RESUMO
The quantification of colocalizing signals in multichannel fluorescence microscopy images depends on the reliable segmentation of corresponding regions of interest, on the selection of appropriate colocalization coefficients, and on a robust statistical criterion to discriminate true from random colocalization. Here, we introduce a confined displacement algorithm based on image correlation spectroscopy in combination with Manders colocalization coefficients M1(ROI) and M2(ROI) to quantify true and random colocalization of a given florescence pattern. We show that existing algorithms based on block scrambling exaggerate the randomization of fluorescent patterns with resulting inappropriately narrow probability density functions and false significance of true colocalization in terms of p values. We further confine our approach to subcellular compartments and show that true and random colocalization can be analysed for model dendrites and for GABA(B) receptor subunits GABA(B)R1/2 in cultured hippocampal neurons. Together, we demonstrate that the confined displacement algorithm detects true colocalization of specific fluorescence patterns down to subcellular levels.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Algoritmos , Animais , Feminino , Hipocampo/citologia , Neurônios/química , Neurônios/citologia , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/análiseRESUMO
GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores de GABA-B/metabolismo , Animais , Encéfalo/metabolismo , Química Encefálica , Células CHO , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Cricetinae , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Agonistas GABAérgicos/farmacologia , Agonistas dos Receptores de GABA-B , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Fosfoproteínas/agonistas , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Fosforilação , Canais de Potássio/metabolismo , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Ratos , Receptores de GABA-B/análise , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The pheromone response pathway of the yeast Saccharomyces cerevisiae is initiated in MATa cells by binding of alpha-factor to the alpha-factor receptor. MATa cells in which the a-factor receptor is inappropriately expressed exhibit reduced pheromone signaling, a phenomenon termed receptor inhibition. In cells undergoing receptor inhibition, activation of the signaling pathway occurs normally at early time points but decreases after prolonged exposure to pheromone. Mutations that suppress the effects of receptor inhibition were obtained in the STE4 gene, which encodes the beta-subunit of the G protein that transmits the pheromone response signal. These mutations mapped to the N terminus and second WD repeat of Ste4p in regions that are not part of its Galpha binding surface. A STE4 allele containing several of these mutations, called STE4(SD13), reversed the signaling defect seen at late times in cells undergoing receptor inhibition but had no effect on the basal activity of the pathway. Moreover, the signaling properties of STE4(SD13) were indistinguishable from those of STE4 in wild-type MATa and MATalpha cells. These results demonstrate that the effect of the STE4(SD13) allele is specific to the receptor inhibition function of STE4. STE4(SD13) suppressed the signaling defect conferred by receptor inhibition in a MATa strain containing a deletion of GPA1, the G protein alpha-subunit gene; however, STE4(SD13) had no effect in a MATalpha strain containing a GPA1 deletion. Suppression of receptor inhibition by STE4(SD13) in a MATa strain containing a GPA1 deletion was unaffected by deletion of STE2, the alpha-factor receptor gene. The results presented here are consistent with a model in which an a-specific gene product other than Ste2p detects the presence of the a-factor receptor and blocks signaling by inhibiting the function of Ste4p.
Assuntos
Proteínas Fúngicas/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP , Subunidades beta da Proteína de Ligação ao GTP , Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP , Feromônios/metabolismo , Receptores de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais , Fatores de Transcrição , Sequência de Aminoácidos , Proteínas Fúngicas/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Proteínas de Ligação ao GTP/genética , Dados de Sequência Molecular , Mutagênese , Fenótipo , Feromônios/genética , Receptores de Fator de Acasalamento , Receptores de Peptídeos/genética , Saccharomyces cerevisiae/genética , Ativação TranscricionalRESUMO
The yeast pheromone response pathway is mediated by two G protein-linked receptors, each of which is expressed only in its specific cell type. The STE3DAF mutation results in inappropriate expression of the a-factor receptor in MATa cells. Expression of this receptor in the inappropriate cell type confers resistance to pheromone-induced G1 arrest, a phenomenon that we have termed receptor inhibition. The ability of STE3DAF cells to cycle in the presence of pheromone was found to correlate with reduced phosphorylation of the cyclin-dependent kinase inhibitor Far1p. Measurement of Fus3p mitogen-activated protein (MAP) kinase activity in wild-type and STE3DAF cells showed that induction of Fus3p activity was the same in both strains at times of up to 1 h after pheromone treatment. However, after 2 or more hours, Fus3p activity declined in STE3DAF cells but remained high in wild-type cells. The level of inducible FUS1 RNA paralleled the changes seen in Fus3p activity. Short-term activation of the Fus3p MAP kinase is therefore sufficient for the early transcriptional induction response to pheromone, but sustained activation is required for cell cycle arrest. Escape from the cell cycle arrest response was not seen in wild-type cells treated with low doses of pheromone, indicating that receptor inhibition is not simply a result of weak signaling but rather acts selectively at late times during the response. STE3DAF was found to inhibit the pheromone response pathway at a step between the G beta subunit and Ste5p, the scaffolding protein that binds the components of the MAP kinase phosphorylation cascade. Overexpression of Ste20p, a kinase thought to act between the G protein and the MAP kinase cascade, suppressed the STE3DAF phenotype. These findings are consistent with a model in which receptor inhibition acts by blocking the signaling pathway downstream of G protein dissociation and upstream of MAP kinase cascade activation, at a step that could directly involve Ste20p.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular , Ciclo Celular , Proteínas Fúngicas/metabolismo , Subunidades beta da Proteína de Ligação ao GTP , Regulação Fúngica da Expressão Gênica , Proteínas Heterotriméricas de Ligação ao GTP , Proteínas Quinases Ativadas por Mitógeno , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Peptídeos/fisiologia , Receptores de Feromônios , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Sequência de Bases , Proteínas Inibidoras de Quinase Dependente de Ciclina , Primers do DNA/química , Proteínas Fúngicas/genética , Proteínas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases , Fator de Acasalamento , Proteínas de Membrana , Peptídeos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Receptores de Fator de Acasalamento , Saccharomyces cerevisiae , Transdução de Sinais , Transcrição GênicaRESUMO
Neuronal GABA(B) receptors regulate calcium and potassium currents via G-protein-coupled mechanisms and play a critical role in long-term inhibition of synaptic transmission in the CNS. Recent studies have demonstrated that assembly of GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits into functional heterodimers is required for coupling to potassium channels in heterologous systems. However whether heterodimerization is required for the coupling of GABA(B) receptors to effector systems in neurons remains to be established. To address this issue, we have studied the coupling of recombinant GABA(B) receptors to endogenous Ca(2+) channels in superior cervical ganglion (SCG) neurons using nuclear microinjection to introduce both sense and antisense expression constructs. Patch-clamp recording from neurons injected with both GABA(B)R1a/1b and GABA(B)R2 cDNAs or with GABA(B)R2 alone produced marked baclofen-mediated inhibition of Ca(2+) channel currents via a pertussis toxin-sensitive mechanism. The actions of baclofen were blocked by CGP62349, a specific GABA(B) antagonist, and were voltage dependent. Interestingly, SCGs were found to express abundantly GABA(B)R1 but not GABA(B)R2 at the protein level. To determine whether heterodimerization of GABA(B)R1 and GABA(B)R2 subunits was required for Ca(2+) inhibition, the GABA(B)R2 expression construct was microinjected with a GABA(B)R1 antisense construct. This resulted in a dramatic decrease in the levels of the endogenous GABA(B)R1 protein and a marked reduction in the inhibitory effects of baclofen on Ca(2+) currents. Therefore our results suggest that in neurons heteromeric assemblies of GABA(B)R1 and GABA(B)R2 are essential to mediate GABAergic inhibition of Ca(2+) channel currents.
Assuntos
Fibras Adrenérgicas/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Receptores de GABA-B , Receptores de GABA/metabolismo , Gânglio Cervical Superior/metabolismo , Animais , Baclofeno/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Dimerização , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Ratos , Receptores de GABA-ARESUMO
GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Subunidades Proteicas , Receptores de GABA-B/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Dimerização , Humanos , Rim/citologia , Rim/metabolismo , Microinjeções , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Ligação Proteica/fisiologia , Ratos , Receptores de GABA/genética , Receptores de GABA/metabolismo , Receptores de GABA-B/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de Proteína , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/metabolismo , TransfecçãoRESUMO
GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.
Assuntos
Líquido Intracelular/metabolismo , Subunidades Proteicas , Transporte Proteico/fisiologia , Receptores de GABA-B/metabolismo , Transdução de Sinais/fisiologia , Motivos de Aminoácidos/fisiologia , Animais , Linhagem Celular , Cricetinae , Dimerização , Proteínas de Ligação ao GTP/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína/fisiologia , Ratos , Receptores de Superfície Celular/metabolismo , Receptores de GABA-B/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Ácido gama-Aminobutírico/metabolismoRESUMO
GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.
Assuntos
Sistema Nervoso Central/metabolismo , Receptores de GABA-B/metabolismo , Animais , Encéfalo/metabolismo , Feminino , Humanos , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/genética , Medula Espinal/metabolismo , Baço/metabolismo , Distribuição Tecidual , Útero/metabolismoRESUMO
A variety of neurological diseases including Huntington's disease (HD), Alzheimer's disease and Parkinson's disease share common neuropathology, primarily featuring the presence of abnormal protein inclusions containing specific misfolded proteins. Mutations leading to expansion of a poly-glutamine track in Huntingtin cause HD, and trigger its misfolding and aggregation. Recent evidence indicates that alterations in the secretory pathway, in particular the endoplasmic reticulum (ER), are emerging features of HD. Although it is not clear how cytoplasmic/nuclear located mutant Huntingtin alters the function of the ER, several reports indicate that mutant Huntingtin affects many essential processes related to the secretory pathway, including inhibition of ER-associated degradation, altered ER/Golgi vesicular trafficking and axonal transport, disrupted autophagy and abnormal ER calcium homeostasis. All these alterations are predicted to have a common pathological outcome associated to disturbance of protein folding and maturation pathways at the ER, generating chronic ER stress and neuronal dysfunction. Here, we review recent evidence involving ER stress in HD pathogenesis and discuss possible therapeutic strategies to target organelle function in the context of disease.
Assuntos
Retículo Endoplasmático/fisiologia , Doença de Huntington/fisiopatologia , Animais , Apoptose , Cálcio/metabolismo , Humanos , Proteína Huntingtina , Doença de Huntington/terapia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Multimerização Proteica , Transporte Proteico , Deficiências na Proteostase , Estresse Fisiológico , Resposta a Proteínas não DobradasRESUMO
Yeast possess two homologs of the synaptobrevin family of vesicle-associated proteins that are proposed to be involved in membrane recognition and to act as receptors for components of the fusion machinery in neurons. We have previously described the yeast homologs, Snc1 and Snc2, and demonstrated that they localize to secretory vesicles and are required for normal secretion. Yeast lacking Snc protein expression accumulate post-Golgi transport vesicles that contain secretory proteins. Therefore, Snc proteins are essential for the fusion of carrier vesicles with the plasma membrane, and this property appears to have been conserved in evolution. We have now examined whether Snc proteins interact with other components of the late secretory pathway in yeast. Here we show that Snc proteins form a tight genetic and physical interaction with a plasma membrane protein, Sec9. Sec9 is the yeast equivalent of SNAP-25, a second receptor protein from neurons that has been shown to interact with synaptobrevin. We suggest, then, that recognition of the plasma membrane by secretory vesicles may involve the formation of a Snc-Sec9 complex and that this interaction has evolved as a fundamental step in secretory processes.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Ligação Proteica , Proteínas R-SNARE , Proteína 25 Associada a SinaptossomaRESUMO
The presence of catalase-containing particles was demonstrated in mouse macrophages of the cell line J774.3. These cells were incubated with clofibrate, a hypolipidemic drug known to cause peroxisome proliferation in rodent hepatocytes. The specific activities of catalase and fatty acid oxidase were significantly increased after 48 h incubation with 2 mM clofibrate. This response proved to be time and dose dependent. Other organelle marker enzymes showed little change in activity. Unexpectedly, clofibrate treatment also produced an increase in a 64-kDa peptide in these cells.
Assuntos
Clofibrato/farmacologia , Macrófagos/enzimologia , Microcorpos/enzimologia , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Indução Enzimática/efeitos dos fármacos , Imunofluorescência , Cinética , Macrófagos/efeitos dos fármacos , CamundongosRESUMO
Yeast possess two homologs of the synaptobrevin family of vesicle-associated membrane proteins that function in membrane recognition and vesicle fusion. Yeast proteins Snc1 and Snc2 localize to secretory vesicles and are required for constitutive exocytosis. They also form a physical complex with a plasma membrane protein, Sec9, which is necessary for vesicle docking and fusion to occur in vivo. Formation of this molecular complex, as a prerequisite for vesicle fusion, appears to have been conserved evolutionarily. Here we demonstrate that Snc proteins undergo a single posttranslational modification with the addition of a palmitate moiety to Cys-95 in Snc1. Modification of Cys-95 (which is located proximal to the transmembrane domain) is rapid, occurs in the endoplasmic reticulum, and is long-lasting. Mutation of Cys-95 to Ser-95 blocks palmitoylation and appears to affect Snc protein stability. This provides evidence that synaptobrevin-like proteins are modified posttranslationally, and we predict that fatty acylation may be common to those found in higher eukaryotes.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/química , Proteínas do Tecido Nervoso/química , Ácidos Palmíticos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas Fúngicas/biossíntese , Expressão Gênica , Genes Fúngicos , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Ácido Palmítico , Fenótipo , Proteínas R-SNARE , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
gamma-Aminobutyric acid type B (GABAB) receptors mediate the transmission of slow and prolonged inhibitory signals in the central nervous system. Two splice variants of GABAB receptors, GABABR1a and GABABR1b, were recently cloned from a mouse cortical and cerebellar cDNA library. As predicted, these receptors belong to the G protein-coupled receptor superfamily. We have used epitope-tagged versions of GABABR1a receptors to study the cellular distribution of these proteins in a variety of non-neuronal and neuronal cell types. Here we report that recombinant GABAB receptors fail to reach the cell surface when expressed in heterologous systems and are retained in the endoplasmic reticulum when introduced into COS cells. In addition, we prove that recombinant GABAB receptors are excluded from the cell surface when overexpressed in ganglion neurons and we further demonstrate that they fail to activate in superior cervical ganglion neurons. Together our observations suggest that recombinant GABAB receptors require additional information for functional targeting to the plasma membrane.
Assuntos
Receptores de GABA-B/metabolismo , Animais , Sequência de Bases , Células COS , Membrana Celular/metabolismo , Cricetinae , Primers do DNA , Retículo Endoplasmático/metabolismo , Gânglios/citologia , Gânglios/metabolismo , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Receptores de GABA-B/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/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.