RESUMO
The modulation of GABAA receptors by protein kinase C is complex and involves effects on both ion channel function and receptor trafficking. Although PKC regulates receptor cell surface expression the mechanism is not well understood. Using immunofluorescence studies in HEK 293 cells, we demonstrate that activation of PKC by the phorbol ester PMA promotes receptor endocytosis and is dependent on the presence of a gamma subunit. This endocytosis is blocked by the dominant negative dynamin mutant K44A indicating that PKC-induced receptor endocytosis involves the dynamin endocytic pathway. Mutation of a dileucine motif within the receptor beta2 subunit inhibits the effect of PKC activation on receptor endocytosis. Using patch clamp analysis, we show that PKC activation produces a robust inhibition of GABA-gated chloride currents in cells expressing wildtype GABAA receptors, but it is ineffective in modulating receptors lacking the dileucine motif. Furthermore, the introduction into the patch pipette of a 10-amino acid peptide corresponding to the dileucine motif present in the receptor beta2 subunit prevents PKC modulation of wildtype recombinant receptors. Furthermore, in cerebral cortical neuronal slices inclusion of this peptide in the patch pipette prevents PKC modulation of native GABAA receptors. Using limited chymotrypsin digestion assays, we also show that PKC increases receptor internalization in primary cultures of cerebral cortical neurons. Lastly, PKC inhibitors do not block constitutive receptor endocytosis or affect GABA-gated chloride currents suggesting that PKC-dependent phosphorylation is not required for GABAA receptor endocytosis but plays a modulatory role in the process.
Assuntos
Endocitose/fisiologia , Leucina/metabolismo , Mutação , Proteína Quinase C/metabolismo , Receptores de GABA-A/metabolismo , Animais , Linhagem Celular , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Dipeptídeos/genética , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Endocitose/efeitos dos fármacos , Feminino , Humanos , Técnicas In Vitro , Leucina/genética , Leucina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/genéticaRESUMO
We describe a new RNA binding protein from Xenopus we have named ePABP2 (embryonic poly(A) binding protein type II). Based on amino acid similarity, ePABP2 is closely related to the ubiquitously expressed nuclear PABP2 protein that directs the elongation of mRNA poly(A) tails during pre-mRNA processing. However, in contrast to known PABP2 proteins, Xenopus ePABP2 is a cytoplasmic protein that is predominantly expressed during the early stages of Xenopus development and in adult ovarian tissue. Biochemical experiments indicate ePABP2 binds poly(A) with specificity and that this binding requires the RRM domain. Mouse and human ePABP2 proteins were also identified and mouse ePABP2 expression is also confined to the earliest stages of mouse development and adult ovarian tissue. We propose that Xenopus ePABP2 is the founding member of a new class of poly(A) binding proteins expressed in vertebrate embryos. Possible roles for this protein in regulating mRNA function in early vertebrate development are discussed.
Assuntos
Citoplasma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Poli A/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Xenopus/embriologia , Xenopus/genética , Envelhecimento/fisiologia , Sequência de Aminoácidos , Animais , Citoplasma/genética , Feminino , Humanos , Camundongos , Dados de Sequência Molecular , Oócitos/citologia , Oócitos/metabolismo , Oogênese/genética , Ovário/citologia , Ovário/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Proteínas de XenopusRESUMO
Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA(A) receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA(A) receptors composed of either alpha1beta2gamma2Lor alpha1beta2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor beta2 subunit that is critical for endocytosis. Internalization of GABAA receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor beta2 subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA(A) receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.
