RESUMO
Phosphatidylinositol 4-phosphate 5-kinases [PtdIns4P5Ks] synthesise the majority of cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and phospholipase D1 (PLD1) synthesises large amounts of phosphatidic acid (PtdOH). The activities of PtdIns4P5Ks and PLDs are thought to be coupled during cell signalling in order to support large simultaneous increases in both PtdIns(4,5)P(2) and PtdOH, since PtdOH activates PtdIns4P5Ks and PLD1 requires PtdIns(4,5)P(2) as a cofactor. However, little is known about the control of such a system. Membrane recruitment of ADP-ribosylation factors (Arfs) activates both PtdIns4P5Ks and PLDs, but it is not known if each enzyme is controlled in series by different Arfs or in parallel by a single form. We show through pull-down and vesicle sedimentation interaction assays that PtdIns4P5K activation may be facilitated by Arf-enhanced membrane association. However PtdIns4P5Ks discriminate poorly between near homogeneously myristoylated Arf1 and Arf6 although examples of all three known active isoforms (mouse alpha>beta, gamma) respond to these G-proteins. Conversely PLD1 genuinely prefers Arf1 and so the two lipid metabolising enzymes are differentially controlled. We propose that isoform selective Arf/PLD interaction and not Arf/PtdIns4P5K will be the critical trigger in the formation of distinct, optimal triples of Arf/PLDs/PtdIns4P5Ks and be the principle regulator of any coupled increases in the signalling lipids PtdIns(4,5)P(2) and PtdOH.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Fosfolipase D/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/genética , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/genética , Animais , Sequência de Bases , Sistema Livre de Células , Primers do DNA/genética , Ativação Enzimática , Guanosina Trifosfato/metabolismo , Células HL-60 , Humanos , Técnicas In Vitro , Cinética , Lipídeos de Membrana/metabolismo , Camundongos , Modelos Biológicos , Ácidos Mirísticos/química , Fosfolipase D/genética , Fosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
The Caenorhabditis elegans neuromuscular junction (NMJ) is one of the best studied synapses in any organism. A variety of genetic screens have identified genes required both for the essential steps of neurotransmitter release from motorneurons as well as the signaling pathways that regulate rates of neurotransmitter release. A number of these regulatory genes encode proteins that converge to regulate neurotransmitter release. In other cases genes are known to regulate signaling at the NMJ but how they act remains unknown. Many of the proteins that regulate activity at the NMJ participate in a network of heterotrimeric G-protein signaling pathways controlling the release of synaptic vesicles and/or dense-core vesicles (DCVs). At least four heterotrimeric G-proteins (Galphaq, Galpha12, Galphao, and Galphas) act within the motorneurons to control the activity of the NMJ. The Galphaq, Galpha12, and Galphao pathways converge to control production and destruction of the lipid-bound second messenger diacylglycerol (DAG) at sites of neurotransmitter release. DAG acts via at least two effectors, MUNC13 and PKC, to control the release of both neurotransmitters and neuropeptides from motorneurons. The Galphas pathway converges with the other three heterotrimeric G-protein pathways downstream of DAG to regulate neuropeptide release. Released neurotransmitters and neuropeptides then act to control contraction of the body-wall muscles to control locomotion. The lipids and proteins involved in these networks are conserved between C. elegans and mammals. Thus, the C. elegans NMJ acts as a model synapse to understand how neuronal activity in the human brain is regulated.