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1.
J Neurosci ; 40(10): 2000-2014, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019829

RESUMO

The activation of neuronal plasma membrane Ca2+ channels stimulates many intracellular responses. Scaffolding proteins can preferentially couple specific Ca2+ channels to distinct downstream outputs, such as increased gene expression, but the molecular mechanisms that underlie the exquisite specificity of these signaling pathways are incompletely understood. Here, we show that complexes containing CaMKII and Shank3, a postsynaptic scaffolding protein known to interact with L-type calcium channels (LTCCs), can be specifically coimmunoprecipitated from mouse forebrain extracts. Activated purified CaMKIIα also directly binds Shank3 between residues 829 and 1130. Mutation of Shank3 residues 949Arg-Arg-Lys951 to three alanines disrupts CaMKII binding in vitro and CaMKII association with Shank3 in heterologous cells. Our shRNA/rescue studies revealed that Shank3 binding to both CaMKII and LTCCs is important for increased phosphorylation of the nuclear CREB transcription factor and expression of c-Fos induced by depolarization of cultured hippocampal neurons. Thus, this novel CaMKII-Shank3 interaction is essential for the initiation of a specific long-range signal from LTCCs in the plasma membrane to the nucleus that is required for activity-dependent changes in neuronal gene expression during learning and memory.SIGNIFICANCE STATEMENT Precise neuronal expression of genes is essential for normal brain function. Proteins involved in signaling pathways that underlie activity-dependent gene expression, such as CaMKII, Shank3, and L-type calcium channels, are often mutated in multiple neuropsychiatric disorders. Shank3 and CaMKII were previously shown to bind L-type calcium channels, and we show here that Shank3 also binds to CaMKII. Our data show that each of these interactions is required for depolarization-induced phosphorylation of the CREB nuclear transcription factor, which stimulates the expression of c-Fos, a neuronal immediate early gene with key roles in synaptic plasticity, brain development, and behavior.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Núcleo Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos , Transdução de Sinais/fisiologia
2.
J Neurochem ; 153(3): 334-345, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31985073

RESUMO

Brain endocannabinoids serve as retrograde neurotransmitters, being synthesized in post-synaptic neurons "on demand" and released to bind pre-synaptic cannabinoid receptors and suppress glutamatergic or GABAergic transmission. The most abundant brain endocannabinoid, 2 arachidonoyl glycerol (2-AG), is primarily synthesized by diacylglycerol lipase-α (DGLα), which is activated by poorly understood mechanisms in response to calcium influx following post-synaptic depolarization and/or the activation of Gq -coupled group 1 metabotropic glutamate receptors. However, the impact of other neurotransmitters and their downstream signaling pathways on synaptic 2-AG signaling has not been intensively studied. Here, we found that DGLα activity in membrane fractions from transfected HEK293T cells was significantly increased by in vitro phosphorylation using cyclic AMP-dependent protein kinase (PKA). Moreover, PKA directly phosphorylated DGLα at Ser798 in vitro. Elevation of cAMP levels in HEK293 cells expressing DGLα increased Ser798 phosphorylation, as detected using a phospho-Ser798-specific antibody, and enhanced DGLα activity; this in situ enhancement of DGLα activity was prevented by mutation of Ser798 to Ala. We investigated the impact of PKA on synaptic 2-AG mobilization in mouse striatal slices by manipulating D1-dopamine receptor (D1R) signaling and assessing depolarization-induced suppression of excitation, a DGLα- and 2-AG-dependent form of short-term synaptic depression. The magnitude of depolarization-enhanced suppression of excitation in direct pathway medium spiny neurons was increased by pre-incubation with a D1R agonist, and this enhancement was blocked by post-synaptic inhibition of PKA. Taken together, these findings provide new molecular insights into the complex mechanisms regulating synaptic endocannabinoid signaling.


Assuntos
Ácidos Araquidônicos/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Lipase Lipoproteica/metabolismo , Receptores de Dopamina D1/metabolismo , Transdução de Sinais/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos
3.
J Neurosci ; 37(8): 2216-2233, 2017 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-28130356

RESUMO

Characterizing the functional impact of novel mutations linked to autism spectrum disorder (ASD) provides a deeper mechanistic understanding of the underlying pathophysiological mechanisms. Here we show that a de novo Glu183 to Val (E183V) mutation in the CaMKIIα catalytic domain, identified in a proband diagnosed with ASD, decreases both CaMKIIα substrate phosphorylation and regulatory autophosphorylation, and that the mutated kinase acts in a dominant-negative manner to reduce CaMKIIα-WT autophosphorylation. The E183V mutation also reduces CaMKIIα binding to established ASD-linked proteins, such as Shank3 and subunits of l-type calcium channels and NMDA receptors, and increases CaMKIIα turnover in intact cells. In cultured neurons, the E183V mutation reduces CaMKIIα targeting to dendritic spines. Moreover, neuronal expression of CaMKIIα-E183V increases dendritic arborization and decreases both dendritic spine density and excitatory synaptic transmission. Mice with a knock-in CaMKIIα-E183V mutation have lower total forebrain CaMKIIα levels, with reduced targeting to synaptic subcellular fractions. The CaMKIIα-E183V mice also display aberrant behavioral phenotypes, including hyperactivity, social interaction deficits, and increased repetitive behaviors. Together, these data suggest that CaMKIIα plays a previously unappreciated role in ASD-related synaptic and behavioral phenotypes.SIGNIFICANCE STATEMENT Many autism spectrum disorder (ASD)-linked mutations disrupt the function of synaptic proteins, but no single gene accounts for >1% of total ASD cases. The molecular networks and mechanisms that couple the primary deficits caused by these individual mutations to core behavioral symptoms of ASD remain poorly understood. Here, we provide the first characterization of a mutation in the gene encoding CaMKIIα linked to a specific neuropsychiatric disorder. Our findings demonstrate that this ASD-linked de novo CAMK2A mutation disrupts multiple CaMKII functions, induces synaptic deficits, and causes ASD-related behavioral alterations, providing novel insights into the synaptic mechanisms contributing to ASD.


Assuntos
Transtorno do Espectro Autista , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Dendritos/metabolismo , Mutação/genética , Transmissão Sináptica/genética , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Transtorno do Espectro Autista/fisiopatologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Células Cultivadas , Cicloeximida/farmacologia , Modelos Animais de Doenças , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Comportamento Exploratório/fisiologia , Feminino , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo
4.
Mol Pharmacol ; 94(6): 1352-1362, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30282777

RESUMO

Ca2+/calmodulin-dependent protein kinase II (CaMKII) and metabotropic glutamate receptor 5 (mGlu5) are critical signaling molecules in synaptic plasticity and learning/memory. Here, we demonstrate that mGlu5 is present in CaMKIIα complexes isolated from mouse forebrain. Further in vitro characterization showed that the membrane-proximal region of the C-terminal domain (CTD) of mGlu5a directly interacts with purified Thr286-autophosphorylated (activated) CaMKIIα However, the binding of CaMKIIα to this CTD fragment is reduced by the addition of excess Ca2+/calmodulin or by additional CaMKIIα autophosphorylation at non-Thr286 sites. Furthermore, in vitro binding of CaMKIIα is dependent on a tribasic residue motif Lys-Arg-Arg (KRR) at residues 866-868 of the mGlu5a-CTD, and mutation of this motif decreases the coimmunoprecipitation of CaMKIIα with full-length mGlu5a expressed in heterologous cells by about 50%. The KRR motif is required for two novel functional effects of coexpressing constitutively active CaMKIIα with mGlu5a in heterologous cells. First, cell-surface biotinylation studies showed that CaMKIIα increases the surface expression of mGlu5a Second, using Ca2+ fluorimetry and single-cell Ca2+ imaging, we found that CaMKIIα reduces the initial peak of mGlu5a-mediated Ca2+ mobilization by about 25% while doubling the relative duration of the Ca2+ signal. These findings provide new insights into the physical and functional coupling of these key regulators of postsynaptic signaling.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Animais , Calmodulina/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Feminino , Células HEK293 , Humanos , Imunoprecipitação/métodos , Masculino , Camundongos , Camundongos Knockout , Ligação Proteica/fisiologia , Transdução de Sinais
5.
J Biol Chem ; 288(31): 22248-56, 2013 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-23782696

RESUMO

Brain-specific angiogenesis inhibitor-1 (BAI1) is an adhesion G protein-coupled receptor that has been studied primarily for its anti-angiogenic and anti-tumorigenic properties. We found that overexpression of BAI1 results in activation of the Rho pathway via a Gα(12/13)-dependent mechanism, with truncation of the BAI1 N terminus resulting in a dramatic enhancement in receptor signaling. This constitutive activity of the truncated BAI1 mutant also resulted in enhanced downstream phosphorylation of ERK as well as increased receptor association with ß-arrestin2 and increased ubiquitination of the receptor. To gain insights into the regulation of BAI1 signaling, we screened the C terminus of BAI1 against a proteomic array of PDZ domains to identify novel interacting partners. These screens revealed that the BAI1 C terminus interacts with a variety of PDZ domains from synaptic proteins, including MAGI-3. Removal of the BAI1 PDZ-binding motif resulted in attenuation of receptor signaling to Rho but had no effect on ERK activation. Conversely, co-expression with MAGI-3 was found to potentiate signaling to ERK by constitutively active BAI1 in a manner that was dependent on the PDZ-binding motif of the receptor. Biochemical fractionation studies revealed that BAI1 is highly enriched in post-synaptic density fractions, a finding consistent with our observations that BAI1 can interact with PDZ proteins known to be concentrated in the post-synaptic density. These findings demonstrate that BAI1 is a synaptic receptor that can activate both the Rho and ERK pathways, with the N-terminal and C-terminal regions of the receptor playing key roles in the regulation of BAI1 signaling activity.


Assuntos
Proteínas Angiogênicas/metabolismo , Densidade Pós-Sináptica/metabolismo , Transdução de Sinais , Proteínas Angiogênicas/fisiologia , Animais , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Camundongos , Domínios PDZ , Ligação Proteica , Receptores Acoplados a Proteínas G
6.
J Biol Chem ; 286(33): 28914-28921, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21708946

RESUMO

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of ß-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with ß-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.


Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Arrestinas/genética , Arrestinas/metabolismo , Córtex Cerebral/anormalidades , Córtex Cerebral/metabolismo , Células HEK293 , Humanos , Mutação , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/genética , Ubiquitinação/genética , beta-Arrestina 2 , beta-Arrestinas
7.
Biol Psychiatry ; 84(4): 304-315, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29458998

RESUMO

BACKGROUND: Endocannabinoid signaling plays an important role in regulating synaptic transmission in the striatum, a brain region implicated as a central node of dysfunction in autism spectrum disorder. Deficits in signaling mediated by the endocannabinoid 2-arachidonoylglycerol (2-AG) have been reported in mouse models of autism spectrum disorder, but a causal role for striatal 2-AG deficiency in phenotypes relevant to autism spectrum disorder has not been explored. METHODS: Using conditional knockout mice, we examined the electrophysiological, biochemical, and behavioral effects of 2-AG deficiency by deleting its primary synthetic enzyme, diacylglycerol lipase α (DGLα), from dopamine D1 receptor-expressing or adenosine A2a receptor-expressing medium spiny neurons (MSNs) to determine the role of 2-AG signaling in striatal direct or indirect pathways, respectively. We then used viral-mediated deletion of DGLα to study the effects of 2-AG deficiency in the ventral and dorsal striatum. RESULTS: Targeted deletion of DGLα from direct-pathway MSNs caused deficits in social interaction, excessive grooming, and decreased exploration of a novel environment. In contrast, deletion from indirect-pathway MSNs had no effect on any measure of behavior examined. Loss of 2-AG in direct-pathway MSNs also led to increased glutamatergic drive, which is consistent with a loss of retrograde feedback inhibition. Subregional DGLα deletion from the dorsal striatum produced deficits in social interaction, whereas deletion from the ventral striatum resulted in repetitive grooming. CONCLUSIONS: These data suggest a role for 2-AG deficiency in social deficits and repetitive behavior, and they demonstrate a key role for 2-AG in regulating striatal direct-pathway MSNs.


Assuntos
Ácidos Araquidônicos/metabolismo , Corpo Estriado/metabolismo , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D1/metabolismo , Comportamento Social , Animais , Ácidos Araquidônicos/deficiência , Transtorno do Espectro Autista/metabolismo , Endocanabinoides/deficiência , Glicerídeos/deficiência , Camundongos , Camundongos Knockout , Transdução de Sinais , Transmissão Sináptica
8.
Protein Sci ; 16(3): 401-10, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17242372

RESUMO

Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.


Assuntos
Arginina/genética , Ácido Aspártico/genética , Coproporfirinogênio Oxidase/química , Alanina/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Catálise , Coproporfirinogênio Oxidase/genética , Coproporfirinogênios/química , Humanos , Cinética , Mesoporfirinas/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Porfirinogênios/química , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos
9.
J Clin Invest ; 125(4): 1497-508, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25751059

RESUMO

Synaptic plasticity is the ability of synapses to modulate the strength of neuronal connections; however, the molecular factors that regulate this feature are incompletely understood. Here, we demonstrated that mice lacking brain-specific angiogenesis inhibitor 1 (BAI1) have severe deficits in hippocampus-dependent spatial learning and memory that are accompanied by enhanced long-term potentiation (LTP), impaired long-term depression (LTD), and a thinning of the postsynaptic density (PSD) at hippocampal synapses. We showed that compared with WT animals, mice lacking Bai1 exhibit reduced protein levels of the canonical PSD component PSD-95 in the brain, which stems from protein destabilization. We determined that BAI1 prevents PSD-95 polyubiquitination and degradation through an interaction with murine double minute 2 (MDM2), the E3 ubiquitin ligase that regulates PSD-95 stability. Restoration of PSD-95 expression in hippocampal neurons in BAI1-deficient mice by viral gene therapy was sufficient to compensate for Bai1 loss and rescued deficits in synaptic plasticity. Together, our results reveal that interaction of BAI1 with MDM2 in the brain modulates PSD-95 levels and thereby regulates synaptic plasticity. Moreover, these results suggest that targeting this pathway has therapeutic potential for a variety of neurological disorders.


Assuntos
Proteínas Angiogênicas/fisiologia , Guanilato Quinases/metabolismo , Hipocampo/fisiopatologia , Deficiências da Aprendizagem/genética , Proteínas de Membrana/metabolismo , Transtornos da Memória/genética , Proteínas do Tecido Nervoso/fisiologia , Plasticidade Neuronal/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Aprendizagem Espacial/fisiologia , Proteínas Angiogênicas/deficiência , Proteínas Angiogênicas/genética , Animais , Encéfalo/irrigação sanguínea , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases/deficiência , Guanilato Quinases/genética , Células HEK293 , Hipocampo/patologia , Humanos , Curva de Aprendizado , Deficiências da Aprendizagem/fisiopatologia , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto/fisiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Neurônios/ultraestrutura , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transmissão Sináptica/fisiologia , Ubiquitinação
10.
Trends Pharmacol Sci ; 35(4): 208-15, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24642458

RESUMO

The brain-specific angiogenesis inhibitors 1-3 (BAI1-3) comprise a subfamily of adhesion G-protein-coupled receptors (GPCRs). These receptors are highly expressed in the brain and were first studied for their ability to inhibit angiogenesis and tumor formation. Subsequently, BAI1 was found to play roles in apoptotic cell phagocytosis and myoblast fusion. Until recently, however, little was known about the physiological importance of the BAI subfamily in the context of normal brain function. Recent work has provided evidence for key roles of BAI1-3 in the regulation of synaptogenesis and dendritic spine formation. In this review, we summarize the current understanding of the BAI subfamily with regard to downstream signaling pathways, physiological actions, and potential importance as novel drug targets in the treatment of psychiatric and neurological diseases.


Assuntos
Proteínas Angiogênicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sinapses/metabolismo , Animais , Humanos
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