RESUMO
Transient receptor potential vanilloids (TRPV1) are non-selective cation channels that sense and transduce inflammatory pain signals. We previously reported that activation of TRPV1 induced the translocation of ß-arrestin2 (ARRB2) from the cytoplasm to the nucleus, raising questions about the functional role of ARRB2 in the nucleus. Here, we determined the ARRB2 nuclear signalosome by conducting a quantitative proteomic analysis of the nucleus-sequestered L395Q ARRB2 mutant, compared to the cytosolic wild-type ARRB2 (WT ARRB2), in a heterologous expression system. We identified clusters of proteins that localize to the nucleolus and are involved in ribosomal biogenesis. Accordingly, L395Q ARRB2 or WT ARRB2 after capsaicin treatment were found to co-localize and interact with the nucleolar marker nucleophosmin (NPM1), treacle protein (TCOF1) and RNA polymerase I (POL I). We further investigated the role of nuclear ARRB2 signaling in regulating neuroplasticity. Using neuroblastoma (neuro2a) cells and dorsal root ganglia (DRG) neurons, we found that L395Q ARRB2 mutant increased POL I activity, inhibited the tumor suppressorp53 (p53) level and caused a decrease in the outgrowth of neurites. Together, our results suggest that the activation of TRPV1 promotes the ARRB2-mediated regulation of ribosomal biogenesis in the nucleolus. The ARRB2-TCOF1-p53 checkpoint signaling pathway might be involved in regulating neurite outgrowth associated with pathological pain conditions.
Assuntos
Nucléolo Celular/metabolismo , Crescimento Neuronal , Ribossomos/metabolismo , Canais de Cátion TRPV/metabolismo , Proteína Supressora de Tumor p53/metabolismo , beta-Arrestina 2/metabolismo , Animais , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Nucleofosmina , Ligação Proteica , Transporte Proteico , Proteômica , RNA Polimerase I/metabolismoRESUMO
BACKGROUND: Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund's Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. RESULTS: We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund's Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. CONCLUSIONS: Our work identified Hsc70 and its ATPase activity as a central cofactor of TRPV1 channel function and points to the role of this stress protein in pain associated with neurodegenerative and/or metabolic disorders, including aging.
Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Neurônios/metabolismo , Canais de Cátion TRPV/metabolismo , Alcaloides/farmacologia , Animais , Anti-Helmínticos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Capsaicina/farmacologia , Células Cultivadas , Césio/farmacologia , Cloretos/farmacologia , Inibidores Enzimáticos/farmacologia , Adjuvante de Freund/toxicidade , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Guanidinas/farmacologia , Células HEK293 , Proteínas de Choque Térmico HSC70/genética , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Camundongos , Neurônios/efeitos dos fármacos , Dor/etiologia , Dor/metabolismo , Dor/patologia , Bloqueadores dos Canais de Potássio/farmacologia , Quinolizinas/farmacologia , Ratos , Canais de Cátion TRPV/genética , MatrinasRESUMO
The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief.
Assuntos
Hiperalgesia/metabolismo , Multimerização Proteica , Canais de Cátion TRPV/metabolismo , Motivos de Aminoácidos , Animais , Artrite Experimental/metabolismo , Artrite Experimental/fisiopatologia , Sítios de Ligação , Deleção de Genes , Células HEK293 , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Ratos , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genéticaRESUMO
Quiescent phases of inflammatory bowel disease (IBD) are often accompanied by chronic abdominal pain. Although the transient receptor potential vanilloid 1 (TRPV1) ion channel has been postulated as an important mediator of visceral hypersensitivity, its functional role in postinflammatory pain remains elusive. This study aimed at establishing the role of TRPV1 in the peripheral sensitization underlying chronic visceral pain in the context of colitis. Wild-type and TRPV1-deficient mice were separated into three groups (control, acute colitis, and recovery), and experimental colitis was induced by oral administration of dextran sulfate sodium (DSS). Recovery mice showed increased chemically and mechanically evoked visceral hypersensitivity 5 wk post-DSS discontinuation, at which point inflammation had completely resolved. Significant changes in nonevoked pain-related behaviors could also be observed in these animals, indicative of persistent discomfort. These behavioral changes correlated with elevated colonic levels of substance P (SP) and TRPV1 in recovery mice, thus leading to the hypothesis that SP could sensitize TRPV1 function. In vitro experiments revealed that prolonged exposure to SP could indeed sensitize capsaicin-evoked currents in both cultured neurons and TRPV1-transfected human embryonic kidney (HEK) cells, a mechanism that involved TRPV1 ubiquitination and subsequent accumulation at the plasma membrane. Importantly, although TRPV1-deficient animals experienced similar disease severity and pain as wild-type mice in the acute phase of colitis, TRPV1 deletion prevented the development of postinflammatory visceral hypersensitivity and pain-associated behaviors. Collectively, our results suggest that chronic exposure of colon-innervating primary afferents to SP could sensitize TRPV1 and thus participate in the establishment of persistent abdominal pain following acute inflammation.
Assuntos
Dor Abdominal/metabolismo , Colite/metabolismo , Colo/inervação , Hiperalgesia/metabolismo , Limiar da Dor , Canais de Cátion TRPV/metabolismo , Dor Visceral/metabolismo , Dor Abdominal/induzido quimicamente , Dor Abdominal/genética , Dor Abdominal/fisiopatologia , Doença Aguda , Animais , Comportamento Animal , Colite/induzido quimicamente , Colite/genética , Colite/fisiopatologia , Sulfato de Dextrana , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Células HEK293 , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hiperalgesia/fisiopatologia , Hiperalgesia/prevenção & controle , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Aferentes/metabolismo , Medição da Dor , Transdução de Sinais , Substância P/metabolismo , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genética , Fatores de Tempo , Transfecção , Dor Visceral/induzido quimicamente , Dor Visceral/genética , Dor Visceral/fisiopatologiaRESUMO
Abstract Sympathetic modulation of cardiac L-type calcium channels is an important mechanism for regulating heart rate and cardiac contractility. At the molecular level, activation of ß-adrenergic receptors (ßAR) increases calcium influx into cardiac myocytes by activating protein kinase A (PKA), leading to subsequent phosphorylation of L-type calcium channels. In the case of the ß2AR, this process is facilitated by the presence of A-Kinase Anchoring Proteins (AKAPs) that serve as scaffolding proteins for the L-type calcium channel and the ß2AR complex. Our work has shown that, in addition to facilitating PKA phosphorylation of the channel, AKAPs also promote an increase in the Cav1.2 channel surface expression. Here we review the molecular mechanisms of ß2AR/AKAP/L-type channel interactions and trafficking.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Canais de Cálcio Tipo L/metabolismo , Transporte Proteico , Receptores Adrenérgicos beta 2/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Canais de Cálcio Tipo L/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Frequência Cardíaca/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Contração Miocárdica/genética , Fosforilação , Receptores Adrenérgicos beta 2/genéticaRESUMO
The anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase known for its oncogenic potential that is involved in the development of the peripheral and central nervous system. ALK receptor ligands ALKAL1 and ALKAL2 were recently found to promote neuronal differentiation and survival. Here, we show that inflammation or injury enhanced ALKAL2 expression in a subset of TRPV1+ sensory neurons. Notably, ALKAL2 was particularly enriched in both mouse and human peptidergic nociceptors, yet weakly expressed in nonpeptidergic, large-diameter myelinated neurons or in the brain. Using a coculture expression system, we found that nociceptors exposed to ALKAL2 exhibited heightened excitability and neurite outgrowth. Intraplantar CFA or intrathecal infusion of recombinant ALKAL2 led to ALK phosphorylation in the lumbar dorsal horn of the spinal cord. Finally, depletion of ALKAL2 in dorsal root ganglia or blocking ALK with clinically available compounds crizotinib or lorlatinib reversed thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury, respectively. Overall, our work uncovers the ALKAL2/ALK signaling axis as a central regulator of nociceptor-induced sensitization. We propose that clinically approved ALK inhibitors used for non-small cell lung cancer and neuroblastomas could be repurposed to treat persistent pain conditions.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Citocinas/metabolismo , Neoplasias Pulmonares , Animais , Humanos , Hiperalgesia/metabolismo , Inflamação/patologia , Ligantes , Camundongos , Dor/tratamento farmacológico , Receptores Proteína Tirosina Quinases , Células Receptoras Sensoriais/metabolismo , Corno Dorsal da Medula Espinal/patologiaRESUMO
Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB1) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB1 receptors (pmCB1), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB1 receptors (mtCB1) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB1 receptor signaling within striatonigral circuits determines multimodal control of behavior.
Assuntos
Encéfalo/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Catalepsia/induzido quimicamente , Membrana Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Nociceptividade/efeitos dos fármacos , Nociceptividade/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
Postoperative shivering and cold hypersensitivity are major side effects of acute and chronic opioid treatments respectively. TRPM8 is a cold and menthol-sensitive channel found in a subset of dorsal root ganglion (DRG) nociceptors. Deletion or inhibition of the TRPM8 channel was found to prevent the cold hyperalgesia induced by chronic administration of morphine. Here, we examined the mechanisms by which morphine was able to promote cold hypersensitivity in DRG neurons and transfected HEK cells. Mice daily injected with morphine for 5 days developed cold hyperalgesia. Treatment with morphine did not alter the expressions of cold sensitive TREK-1, TRAAK and TRPM8 in DRGs. However, TRPM8-expressing DRG neurons isolated from morphine-treated mice exhibited hyperexcitability. Sustained morphine treatment in vitro sensitized TRPM8 responsiveness to cold or menthol and reduced activation-evoked desensitization of the channel. Blocking phospholipase C (PLC) as well as protein kinase C beta (PKCß), but not protein kinase A (PKA) or Rho-associated protein kinase (ROCK), restored channel desensitization. Identification of two PKC phosphorylation consensus sites, S1040 and S1041, in the TRPM8 and their site-directed mutation were able to prevent the MOR-induced reduction in TRPM8 desensitization. Our results show that activation of MOR by morphine 1) promotes hyperexcitability of TRPM8-expressing neurons and 2) induces a PKCß-mediated reduction of TRPM8 desensitization. This MOR-PKCß dependent modulation of TRPM8 may underlie the onset of cold hyperalgesia caused by repeated administration of morphine. Our findings point to TRPM8 channel and PKCß as important targets for opioid-induced cold hypersensitivity.
Assuntos
Morfina/farmacologia , Proteína Quinase C beta/metabolismo , Receptores Opioides mu/metabolismo , Transdução de Sinais , Canais de Cátion TRPM/metabolismo , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Hiperalgesia/patologia , Masculino , Mentol , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Synapses established during central nervous system development can be modified through synapse elimination and formation. These processes are, in part, activity dependent and require regulated trafficking of post-synaptic components. Here, we investigate the activity-driven remodeling of cultured rat hippocampal neurons at 14 days in vitro, focusing on the post-synaptic proteins PSD-95, Shank, neuroligin (NL)1 and actin. Using live imaging and photoconductive stimulation, we found that high-frequency activity altered the trajectory, but not velocity, of PSD-95-GFP and Shank-YFP clusters, whereas it reduced the speed and increased the number of NL1 clusters. Actin-CFP reorganized into puncta following activity and approximately 50% of new puncta colocalized with NL1 clusters. Actin reorganization was enhanced by the overexpression of NL1 and decreased by the expression of an NL1 mutant, NL1-R473C. These results demonstrate activity-dependent changes that may result in the formation of new post-synaptic sites and suggest that NL1 modulates actin reorganization. The results also suggest that a common mechanism underlies both the developmental and activity-dependent remodeling of excitatory synapses.
Assuntos
Actinas/metabolismo , Molécula de Adesão de Leucócito Ativado/metabolismo , Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Actinas/genética , Molécula de Adesão de Leucócito Ativado/genética , Análise de Variância , Animais , Animais Recém-Nascidos , Contagem de Células/métodos , Proteína 4 Homóloga a Disks-Large , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica/efeitos da radiação , Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Luminescentes/genética , Proteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso , Estimulação Luminosa/métodos , Transporte Proteico/fisiologia , Transporte Proteico/efeitos da radiação , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Ratos , Ratos Sprague-Dawley , Transfecção/métodosRESUMO
Pain and inflammation are inherently linked responses to injury, infection, or chronic diseases. Given that acute inflammation in humans or mice enhances the analgesic properties of opioids, there is much interest in determining the inflammatory transducers that prime opioid receptor signaling in primary afferent nociceptors. Here, we found that activation of the transient receptor potential vanilloid type 1 (TRPV1) channel stimulated a mitogen-activated protein kinase (MAPK) signaling pathway that was accompanied by the shuttling of the scaffold protein ß-arrestin2 to the nucleus. The nuclear translocation of ß-arrestin2 in turn prevented its recruitment to the µ-opioid receptor (MOR), the subsequent internalization of agonist-bound MOR, and the suppression of MOR activity that occurs upon receptor desensitization. Using the complete Freund's adjuvant (CFA) inflammatory pain model to examine the role of TRPV1 in regulating endogenous opioid analgesia in mice, we found that naloxone methiodide (Nal-M), a peripherally restricted, nonselective, and competitive opioid receptor antagonist, slowed the recovery from CFA-induced hypersensitivity in wild-type, but not TRPV1-deficient, mice. Furthermore, we showed that inflammation prolonged morphine-induced antinociception in a mouse model of opioid receptor desensitization, a process that depended on TRPV1. Together, our data reveal a TRPV1-mediated signaling pathway that serves as an endogenous pain-resolution mechanism by promoting the nuclear translocation of ß-arrestin2 to minimize MOR desensitization. This previously uncharacterized mechanism may underlie the peripheral opioid control of inflammatory pain. Dysregulation of the TRPV1-ß-arrestin2 axis may thus contribute to the transition from acute to chronic pain.
Assuntos
Dor Aguda/metabolismo , Analgésicos Opioides/farmacologia , Dor Crônica/metabolismo , Naloxona/análogos & derivados , Antagonistas de Entorpecentes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Dor Aguda/induzido quimicamente , Dor Aguda/tratamento farmacológico , Dor Aguda/genética , Analgesia , Animais , Dor Crônica/induzido quimicamente , Dor Crônica/tratamento farmacológico , Dor Crônica/genética , Modelos Animais de Doenças , Adjuvante de Freund/efeitos adversos , Adjuvante de Freund/farmacologia , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Naloxona/farmacologia , Compostos de Amônio Quaternário/farmacologia , Transdução de Sinais/genética , Canais de Cátion TRPV/genética , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismoRESUMO
Rem2 belongs to the RGK family of small GTPases whose members are known to interact with the voltage gated calcium channel beta subunit, and to inhibit or abolish calcium currents. To identify the underlying functional domains of Rem2, we created several N- or C-terminally truncated Rem2 proteins and examined their abilities to interact with the Ca(v) beta subunit and to regulate the activities of Ca(v)2.2 N-type calcium channels. Confocal imaging of Rem2 in tsA-201 cells revealed that it contains a membrane-targeting signal in its C-terminus, consistent with previous studies. Co-precipitation assays showed that Ca(v) beta(3) interaction depends on Rem2 residues 1-123. Only Rem2 proteins that targeted the cell membrane as well as bound the beta subunit were able to reduce whole cell calcium currents.
Assuntos
Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/metabolismo , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Rim/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Relação Estrutura-AtividadeRESUMO
The neuroligins are a family of postsynaptic transmembrane proteins that associate with presynaptic partners, the beta-neurexins. Neurexins and neuroligins play a critical role in initiating formation and differentiation of synaptic junctions. A recent study reported that a mutation of neuroligin-3 (NL3), an X-linked gene, was found in siblings with autistic spectrum disorder in which two affected brothers had a point mutation that substituted a Cys for Arg451. To characterize the mutation at the biochemical level, we analyzed expression and activity of the mutated protein. Mass spectrometry comparison of the disulfide bonding pattern between the native and the mutated proteins indicates the absence of aberrant disulfide bonding, suggesting that the secondary structure of the mutated protein is conserved. However, the mutation separately affects protein expression and activity. The Cys mutation causes defective neuroligin trafficking, leading to retention of the protein in the endoplasmic reticulum. This, in turn, decreases the delivery of NL3 to the cell surface. Also, the small fraction of protein that reaches the cell membrane lacks or has markedly diminished beta-neurexin-1 (NX1beta) binding activity. Other substitutions for Arg451 allow for normal cellular expression but diminished affinity for NX1beta. Our findings reveal a cellular phenotype and loss of function for a congenital mutation associated with autistic spectrum disorders.
Assuntos
Transtorno Autístico/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional/genética , Substituição de Aminoácidos , Animais , Moléculas de Adesão Celular Neuronais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Expressão Gênica , Humanos , Immunoblotting , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas do Tecido Nervoso/química , Ligação Proteica/genética , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Solubilidade , Ressonância de Plasmônio de Superfície , TransfecçãoRESUMO
BACKGROUND: Rem2 is a small monomeric GTP-binding protein of the RGK family, whose known functions are modulation of calcium channel currents and alterations of cytoskeletal architecture. Rem2 is the only RGK protein found predominantly in the brain, where it has been linked to synaptic development. We wished to determine the effect of neuronal activity on the subcellular distribution of Rem2 and its interacting partners. RESULTS: We show that Rem2 undergoes activity-and N-Methyl-D-Aspartate Receptor (NMDAR)-dependent translocation in rat hippocampal neurons. This redistribution of Rem2, from a diffuse pattern to one that is highly punctate, is dependent on Ca(2+) influx, on binding to calmodulin (CaM), and also involves an auto-inhibitory domain within the Rem2 distal C-terminus region. We found that Rem2 can bind to Ca(2+)/CaM-dependent protein kinase IIα (CaMKII) a in Ca(2+)/CaM-dependent manner. Furthermore, our data reveal a spatial and temporal correlation between NMDAR-dependent clustering of Rem2 and CaMKII in neurons, indicating co-assembly and co-trafficking in neurons. Finally, we show that inhibiting CaMKII aggregation in neurons and HEK cells reduces Rem2 clustering, and that Rem2 affects the baseline distribution of CaMKII in HEK cells. CONCLUSIONS: Our data suggest a novel function for Rem2 in co-trafficking with CaMKII, and thus potentially expose a role in neuronal plasticity.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , DNA/metabolismo , Epitopos/química , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Hipocampo/metabolismo , Humanos , Microscopia de Fluorescência/métodos , Plasticidade Neuronal , Neurônios/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
The RGK family of proteins, small GTPases of the Ras superfamily, are known to regulate calcium currents. It is commonly thought that this is due to an interaction with the Cavß subunit, however, the mechanism of this inhibition is unclear. There have been conflicting reports of whether RGK proteins can affect channel trafficking or whether they reduce calcium currents by interacting with channels at the membrane. In the last year, several studies have emerged which explore the intricacies of RGK protein interaction with the channel itself and the importance of the Cavß subunit for this interaction, in addition to providing some tantalizing suggestions for the mechanism by which RGK proteins reduce or eliminate calcium currents. In this review, we present an overview of these recent advances and suggest a model that may synthesize these latest works.
Assuntos
Canais de Cálcio/metabolismo , Ativação do Canal Iônico , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Canais de Cálcio/química , Humanos , Potenciais da Membrana , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Relação Estrutura-Atividade , Proteínas ras/químicaRESUMO
Neuroligins 1-4 are postsynaptic transmembrane proteins capable of initiating presynaptic maturation via interactions with beta-neurexin. Both neuroligins and beta-neurexins have alternatively spliced inserts in their extracellular domains. Using analytical ultracentrifugation, we determined that the extracellular domains of the neuroligins sediment as dimers, whereas the extracellular domains of the beta-neurexins appear monomeric. Sedimentation velocity experiments of titrated stoichiometry ratios of beta-neurexin and neuroligin suggested a 2:2 complex formation. The recognition properties of individual neuroligins toward beta-neurexin-1 (NX1beta), along with the influence of their splice inserts, were explored by surface plasmon resonance and affinity chromatography. Different neuroligins display a range of NX1beta affinities spanning more than 2 orders of magnitude. Whereas splice insert 4 in beta-neurexin appears to act only as a modulator of the neuroligin/beta-neurexin association, splice insert B in neuroligin-1 (NL1) is the key element regulating the NL1/NX1beta binding. Our data indicate that gene selection, mRNA splicing, and post-translational modifications combine to give rise to a controlled neuroligin recognition code with a rank ordering of affinities for particular neurexins that is conserved for the neuroligins across mammalian species.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Clonagem Molecular , DNA Complementar/genética , Variação Genética , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Plasmídeos , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Ratos , Ressonância de Plasmônio de SuperfícieRESUMO
Neuroligins, proteins of the alpha/beta-hydrolase fold family, are found as postsynaptic transmembrane proteins whose extracellular domain associates with presynaptic partners, proteins of the neurexin family. To characterize the molecular basis of neuroligin interaction with neurexin-beta, we expressed five soluble and exportable forms of neuroligin-1 from recombinant DNA sources, by truncating the protein before the transmembrane span near its carboxyl terminus. The extracellular domain of functional neuroligin-1 associates as a dimer when analyzed by sedimentation equilibrium. By surface plasmon resonance, we established that soluble neuroligins-1 bind neurexin-1beta, but the homologous alpha/beta-hydrolase fold protein, acetylcholinesterase, failed to associate with the neurexins. Neuroligin-1 has a unique N-linked glycosylation pattern in the neuroligin family, and glycosylation and its processing modify neuroligin activity. Incomplete processing of the protein and enzymatic removal of the oligosaccharides chain or the terminal sialic acids from neuroligin-1 enhance its activity, whereas deglycosylation of neurexin-1beta did not alter its association capacity. In particular, the N-linked glycosylation at position 303 appears to be a major determinant in modifying the association with neurexin-1beta. We show here that glycosylation processing of neuroligin, in addition to mRNA splicing and gene selection, contributes to the specificity of the neurexin-beta/neuroligin-1 association.
Assuntos
Proteínas de Membrana/química , Proteínas do Tecido Nervoso/química , Proteínas Recombinantes/química , Acetilcolinesterase/química , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Cálcio/metabolismo , Cátions , Moléculas de Adesão Celular Neuronais , Linhagem Celular , Membrana Celular/metabolismo , Técnicas de Cocultura , DNA/metabolismo , DNA Complementar/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Glicosilação , Humanos , Immunoblotting , Imuno-Histoquímica , Íons , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Plasmídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ratos , Ressonância de Plasmônio de Superfície , Fatores de Tempo , TransfecçãoRESUMO
Neuroligins (NLs) are a family of transmembrane proteins that function in synapse formation and/or remodeling by interacting with beta-neurexins (beta-NXs) to form heterophilic cell adhesions. The large N-terminal extracellular domain of NLs, required for beta-NX interactions, has sequence homology to the alpha/beta hydrolase fold superfamily of proteins. By peptide mapping and mass spectrometric analysis of a soluble recombinant form of NL1, several structural features of the extracellular domain have been established. Of the nine cysteine residues in NL1, eight are shown to form intramolecular disulfide bonds. Disulfide pairings of Cys 117 to Cys 153 and Cys 342 to Cys 353 are consistent with disulfide linkages that are conserved among the family of alpha/beta hydrolase proteins. The disulfide bond between Cys 172 and Cys 181 occurs within a region of the protein encoded by an alternatively spliced exon. The disulfide pairing of Cys 512 and Cys 546 in NL1 yields a structural motif unique to the NLs, since these residues are highly conserved. The potential N-glycosylation sequons in NL1 at Asn 109, Asn 303, Asn 343, and Asn 547 are shown occupied by carbohydrate. An additional consensus sequence for N-glycosylation at Asn 662 is likely occupied. Analysis of N-linked oligosaccharide content by mass matching paradigms reveals significant microheterogeneous populations of complex glycosyl moieties. In addition, O-linked glycosylation is observed in the predicted stalk region of NL1, prior to the transmembrane spanning domain. From predictions based on sequence homology of NL1 to acetylcholinesterase and the molecular features of NL1 established from mass spectrometric analysis, a novel topology model for NL three-dimensional structure has been constructed.