RESUMO
A disintegrin and metalloproteinase 10 (ADAM10) is the major α-secretase that catalyzes the amyloid precursor protein (APP) ectodomain shedding in the brain and prevents amyloid formation. Its activity depends on correct intracellular trafficking and on synaptic membrane insertion. Here, we describe that in hippocampal neurons the synapse-associated protein-97 (SAP97), an excitatory synapse scaffolding element, governs ADAM10 trafficking from dendritic Golgi outposts to synaptic membranes. This process is mediated by a previously uncharacterized protein kinase C phosphosite in SAP97 SRC homology 3 domain that modulates SAP97 association with ADAM10. Such mechanism is essential for ADAM10 trafficking from the Golgi outposts to the synapse, but does not affect ADAM10 transport from the endoplasmic reticulum. Notably, this process is altered in Alzheimer's disease brains. These results help in understanding the mechanism responsible for the modulation of ADAM10 intracellular path, and can constitute an innovative therapeutic strategy to finely tune ADAM10 shedding activity towards APP.
Assuntos
Proteínas ADAM/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismo , Proteínas ADAM/química , Proteína ADAM10 , Proteínas Adaptadoras de Transdução de Sinal/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/química , Animais , Células COS , Chlorocebus aethiops , Proteína 1 Homóloga a Discs-Large , Ativação Enzimática , Células HEK293 , Humanos , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Fosfotreonina/metabolismo , Densidade Pós-Sináptica/metabolismo , Ligação Proteica , Ratos , Sinapses/metabolismoRESUMO
Genetic mutations of SHANK3 have been reported in patients with intellectual disability, autism spectrum disorder (ASD) and schizophrenia. At the synapse, Shank3/ProSAP2 is a scaffolding protein that connects glutamate receptors to the actin cytoskeleton via a chain of intermediary elements. Although genetic studies have repeatedly confirmed the association of SHANK3 mutations with susceptibility to psychiatric disorders, very little is known about the neuronal consequences of these mutations. Here, we report the functional effects of two de novo mutations (STOP and Q321R) and two inherited variations (R12C and R300C) identified in patients with ASD. We show that Shank3 is located at the tip of actin filaments and enhances its polymerization. Shank3 also participates in growth cone motility in developing neurons. The truncating mutation (STOP) strongly affects the development and morphology of dendritic spines, reduces synaptic transmission in mature neurons and also inhibits the effect of Shank3 on growth cone motility. The de novo mutation in the ankyrin domain (Q321R) modifies the roles of Shank3 in spine induction and morphology, and actin accumulation in spines and affects growth cone motility. Finally, the two inherited mutations (R12C and R300C) have intermediate effects on spine density and synaptic transmission. Therefore, although inherited by healthy parents, the functional effects of these mutations strongly suggest that they could represent risk factors for ASD. Altogether, these data provide new insights into the synaptic alterations caused by SHANK3 mutations in humans and provide a robust cellular readout for the development of knowledge-based therapies.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/genética , Dendritos/ultraestrutura , Espinhas Dendríticas/genética , Mutação/genética , Neurônios/citologia , Animais , Transtorno Autístico/genética , Linhagem Celular Transformada/citologia , Células Cultivadas , Chlorocebus aethiops , Dendritos/genética , Espinhas Dendríticas/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Microscopia Confocal , Proteínas do Tecido Nervoso , Transfecção , Tubulina (Proteína)/metabolismoRESUMO
Both postsynaptic density and presynaptic active zone are structural matrix containing scaffolding proteins that are involved in the organization of the synapse. Little is known about the functional role of these proteins in the signaling of presynaptic receptors. Here we show that the interaction of the presynaptic metabotropic glutamate (mGlu) receptor subtype, mGlu7a, with the postsynaptic density-95 disc-large zona occludens 1 (PDZ) domain-containing protein, PICK1, is required for specific inhibition of P/Q-type Ca(2+) channels, in cultured cerebellar granule neurons. Furthermore, we show that activation of the presynaptic mGlu7a receptor inhibits synaptic transmission and this effect also requires the presence of PICK1. These results indicate that the scaffolding protein, PICK1, plays an essential role in the control of synaptic transmission by the mGlu7a receptor complex.
Assuntos
Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transmissão Sináptica/fisiologia , Aminobutiratos/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Proteínas de Ciclo Celular , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Maleato de Dizocilpina/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Técnicas de Patch-Clamp , Receptores de Glutamato Metabotrópico/genética , Transmissão Sináptica/efeitos dos fármacos , Sinaptofisina/metabolismo , ômega-Agatoxina IVA/farmacologia , ômega-Conotoxina GVIA/farmacologiaRESUMO
There is accumulating evidence that the specificity of the transduction cascades activated by G protein-coupled receptors cannot solely depend on the nature of the coupled G protein. To identify additional structural determinants, we studied two metabotropic glutamate (mGlu) receptors, the mGlu2 and mGlu7 receptors, that are both coupled to G(o) proteins but are known to affect different effectors in neurons. Thus, the mGlu2 receptor selectively blocks N- and L-type Ca(2+) channels via a protein kinase C-independent pathway, whereas the mGlu7 receptor selectively blocks P/Q-type Ca(2+) channels via a protein kinase C-dependent pathway, and both effects are pertussis toxin-sensitive. We examined the role of the C-terminal domain of these receptors in this coupling. Chimeras were constructed by exchanging the C terminus of these receptors and transfected into neurons. Different chimeric receptors bearing the C terminus of mGlu7 receptor blocked selectively P/Q-type Ca(2+) channels, whereas chimeras bearing the C terminus of mGlu2 receptor selectively blocked N- and L-type Ca(2+) channels. These results show that the C terminus of mGlu2 and mGlu7 receptors is a key structural determinant that allows these receptors to select a specific signaling pathway in neurons.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Proteínas de Ligação ao GTP/metabolismo , Camundongos , Receptores de Glutamato Metabotrópico/químicaRESUMO
Although presynaptic localization of mGluR7 is well established, the mechanism by which the receptor may control Ca(2+) channels in neurons is still unknown. We show here that cultured cerebellar granule cells express native metabotropic glutamate receptor type 7 (mGluR7) in neuritic processes, whereas transfected mGluR7 was also expressed in cell bodies. This allowed us to study the effect of the transfected receptor on somatic Ca(2+) channels. In transfected neurons, mGuR7 selectively inhibited P/Q-type Ca(2+) channels. The effect was mimicked by GTPgammaS and blocked by pertussis toxin (PTX) or a selective antibody raised against the G-protein alphao subunit, indicating the involvement of a G(o)-like protein. The mGuR7 effect did not display the characteristics of a direct interaction between G-protein betagamma subunits and the alpha1A Ca(2+) channel subunit, but was abolished by quenching betagamma subunits with specific intracellular peptides. Intracellular dialysis of G-protein betagamma subunits did not mimic the action of mGluR7, suggesting that both G-protein betagamma and alphao subunits were required to mediate the effect. Inhibition of phospholipase C (PLC) blocked the inhibitory action of mGluR7, suggesting that a coincident activation of PLC by the G-protein betagamma with alphao subunits was required. The Ca(2+) chelator BAPTA, as well as inhibition of either the inositol trisphosphate (IP(3)) receptor or protein kinase C (PKC) abolished the mGluR7 effect. Moreover, activation of native mGluR7 induced a PTX-dependent IP(3) formation. These results indicated that IP(3)-mediated intracellular Ca(2+) release was required for PKC-dependent inhibition of the Ca(2+) channels. Possible control of synaptic transmission by the present mechanisms is discussed.
Assuntos
Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/metabolismo , Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Anticorpos/farmacologia , Bário/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Líquido Intracelular/metabolismo , Camundongos , Neuritos/metabolismo , Neurônios/citologia , Técnicas de Patch-Clamp , Toxina Pertussis , Receptores de Glutamato Metabotrópico/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Metabotropic glutamate receptors (mGlu receptors), the Ca2+-sensing receptor, gamma-aminobutyric acid type B receptors, and one group of pheromone receptors constitute a unique family (also called family 3) of heptahelical receptors. This original family shares no sequence similarity with any other G protein-coupled receptors. The identification and comparison of the molecular determinants of receptor/G protein coupling within the different receptor families may help identify general rules involved in this protein/protein interaction. In order to detect possible contact sites important for coupling selectivity between family 3 receptors and the G protein alpha-subunits, we examined the coupling of the cyclase-inhibiting mGlu2 and mGlu4 receptors to chimeric alphaq-subunits bearing the 5 extreme C-terminal amino acid residues of either Galphai, Galphao, or Galphaz. Whereas mGlu4 receptor activated all three chimeric G proteins, mGlu2 receptor activated Galphaqi and Galphaqo but not Galphaqz. The mutation of isoleucine -4 of Galphaqz into cysteine was sufficient to recover coupling of the mutant G protein to mGlu2 receptor. Moreover, the mutation of cysteine -4 of Galphaqo into isoleucine was sufficient to suppress the coupling to mGlu2 receptor. Mutations at positions -5 and -1 had an effect on coupling efficiency, but not selectivity. Our results emphasize the importance of the residue -4 of the alpha-subunits in their specific interaction to heptahelical receptors by extending this finding on the third family of G protein-coupled receptors.