeast two hybrid system to identify proteins that interact with the C-terminus of GABA(B)R1. We report a direct association between GABA(B) receptors and two members of the 14-3-3 protein family, 14-3-3eta and 14-3-3zeta. We demonstrate that the C-terminus of GABA(B)R1 associates with 14-3-3zeta in rat brain preparations and tissue cultured cells, that they codistribute after rat brain fractionation, colocalize in neurons, and that the binding site overlaps partially with the coiled-coil domain of GABA(B)R1. Furthermore we show a reduced interaction between the C-terminal domains of GABA(B)R1 and GABA(B)R2 in the presence of 14-3-3. The results strongly suggest that GABA(B)R1 and 14-3-3 associate in the nervous system and begin to reveal the signaling complexities of the GABA(B)R1/GABA(B)R2 receptor heterodimer.
Assuntos
Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Transdução de Sinais/fisiologia , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas 14-3-3 , Animais , Química Encefálica/fisiologia , Células COS , Fracionamento Celular , Expressão Gênica/fisiologia , Hipocampo/citologia , Técnicas In Vitro , Neurônios/citologia , Neurônios/metabolismo , Estrutura Terciária de Proteína , Ratos , Receptores de GABA-B/química , Sinapses/metabolismo , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Type A gamma-aminobutyric acid receptors (GABA(A)), the major sites of fast synaptic inhibition in the brain, are believed to be composed predominantly of alpha, beta, and gamma subunits. Although cell surface expression is essential for GABA(A) receptor function, little is known regarding its regulation. To address this issue, the membrane stability of recombinant alpha(1)beta(2) or alpha(1)beta(2)gamma(2) receptors was analyzed in human embryonic kidney cells. Alpha(1)beta(2)gamma(2) but not alpha(1)beta(2) receptors were found to recycle constitutively between the cell surface and a microtubule-dependent, perinuclear endosomal compartment. Similar GABA(A) receptor endocytosis was also seen in cultured hippocampal and cortical neurons. GABA(A) receptor surface levels were reduced upon protein kinase C (PKC) activation. Like basal endocytosis, this response required the gamma(2) subunit but not receptor phosphorylation. Although inhibiting PKC activity did not block alpha(1)beta(2)gamma(2) receptor endocytosis, it did prevent receptor down-regulation, suggesting that PKC activity may block alpha(1)beta(2)gamma(2) receptor recycling to the cell surface. In agreement with this observation, blocking recycling from endosomes with wortmannin selectively reduced surface levels of gamma(2)-containing receptors. Together, our results demonstrate that the surface stability of GABA(A) receptors can be dynamically and specifically regulated, enabling neurons to modulate cell surface receptor number upon the appropriate cues.
Assuntos
Proteína Quinase C/metabolismo , Receptores de GABA-A/metabolismo , Transdução de Sinais , Linhagem Celular , Humanos , Receptores de GABA-A/química , Transmissão SinápticaRESUMO
GABA receptors (GABA(A)) are the major sites of fast synaptic inhibition in the brain and can be assembled from five subunit classes: alpha, beta, gamma, delta, and epsilon. Receptor function can be regulated by direct phosphorylation of beta and gamma2 subunits, but how kinases are targeted to GABA(A) receptors is unknown. Here we show that protein kinase C-betaII (PKC-betaII) is capable of directly binding to the intracellular domain of the receptor beta1 and beta3 subunits, but not to those of the alpha1 or gamma2 subunits. Moreover, associating PKC-betaII is capable of specifically phosphorylating serine 409 in beta1 subunit and serines 408/409 within the beta3 subunit, key residues for modulating GABA(A) receptor function. The receptor for activated C kinase (RACK-1) was found also to bind to the beta1 subunit intracellular domain, but PKC binding appeared to be independent of this protein. Using immunoprecipitation, the association of PKC isoforms and RACK-1 with neuronal GABA(A) receptors was seen. Furthermore, PKC isoforms associating with neuronal receptors were capable of phosphorylating the receptor beta3 subunit. Together, these observations suggest GABA(A) receptors are intimately associated with PKC isoforms via a direct interaction with receptor beta subunits. This interaction may serve to localize PKC activity to GABA(A) receptors in neurons allowing the rapid regulation of receptor activity by cell-signaling pathways that modify PKC activity.
Assuntos
Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Animais , Sítios de Ligação , Isoenzimas/isolamento & purificação , Substâncias Macromoleculares , Fragmentos de Peptídeos/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Fosforilação , Proteína Quinase C/isolamento & purificação , Proteína Quinase C beta , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-Dawley , Receptores de Quinase C Ativada , Receptores de Superfície Celular/isolamento & purificação , Receptores de GABA-A/isolamento & purificaçãoRESUMO
The expression of alpha and beta gamma-aminobutyric acid type A receptor subunits produces GABA-gated channels which require the incorporation of either the gamma2 or gamma3 subunit for benzodiazepine modulation. Here we examine the role of the gamma2 subunit splice variants, gamma2S and gamma2L which differ by eight amino acids in the major intracellular domain, in mediating cell surface expression. Using immunocytochemistry we have demonstrated that when expressed alone, the gamma2S subunit can access the cell surface and internalize constitutively. In contrast, alpha1, beta2 and gamma2L are retained predominantly in the endoplasmic reticulum (ER) when expressed alone. Replacing the insert which differentiates gamma2L from gamma2S (LLRMFSFK) with eight alanines produces a phenotype identical to gamma2S. Both gamma2 subunits fail to produce high molecular weight oligomers observed for alpha1beta2 and alpha1beta2gamma2 heterooligomers and do not form functional ion channels. Surface expression of gamma2S is repressed upon the coexpression of alpha1 or beta2 subunits, resulting in ER-retained heterooligomers, suggesting that homomeric gamma2S is unlikely to occur in vivo. However, its independent maturation to surface competence and preferential assembly with alpha and beta subunits may ensure the production of functional benzodiazepine-sensitive receptors. Furthermore, the presence of the gamma2 subunit appears to confer an endocytotic capacity to these heterooligomeric receptors.