RESUMEN
GABAB receptors (GBRs), the G protein-coupled receptors for GABA, regulate synaptic transmission throughout the brain. A main synaptic function of GBRs is the gating of Cav2.2-type Ca2+ channels. However, the cellular compartment where stable GBR/Cav2.2 signaling complexes form remains unknown. In this study, we demonstrate that the vesicular protein synaptotagmin-11 (Syt11) binds to both the auxiliary GBR subunit KCTD16 and Cav2.2 channels. Through these dual interactions, Syt11 recruits GBRs and Cav2.2 channels to post-Golgi vesicles, thus facilitating assembly of GBR/Cav2.2 signaling complexes. In addition, Syt11 stabilizes GBRs and Cav2.2 channels at the neuronal plasma membrane by inhibiting constitutive internalization. Neurons of Syt11 knockout mice exhibit deficits in presynaptic GBRs and Cav2.2 channels, reduced neurotransmitter release, and decreased GBR-mediated presynaptic inhibition, highlighting the critical role of Syt11 in the assembly and stable expression of GBR/Cav2.2 complexes. These findings support that Syt11 acts as a vesicular scaffold protein, aiding in the assembly of signaling complexes from low-abundance components within transport vesicles. This mechanism enables insertion of pre-assembled functional signaling units into the synaptic membrane.
Asunto(s)
Ratones Noqueados , Transducción de Señal , Sinaptotagminas , Animales , Sinaptotagminas/metabolismo , Sinaptotagminas/genética , Ratones , Humanos , Neuronas/metabolismo , Transmisión Sináptica , Receptores de GABA-B/metabolismo , Receptores de GABA-B/genética , Terminales Presinápticos/metabolismo , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo N/genética , Aparato de Golgi/metabolismo , Unión Proteica , Células HEK293RESUMEN
GABAB receptors are the G-protein coupled receptors for the main inhibitory neurotransmitter in the brain, GABA. GABAB receptors were shown to associate with homo-oligomers of auxiliary KCTD8, KCTD12, KCTD12b, and KCTD16 subunits (named after their T1 K+-channel tetramerization domain) that regulate G-protein signaling of the receptor. Here we provide evidence that GABAB receptors also associate with hetero-oligomers of KCTD subunits. Coimmunoprecipitation experiments indicate that two-thirds of the KCTD16 proteins in the hippocampus of adult mice associate with KCTD12. We show that the KCTD proteins hetero-oligomerize through self-interacting T1 and H1 homology domains. Bioluminescence resonance energy transfer measurements in live cells reveal that KCTD12/KCTD16 hetero-oligomers associate with both the receptor and the G-protein. Electrophysiological experiments demonstrate that KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties on G-protein-activated Kir3 currents. During prolonged receptor activation (one min) KCTD12/KCTD16 hetero-oligomers produce moderately desensitizing fast deactivating K+ currents, whereas KCTD12 and KCTD16 homo-oligomers produce strongly desensitizing fast deactivating currents and nondesensitizing slowly deactivating currents, respectively. During short activation (2 s) KCTD12/KCTD16 hetero-oligomers produce nondesensitizing slowly deactivating currents. Electrophysiological recordings from hippocampal neurons of KCTD knock-out mice are consistent with these findings and indicate that KCTD12/KCTD16 hetero-oligomers increase the duration of slow IPSCs. In summary, our data demonstrate that simultaneous assembly of distinct KCTDs at the receptor increases the molecular and functional repertoire of native GABAB receptors and modulates physiologically induced K+ current responses in the hippocampus. SIGNIFICANCE STATEMENT: The KCTD proteins 8, 12, and 16 are auxiliary subunits of GABAB receptors that differentially regulate G-protein signaling of the receptor. The KCTD proteins are generally assumed to function as homo-oligomers. Here we show that the KCTD proteins also assemble hetero-oligomers in all possible dual combinations. Experiments in live cells demonstrate that KCTD hetero-oligomers form at least tetramers and that these tetramers directly interact with the receptor and the G-protein. KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties to GABAB receptor-induced Kir3 currents in heterologous cells. KCTD12/KCTD16 hetero-oligomers are abundant in the hippocampus, where they prolong the duration of slow IPSCs in pyramidal cells. Our data therefore support that KCTD hetero-oligomers modulate physiologically induced K+ current responses in the brain.
Asunto(s)
Canales de Potasio/genética , Canales de Potasio/metabolismo , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Animales , Química Encefálica/genética , Células CHO , Cricetinae , Cricetulus , Fenómenos Electrofisiológicos/genética , Potenciales Postsinápticos Excitadores/genética , Femenino , Cinética , Masculino , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Receptores Acoplados a Proteínas G/metabolismo , Receptores KIR/metabolismoRESUMEN
GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. They are expressed in almost all neurons of the brain, where they regulate synaptic transmission and signal propagation by controlling the activity of voltage-gated calcium (Ca(v)) and inward-rectifier potassium (K(ir)) channels. Molecular cloning revealed that functional GABA(B) receptors are formed by the heteromeric assembly of GABA(B1) with GABA(B2) subunits. However, cloned GABA(B(1,2)) receptors failed to reproduce the functional diversity observed with native GABA(B) receptors. Here we show by functional proteomics that GABA(B) receptors in the brain are high-molecular-mass complexes of GABA(B1), GABA(B2) and members of a subfamily of the KCTD (potassium channel tetramerization domain-containing) proteins. KCTD proteins 8, 12, 12b and 16 show distinct expression profiles in the brain and associate tightly with the carboxy terminus of GABA(B2) as tetramers. This co-assembly changes the properties of the GABA(B(1,2)) core receptor: the KCTD proteins increase agonist potency and markedly alter the G-protein signalling of the receptors by accelerating onset and promoting desensitization in a KCTD-subtype-specific manner. Taken together, our results establish the KCTD proteins as auxiliary subunits of GABA(B) receptors that determine the pharmacology and kinetics of the receptor response.
Asunto(s)
Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Conductividad Eléctrica , Agonistas de Receptores GABA-B , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Cinética , Ratones , Neuronas/metabolismo , Oocitos/metabolismo , Potasio/metabolismo , Canales de Potasio/metabolismo , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Transducción de Señal , XenopusRESUMEN
GABA(B) receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA(B) receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA(B1a), GABA(B1b), and GABA(B2) form fully functional heteromeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA(B) receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA(B) receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA(B) receptor-mediated K(+) current response. In summary, our experiments support that the up-regulation of functional GABA(B) receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.
Asunto(s)
Hipocampo/metabolismo , Neuronas/metabolismo , Canales de Potasio/metabolismo , Potasio/metabolismo , Multimerización de Proteína/fisiología , Receptores de GABA-B/metabolismo , Transducción de Señal/fisiología , Animales , Células COS , Membrana Celular/genética , Membrana Celular/metabolismo , Chlorocebus aethiops , Hipocampo/citología , Ratones , Ratones Noqueados , Neuronas/citología , Canales de Potasio/genética , Receptores de GABA-B/genéticaRESUMEN
UNLABELLED: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCC) are the most common liver tumors and a leading cause for cancer-related death in men. Notch2 regulates cellular differentiation in the developing and adult liver. Although aberrant Notch signaling is implicated in various cancers, it is still unclear whether Notch2 regulates proliferation and differentiation in liver carcinogenesis and thereby contributes to HCC and CCC formation. Here, we investigated the oncogenic potential of constitutive Notch2 signaling in the liver. We show that liver-specific expression of the intracellular domain of Notch2 (N2ICD) in mice is sufficient to induce HCC formation and biliary hyperplasia. Specifically, constitutive N2ICD signaling in the liver leads to up-regulation of pro-proliferative genes and proliferation of hepatocytes and biliary epithelial cells (BECs). Using the diethylnitrosamine (DEN) HCC carcinogenesis model, we further show that constitutive Notch2 signaling accelerates DEN-induced HCC formation. DEN-induced HCCs with constitutive Notch2 signaling (DEN(N2ICD) HCCs) exhibit a marked increase in size, proliferation, and expression of pro-proliferative genes when compared with HCCs from DEN-induced control mice (DEN(ctrl) HCCs). Moreover, DEN(N2ICD) HCCs exhibit increased Sox9 messenger RNA (mRNA) levels and reduced Albumin and Alpha-fetoprotein mRNA levels, indicating that they are less differentiated than DEN(ctrl) HCCs. Additionally, DEN(N2ICD) mice develop large hepatic cysts, dysplasia of the biliary epithelium, and eventually CCC. CCC formation in patients and DEN(N2ICD) mice is accompanied by re-expression of hepatocyte nuclear factor 4α(HNF4α), possibly indicating dedifferentiation of BECs. CONCLUSION: Our data establish an oncogenic role for constitutive Notch2 signaling in liver cancer development.
Asunto(s)
Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/fisiopatología , Dietilnitrosamina/efectos adversos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/fisiopatología , Receptor Notch2/fisiología , Transducción de Señal/fisiología , Animales , Neoplasias de los Conductos Biliares/fisiopatología , Conductos Biliares Intrahepáticos , Carcinoma Hepatocelular/metabolismo , Diferenciación Celular , Proliferación Celular , Colangiocarcinoma/fisiopatología , Modelos Animales de Enfermedad , Femenino , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Receptor Notch2/genéticaRESUMEN
GABAB receptors (GBRs) are G protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GBRs regulate fast synaptic transmission by gating Ca2+ and K+ channels via the Gßγ subunits of the activated G protein. It has been demonstrated that auxiliary GBR subunits, the KCTD proteins, shorten onset and rise time and increase desensitization of receptor-induced K+ currents. KCTD proteins increase desensitization of K+ currents by scavenging Gßγ from the channel, yet the mechanism responsible for the rapid activation of K+ currents has remained elusive. In this study, we demonstrate that KCTD proteins preassemble Gßγ at GBRs. The preassembly obviates the need for diffusion-limited G protein recruitment to the receptor, thereby accelerating G protein activation and, as a result, K+ channel activation. Preassembly of Gßγ at the receptor relies on the interaction of KCTD proteins with a loop protruding from the seven-bladed propeller of Gß subunits. The binding site is shared between Gß1 and Gß2, limiting the interaction of KCTD proteins to these particular Gß isoforms. Substituting residues in the KCTD binding site of Gß1 with those from Gß3 hinders the preassembly of Gßγ with GBRs, delays onset and prolongs rise time of receptor-activated K+ currents. The KCTD-Gß interface, therefore, represents a target for pharmacological modulation of channel gating by GBRs.
RESUMEN
Adherens junction-associated protein 1 (AJAP1) has been implicated in brain diseases; however, a pathogenic mechanism has not been identified. AJAP1 is widely expressed in neurons and binds to γ-aminobutyric acid type B receptors (GBRs), which inhibit neurotransmitter release at most synapses in the brain. Here, we show that AJAP1 is selectively expressed in dendrites and trans-synaptically recruits GBRs to presynaptic sites of neurons expressing AJAP1. We have identified several monoallelic AJAP1 variants in individuals with epilepsy and/or neurodevelopmental disorders. Specifically, we show that the variant p.(W183C) lacks binding to GBRs, resulting in the inability to recruit them. Ultrastructural analysis revealed significantly decreased presynaptic GBR levels in Ajap1-/- and Ajap1W183C/+ mice. Consequently, these mice exhibited reduced GBR-mediated presynaptic inhibition at excitatory and inhibitory synapses, along with impaired synaptic plasticity. Our study reveals that AJAP1 enables the postsynaptic neuron to regulate the level of presynaptic GBR-mediated inhibition, supporting the clinical relevance of loss-of-function AJAP1 variants.
Asunto(s)
Neurotransmisores , Sinapsis , Transmisión Sináptica , Animales , Femenino , Humanos , Masculino , Ratones , Alelos , Epilepsia/metabolismo , Epilepsia/genética , Epilepsia/patología , Mutación con Pérdida de Función , Ratones Noqueados , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Plasticidad Neuronal , Neuronas/metabolismo , Neurotransmisores/metabolismo , Sinapsis/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismoRESUMEN
GABA(B) receptors assemble from principle and auxiliary subunits. The principle subunits GABA(B1) and GABA(B2) form functional heteromeric GABA(B(1,2)) receptors that associate with homotetramers of auxiliary KCTD8, -12, -12b, or -16 (named after their K(+) channel tetramerization domain) subunits. These auxiliary subunits constitute receptor subtypes with distinct functional properties. KCTD12 and -12b generate desensitizing receptor responses while KCTD8 and -16 generate largely non-desensitizing receptor responses. The structural elements of the KCTDs underlying these differences in desensitization are unknown. KCTDs are modular proteins comprising a T1 tetramerization domain, which binds to GABA(B2), and a H1 homology domain. KCTD8 and -16 contain an additional C-terminal H2 homology domain that is not sequence-related to the H1 domains. No functions are known for the H1 and H2 domains. Here we addressed which domains and sequence motifs in KCTD proteins regulate desensitization of the receptor response. We found that the H1 domains in KCTD12 and -12b mediate desensitization through a particular sequence motif, T/NFLEQ, which is not present in the H1 domains of KCTD8 and -16. In addition, the H2 domains in KCTD8 and -16 inhibit desensitization when expressed C-terminal to the H1 domains but not when expressed as a separate protein in trans. Intriguingly, the inhibitory effect of the H2 domain is sequence-independent, suggesting that the H2 domain sterically hinders desensitization by the H1 domain. Evolutionary analysis supports that KCTD12 and -12b evolved desensitizing properties by liberating their H1 domains from antagonistic H2 domains and acquisition of the T/NFLEQ motif.
Asunto(s)
Evolución Molecular , Subunidades de Proteína/metabolismo , Proteínas/metabolismo , Receptores de GABA-B/metabolismo , Secuencias de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Proteínas/genética , Receptores de GABA-B/genéticaRESUMEN
Amyloid-ß precursor protein (APP) regulates neuronal activity through the release of secreted APP (sAPP) acting at cell surface receptors. APP and sAPP were reported to bind to the extracellular sushi domain 1 (SD1) of GABAB receptors (GBRs). A 17 amino acid peptide (APP17) derived from APP was sufficient for SD1 binding and shown to mimic the inhibitory effect of sAPP on neurotransmitter release and neuronal activity. The functional effects of APP17 and sAPP were similar to those of the GBR agonist baclofen and blocked by a GBR antagonist. These experiments led to the proposal that sAPP activates GBRs to exert its neuronal effects. However, whether APP17 and sAPP influence classical GBR signaling pathways in heterologous cells was not analyzed. Here, we confirm that APP17 binds to GBRs with nanomolar affinity. However, biochemical and electrophysiological experiments indicate that APP17 does not influence GBR activity in heterologous cells. Moreover, APP17 did not regulate synaptic GBR localization, GBR-activated K+ currents, neurotransmitter release, or neuronal activity in vitro or in vivo. Our results show that APP17 is not a functional GBR ligand and indicate that sAPP exerts its neuronal effects through receptors other than GBRs.
Asunto(s)
Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Neuronas/metabolismo , Neurotransmisores/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
We have previously identified a protein, consisting of seven WD-repeats, forming a putative beta-propeller, and an FYVE domain, ProF, which is highly expressed in 3T3-L1 cells, a cell line that can be differentiated into adipocytes. We recently found ProF to interact with the kinases Akt and protein kinase Czeta. Here we demonstrate that ProF is a positive regulator of adipogenesis. Knockdown of ProF by RNA interference leads to decreased adipocyte differentiation. This is shown by reduced lipid accumulation, decreased expression of the differentiation markers PPARgamma and C/EBPalpha, and reduced glucose uptake in differentiated cells. Furthermore, ProF overexpression leads to increased adipogenesis. ProF binds to the transcription factor Foxo1 (Forkhead box O1), a negative regulator of insulin action and adipogenesis, and facilitates the phosphorylation and thus inactivation of Foxo1 by Akt. Additionally, dominant-negative Foxo1 restores adipogenesis in ProF knockdown cells. Thus, ProF modulates Foxo1 phosphorylation by Akt, promoting adipocyte differentiation. Furthermore, ProF might be involved in metabolic disorders such as diabetes.
Asunto(s)
Adipogénesis/fisiología , Proteínas Portadoras/fisiología , Factores de Transcripción Forkhead/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis/genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Humanos , Immunoblotting , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
GABAB receptors (GBRs), the G protein-coupled receptors for the inhibitory neurotransmitter γ-aminobutyric acid (GABA), activate Go/i-type G proteins that regulate adenylyl cyclase, Ca2+ channels, and K+ channels. GBR signaling to enzymes and ion channels influences neuronal activity, plasticity processes, and network activity throughout the brain. GBRs are obligatory heterodimers composed of GB1a or GB1b subunits with a GB2 subunit. Heterodimeric GB1a/2 and GB1b/2 receptors represent functional units that associate in a modular fashion with regulatory, trafficking, and effector proteins to generate receptors with distinct physiological functions. This review summarizes current knowledge on the structure, organization, and functions of multi-protein GBR complexes.
Asunto(s)
Receptores de GABA-B , Receptores de GABA , Neuronas , Transducción de Señal , Ácido gamma-AminobutíricoRESUMEN
The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation.
Asunto(s)
Canales de Calcio Tipo R/metabolismo , Proteínas de Transporte de Catión/metabolismo , Habénula/metabolismo , Terminales Presinápticos/metabolismo , Receptores de GABA/metabolismo , Animales , Canales de Calcio Tipo R/genética , Proteínas de Transporte de Catión/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de GABA/genética , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Sinapsis/genética , Sinapsis/metabolismoRESUMEN
For a long time metabotropic glutamate receptors (mGluRs) were thought to regulate neuronal functions as obligatory homodimers. Recent reports, however, indicate the existence of heterodimers between group-II and -III mGluRs in the brain, which differ from the homodimers in their signal transduction and sensitivity to negative allosteric modulators (NAMs). Whether the group-I mGluRs, mGlu1 and mGlu5, form functional heterodimers in the brain is still a matter of debate. We now show that mGlu1 and mGlu5 co-purify from brain membranes and hippocampal tissue and co-localize in cultured hippocampal neurons. Complementation assays with mutants deficient in agonist-binding or G protein-coupling reveal that mGlu1/5 heterodimers are functional in heterologous cells and transfected cultured hippocampal neurons. In contrast to heterodimers between group-II and -III mGluRs, mGlu1/5 receptors exhibit a symmetric signal transduction, with both protomers activating G proteins to a similar extent. NAMs of either protomer in mGlu1/5 receptors partially inhibit signaling, showing that both protomers need to be able to reach an active conformation for full receptor activity. Complete heterodimer inhibition is observed when both protomers are locked in their inactive state by a NAM. In summary, our data show that mGlu1/5 heterodimers exhibit a symmetric signal transduction and thus intermediate signaling efficacy and kinetic properties. Our data support the existence of mGlu1/5 heterodimers in neurons and highlight differences in the signaling transduction of heterodimeric mGluRs that influence allosteric modulation.
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Hipocampo/metabolismo , Neuronas/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Regulación Alostérica , Animales , Encéfalo/metabolismo , Cromatografía Liquida , Hipocampo/citología , Ratones , Ratones Noqueados , Multimerización de Proteína , Receptor del Glutamato Metabotropico 5/genética , Receptores de Glutamato Metabotrópico/genética , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
c-Src is a non-receptor tyrosine kinase involved in regulating cell proliferation, cell migration and cell invasion and is tightly controlled by reversible phosphorylation on regulatory sites and through protein-protein interactions. The interaction of c-Src with PDZ proteins was recently identified as novel mechanism to restrict c-Src function. The objective of this study was to identify and characterise PDZ proteins that interact with c-Src to control its activity. By PDZ domain array screen, we identified the interaction of c-Src with the PDZ protein Membrane Protein Palmitoylated 2 (MPP2), a member of the Membrane-Associated Guanylate Kinase (MAGUK) family, to which also the Discs large (Dlg) tumour suppressor protein belongs. The function of MPP2 has not been established and the functional significance of the MPP2 c-Src interaction is not known. We found that in non-transformed breast epithelial MCF-10A cells, endogenous MPP2 associated with the cytoskeleton in filamentous structures, which partially co-localised with microtubules and c-Src. MPP2 and c-Src interacted in cells, where c-Src kinase activity promoted increased interaction of c-Src with MPP2. We furthermore found that MPP2 was able to negatively regulate c-Src kinase activity in cells, suggesting that the functional significance of the MPP2-c-Src interaction is to restrict Src activity. Consequently, the c-Src-dependent disorganisation of the cortical actin cytoskeleton of epithelial cells expressing c-Src was suppressed by MPP2. In conclusion we demonstrate here that MPP2 interacts with c-Src in cells to control c-Src activity and morphological function.
Asunto(s)
Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS/metabolismo , Línea Celular , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/genética , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/química , Humanos , Riñón/embriología , Microtúbulos/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas pp60(c-src)/química , Alineación de Secuencia , TransfecciónRESUMEN
GABAB receptors (GBRs), the G protein-coupled receptors for the neurotransmitter γ-aminobutyric acid (GABA), regulate synaptic transmission at most synapses in the brain. Proteomic approaches revealed that native GBR complexes assemble from an inventory of ~30 proteins that provide a molecular basis for the functional diversity observed with these receptors. Studies with reconstituted GBR complexes in heterologous cells and complementary knockout studies have allowed to identify cellular and physiological functions for obligate and several non-obligate receptor components. It emerges that modular association of receptor components in space and time generates a variety of multiprotein receptor complexes with different localizations, kinetic properties and effector channels. This article summarizes current knowledge on the organizing principle of GBR complexes. We further discuss unanticipated receptor functions, links to disease and opportunities for drug discovery arising from the identification of novel receptor components.
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Receptores de GABA-B/metabolismo , Receptores de GABA-B/fisiología , Animales , Encéfalo/metabolismo , Membrana Celular , Agonistas de Receptores GABA-B/farmacología , Antagonistas de Receptores de GABA-B/farmacología , Proteómica , Receptores Acoplados a Proteínas G , Receptores de GABA-B/químicaRESUMEN
Targeting multiprotein receptor complexes, rather than receptors directly, is a promising concept in drug discovery. This is particularly relevant to the GABAB receptor complex, which plays a prominent role in many brain functions and diseases. Here, we provide the first studies targeting a key protein-protein interaction of the GABAB receptor complex-the interaction with KCTD proteins. By employing the µSPOT technology, we first defined the GABAB receptor-binding epitope mediating the KCTD interaction. Subsequently, we developed a highly potent peptide-based inhibitor that interferes with the KCTD/GABAB receptor complex and efficiently isolates endogenous KCTD proteins from mouse brain lysates. X-ray crystallography and SEC-MALS revealed inhibitor induced oligomerization of KCTD16 into a distinct hexameric structure. Thus, we provide a template for modulating the GABAB receptor complex, revealing a fundamentally novel approach for targeting GABAB receptor-associated neuropsychiatric disorders.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Péptidos/metabolismo , Receptores de GABA-B/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Encéfalo/metabolismo , Cristalografía por Rayos X , Polarización de Fluorescencia , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Péptidos/química , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Receptores de GABA-B/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
GABAB receptors (GBRs) are key regulators of synaptic release but little is known about trafficking mechanisms that control their presynaptic abundance. We now show that sequence-related epitopes in APP, AJAP-1 and PIANP bind with nanomolar affinities to the N-terminal sushi-domain of presynaptic GBRs. Of the three interacting proteins, selectively the genetic loss of APP impaired GBR-mediated presynaptic inhibition and axonal GBR expression. Proteomic and functional analyses revealed that APP associates with JIP and calsyntenin proteins that link the APP/GBR complex in cargo vesicles to the axonal trafficking motor. Complex formation with GBRs stabilizes APP at the cell surface and reduces proteolysis of APP to Aß, a component of senile plaques in Alzheimer's disease patients. Thus, APP/GBR complex formation links presynaptic GBR trafficking to Aß formation. Our findings support that dysfunctional axonal trafficking and reduced GBR expression in Alzheimer's disease increases Aß formation.
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Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Transporte Axonal , Receptores de GABA-B/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Animales , Axones/metabolismo , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Dendritas/metabolismo , Epítopos/metabolismo , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Cinesinas/metabolismo , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Estabilidad Proteica , Proteómica , Transducción de Señal , Sinapsis/metabolismoRESUMEN
We have recently identified a protein, consisting of seven WD repeats, presumably forming a beta-propeller, and a domain identified in Fab1p, YOTB, VAC1p, and EEA1 (FYVE) domain, ProF. The FYVE domain targets the protein to vesicular membranes, while the WD repeats allow binding of the activated kinases Akt and protein kinase (PK)Czeta. Here, we describe the vesicle-associated membrane protein 2 (VAMP2) as interaction partner of ProF. The interaction is demonstrated with overexpressed and endogenous proteins in mammalian cells. ProF and VAMP2 partially colocalize on vesicular structures with PKCzeta and the proteins form a ternary complex. VAMP2 can be phosphorylated by activated PKCzeta in vitro and the presence of ProF increases the PKCzeta-dependent phosphorylation of VAMP2 in vitro. ProF is an adaptor protein that brings together a kinase with its substrate. VAMP2 is known to regulate docking and fusion of vesicles and to play a role in targeting vesicles to the plasma membrane. The complex may be involved in vesicle cycling in various secretory pathways.
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Proteínas Portadoras/metabolismo , Proteína Quinasa C/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Células 3T3 , Animales , Células COS , Chlorocebus aethiops , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Ratones , Microscopía Confocal , Fosforilación , Unión Proteica , Especificidad por Sustrato , Técnicas del Sistema de Dos HíbridosRESUMEN
WD (tryptophan-aspartic acid dipeptide)-repeat proteins play a central role in signal transduction cascades by co-ordinating the interaction of key signalling molecules. We identified a novel propeller-FYVE [domain identified in Fab1p, YOTB, Vac1p and EEA1 (early endosome antigen 1)] protein, ProF, which is expressed in various cell lines and tissues and consists of seven WD-repeats and a FYVE domain. WD-repeat proteins offer a platform for protein-protein interactions by folding into a seven-bladed propeller-like structure, while the FYVE domain binds to phosphatidylinositol 3-phosphate present mainly on intracellular membranes. The ProF protein partially co-localizes with EEA1 on vesicular structures and binds to the protein kinases Akt and PKCzeta/lambda (protein kinase Czeta/lambda) via its WD-repeat propeller. ProF interacts more strongly with the kinases after hormonal stimulation. Endogenously expressed ProF and the two kinases interact in brain and in the preadipocyte cell line 3T3-L1, suggesting a role in secretory vesicular processes. In summary, we describe a new binding partner for kinases, located on vesicular structures in specialized cells, which may play a role for the spatial organization of signalling cascades.
Asunto(s)
Proteínas Portadoras/metabolismo , Isoenzimas/metabolismo , Proteína Oncogénica v-akt/metabolismo , Proteína Quinasa C/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/aislamiento & purificación , Línea Celular , Chlorocebus aethiops , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Ratas , Transducción de SeñalRESUMEN
GABAB receptors, the most abundant inhibitory G protein-coupled receptors in the mammalian brain, display pronounced diversity in functional properties, cellular signaling and subcellular distribution. We used high-resolution functional proteomics to identify the building blocks of these receptors in the rodent brain. Our analyses revealed that native GABAB receptors are macromolecular complexes with defined architecture, but marked diversity in subunit composition: the receptor core is assembled from GABAB1a/b, GABAB2, four KCTD proteins and a distinct set of G-protein subunits, whereas the receptor's periphery is mostly formed by transmembrane proteins of different classes. In particular, the periphery-forming constituents include signaling effectors, such as Cav2 and HCN channels, and the proteins AJAP1 and amyloid-ß A4, both of which tightly associate with the sushi domains of GABAB1a. Our results unravel the molecular diversity of GABAB receptors and their postnatal assembly dynamics and provide a roadmap for studying the cellular signaling of this inhibitory neurotransmitter receptor.