D5 dopamine receptors are required for dopaminergic activation of phospholipase C.

Dopamine activates phospholipase C in discrete regions of the mammalian brain, and this action is believed to be mediated through a D(1)-like receptor. Although multiple lines of evidence exclude a role for the D(1) subtype of D(1)-like receptors in the phosphoinositide response, the D(5) subtype has not been similarly examined. Here, mice lacking D(5) dopamine receptors were tested for dopamine agonist-induced phosphoinositide signaling both in vitro and in vivo. The results show that hippocampal, cortical, and striatal tissues of D(5) receptor knockout mice significantly or completely lost the ability to produce inositol phosphate or diacylglycerol messengers after stimulation with dopamine or several selective D(1)-like receptor agonists. Moreover, endogenous inositol-1,4,5-trisphosphate stimulation by the phospholipase C-selective D(1)-like agonist 3-methyl-6-chloro-7,8-dihydroxy-1-[3methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959) was robust in wild-type animals but undetectable in the D(5) receptor mutants. Hence, D(5) receptors are required for dopamine and selective D(1)-like agonists to induce phospholipase C-mediated phosphoinositide signaling in the mammalian brain.

Trans-Golgi network and endosome dynamics connect ceramide homeostasis with regulation of the unfolded protein response and TOR signaling in yeast.

Synthetic genetic array analyses identify powerful genetic interactions between a thermosensitive allele (sec14-1(ts)) of the structural gene for the major yeast phosphatidylinositol transfer protein (SEC14) and a structural gene deletion allele (tlg2Delta) for the Tlg2 target membrane-soluble N-ethylmaleimide-sensitive factor attachment protein receptor. The data further demonstrate Sec14 is required for proper trans-Golgi network (TGN)/endosomal dynamics in yeast. Paradoxically, combinatorial depletion of Sec14 and Tlg2 activities elicits trafficking defects from the endoplasmic reticulum, and these defects are accompanied by compromise of the unfolded protein response (UPR). UPR failure occurs downstream of Hac1 mRNA splicing, and it is further accompanied by defects in TOR signaling. The data link TGN/endosomal dynamics with ceramide homeostasis, UPR activity, and TOR signaling in yeast, and they identify the Sit4 protein phosphatase as a primary conduit through which ceramides link to the UPR. We suggest combinatorial Sec14/Tlg2 dysfunction evokes inappropriate turnover of complex sphingolipids in endosomes. One result of this turnover is potentiation of ceramide-activated phosphatase-mediated down-regulation of the UPR. These results provide new insight into Sec14 function, and they emphasize the TGN/endosomal system as a central hub for homeostatic regulation in eukaryotes.

Functional anatomy of phospholipid binding and regulation of phosphoinositide homeostasis by proteins of the sec14 superfamily.

Sec14, the major yeast phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein, regulates essential interfaces between lipid metabolism and membrane trafficking from the trans-Golgi network (TGN). How Sec14 does so remains unclear. We report that Sec14 binds PtdIns and PtdCho at distinct (but overlapping) sites, and both PtdIns- and PtdCho-binding activities are essential Sec14 activities. We further show both activities must reside within the same molecule to reconstitute a functional Sec14 and for effective Sec14-mediated regulation of phosphoinositide homeostasis in vivo. This regulation is uncoupled from PtdIns-transfer activity and argues for an interfacial presentation mode for Sec14-mediated potentiation of PtdIns kinases. Such a regulatory role for Sec14 is a primary counter to action of the Kes1 sterol-binding protein that antagonizes PtdIns 4-OH kinase activity in vivo. Collectively, these findings outline functional mechanisms for the Sec14 superfamily and reveal additional layers of complexity for regulating phosphoinositide homeostasis in eukaryotes.

Diverse antidepressants increase CDP-diacylglycerol production and phosphatidylinositide resynthesis in depression-relevant regions of the rat brain.

BACKGROUND: Major depression is a serious mood disorder affecting millions of adults and children worldwide. While the etiopathology of depression remains obscure, antidepressant medications increase synaptic levels of monoamine neurotransmitters in brain regions associated with the disease. Monoamine transmitters activate multiple signaling cascades some of which have been investigated as potential mediators of depression or antidepressant drug action. However, the diacylglycerol arm of phosphoinositide signaling cascades has not been systematically investigated, even though downstream targets of this cascade have been implicated in depression. With the ultimate goal of uncovering the primary postsynaptic actions that may initiate cellular antidepressive signaling, we have examined the antidepressant-induced production of CDP-diacylglycerol which is both a product of diacylglycerol phosphorylation and a precursor for the synthesis of physiologically critical glycerophospholipids such as the phosphatidylinositides. For this, drug effects on [3H]cytidine-labeled CDP-diacylglycerol and [3H]inositol-labeled phosphatidylinositides were measured in response to the tricyclics desipramine and imipramine, the selective serotonin reuptake inhibitors fluoxetine and paroxetine, the atypical antidepressants maprotiline and nomifensine, and several monoamine oxidase inhibitors. RESULTS: Multiple compounds from each antidepressant category significantly stimulated [3H]CDP-diacylglycerol accumulation in cerebrocortical, hippocampal, and striatal tissues, and also enhanced the resynthesis of inositol phospholipids. Conversely, various antipsychotics, anxiolytics, and non-antidepressant psychotropic agents failed to significantly induce CDP-diacylglycerol or phosphoinositide synthesis. Drug-induced CDP-diacylglycerol accumulation was independent of lithium and only partially dependent on phosphoinositide hydrolysis, thus indicating that antidepressants can mobilize CDP-diacylglycerol from additional pools lying outside of the inositol cycle. Further, unlike direct serotonergic, muscarinic, or alpha-adrenergic agonists that elicited comparable or lower effects on CDP-diacylglycerol versus inositol phosphates, the antidepressants dose-dependently induced significantly greater accumulations of CDP-diacylglycerol. CONCLUSION: Chemically divergent antidepressant agents commonly and significantly enhanced the accumulation of CDP-diacylglycerol. The latter is not only a derived product of phosphoinositide hydrolysis but is also a crucial intermediate in the biosynthesis of several signaling substrates. Hence, altered CDP-diacylglycerol signaling might be implicated in the pathophysiology of depression or the mechanism of action of diverse antidepressant medications.

The Sec14-superfamily and the regulatory interface between phospholipid metabolism and membrane trafficking.

A central principle of signal transduction is the appropriate control of the process so that relevant signals can be detected with fine spatial and temporal resolution. In the case of lipid-mediated signaling, organization and metabolism of specific lipid mediators is an important aspect of such control. Herein, we review the emerging evidence regarding the roles of Sec14-like phosphatidylinositol transfer proteins (PITPs) in the action of intracellular signaling networks; particularly as these relate to membrane trafficking. Finally, we explore developing ideas regarding how Sec14-like PITPs execute biological function. As Sec14-like proteins define a protein superfamily with diverse lipid (or lipophile) binding capabilities, it is likely these under-investigated proteins will be ultimately demonstrated as a ubiquitously important set of biological regulators whose functions influence a large territory in the signaling landscape of eukaryotic cells.

Tandem regulation of phosphoinositide signaling and acute behavioral effects induced by antidepressant agents in rats.

RATIONALE: Antidepressants increase synaptic monoamine concentrations, but the subsequent signaling events that produce the beneficial clinical effects remain unclear. Diverse antidepressants increase CDP-diacylglycerol, a crucial step in phosphoinositide signaling. Serotonin 5HT(2) receptors, implicated in depression or the actions of some antidepressants, signal through phosphoinositide hydrolysis. Thus, cross talk between antidepressant-induced CDP-diacylglycerol and 5HT(2) signaling could contribute to the antidepressant mechanism. OBJECTIVE: The objective of the study was to test the hypotheses that antidepressants enhance net signaling via 5HT(2) receptors by augmenting the supply of phosphoinositide substrates and that this action contributes to the behavioral effects of the drugs. MATERIALS AND METHODS: Brain slices pre-labeled with [(3)H]inositol in the presence of various antidepressant concentrations were washed and incubated with the 5HT(2) agonist, alpha-methylserotonin, followed by measuring phosphoinositide synthesis and inositol phosphate accumulation. Further, rats administered antidepressants after pretreatment with neomycin to inhibit metabolic utilization of phosphoinositides were behaviorally evaluated in the forced swim test. RESULTS: Diverse antidepressants significantly enhanced phosphoinositide synthesis. While alpha-methylserotonin increased inositol phosphate accumulation, this effect was significantly accentuated in hippocampal or cortical tissues pre-incubated in the presence of imipramine, desipramine, fluoxetine, paroxetine, or maprotiline. Drug-induced behavioral antidepressant effects were reversed by neomycin pretreatment, whereas neomycin alone did not alter basal immobility times. CONCLUSIONS: Antidepressants probably exert tandem neurochemical effects by increasing synaptic monoamine concentrations and by producing phosphoinositides used in 5HT(2) receptor signaling. This combination of actions may constitute the mechanism of at least the acute behavioral effects of the drugs and could implicate aberrant neurolipid signaling in the pathophysiology of depression.

Nonclassical PITPs activate PLD via the Stt4p PtdIns-4-kinase and modulate function of late stages of exocytosis in vegetative yeast.

Phospholipase D (PLD) is a PtdCho-hydrolyzing enzyme that plays central signaling functions in eukaryotic cells. We previously demonstrated that action of a set of four nonclassical and membrane-associated Sec14p-like phosphatidylinositol transfer proteins (PITPs) is required for optimal activation of yeast PLD in vegetative cells. Herein, we focus on mechanisms of Sfh2p and Sfh5p function in this regulatory circuit. We describe several independent lines of in vivo evidence to indicate these SFH PITPs regulate PLD by stimulating PtdIns-4,5-P2 synthesis and that this stimulated PtdIns-4,5-P2 synthesis couples to action of the Stt4p PtdIns 4-kinase. Furthermore, we provide genetic evidence to suggest that specific subunits of the yeast exocyst complex (i.e. a component of the plasma membrane vesicle docking machinery) and the Sec9p plasma membrane t-SNARE are regulated by PtdIns(4,5)P2 and that Sfh5p helps regulate this interface in vivo. The collective in vivo and biochemical data suggest SFH-mediated stimulation of Stt4p activity is indirect, most likely via a substrate delivery mechanism.