The effect of membrane domains on the G protein–phospholipase Cβ signaling pathway

The plasma membrane serves as a barrier to limit the exit and entry of components into and out of the cell, offering protection from the external environment. Communication between the cell and the external environment is mediated by multiple signaling pathways. While the plasma membrane was historically viewed as a lipid bilayer with freely diffusing proteins, the last decade has shown that the lipids and proteins in the plasma membrane are organized in a non-random manner, and that this organization can direct and modify various signaling pathways in the cell. In this review, we qualitatively discuss the ways that membrane domains can affect cell signaling. We then focus on how membrane domains can affect a specific signaling pathway – the G protein–phospholipase Cβ pathway and show how membrane domains can play an active role in directing or redirecting G protein signals.     

磷脂酶D的细胞信号转导作用

磷脂酶D(PLD)是一类重要的跨膜信号转导酶类.分别由一个基因家族的不同成员编码.植物PLD的总体域结构相似,只是不同类型之间在某些单元上有重要差异.它们各具独特的生物化学特性.不同的PLD在不同的胁迫类型启动的特定的细胞过程中执行独特的细胞信号转导功能.PLD与其它磷脂酶及Ca2 信使之间有交互作用,形成复杂的信号转导网络.这一网络在不同植物种类、器官、组织和细胞类型中表现出特异性.文章最后讨论了PLD研究中有待揭示的问题并展望了今后的发展方向.     

腺苷-磷酸激活的蛋白激酶在脂类营养代谢中的研究及应用

腺苷-磷酸激活的蛋白激酶（AMP-activated protein kinase,AMPK）是公认的重要能量感受酶。其作用与多个代谢途径有关,尤其在脂类营养代谢过程中发挥着关键的调控作用。AMPK对脂质代谢的调控通过多个信号通路进行,涉及到骨骼肌、肝脏、乳腺等多个组织。对AMPK调控脂类营养代谢机理的研究为2型糖尿病、脂肪肝、肥胖症、癌症等多种疾病的治疗提供了靶点,但AMPK在奶牛乳腺组织的研究较少,其在提高奶牛生产性能方面潜能巨大。     

A high-cholesterol diet increases the association between caveolae and insulin receptors in rat liver

Caveolin-1, a component of caveolae, regulates signaling pathway compartmentalization by interacting with tyrosine (Tyr) kinase receptors and their substrates. Perturbations in caveolae lipid composition have been shown in vitro to displace proteins from lipid microdomains, thereby altering their functionality and subsequent downstream signaling. The role of caveolin-1 in insulin receptor (IR) signaling has been widely investigated in vitro mainly in 3T3-L1 adipocyte cells. However, in vivo experiments investigating this connection in liver tissue have not been carried out. The objective of the present study was to investigate the effects of a high-cholesterol diet on caveolin-1 expression and IR localization and activity in the rat liver. Compared with a standard diet, rats fed with diet rich in cholesterol significantly altered liver caveolae by increasing both caveolin-1 (66%, P < 0.05) and caveolin-2 (55%, P < 0.05) expression while caveolin-1 mRNA levels were reduced. Concomitantly, a 25% increase in localization of the caveolae-resident signaling protein IR was observed. The distribution of caveolar and noncaveolar phosphorylated IR was unaffected but insulin-induced IR activation was significantly enhanced following consumption of the high-cholesterol diet (120%, P < 0.001). However, the downstream molecules IRS-1 and Akt have shown impaired activity in cholesterol-fed rats suggesting insulin resistance condition. Insulin stimulation failed to induce Tyr phosphorylation of caveolin-1 in cholesterol-fed rats. These findings suggest a mechanism by which a high-cholesterol diet altered caveolin-1 expression in vivo accompanied by altered IR localization and activity.     

Do annexins participate in lipid messenger mediated intracellular signaling? A question revisited

Abstract

Annexins are physiologically important proteins that play a role in calcium buffering but also influence membrane structure, participate in Ca²⁺-dependent membrane repair events and in remodelling of the cytoskeleton. Thirty years ago several peptides isolated from lung perfusates, peritoneal leukocytes, neutrophiles and renal cells were proven inhibitory to the activity of phospholipase A₂. Those peptides were found to derive from structurally related proteins: annexins AnxA1 and AnxA2. These findings raised the question whether annexins may participate in regulation of the production of lipid second messengers and, therefore, modulate numerous lipid mediated signaling pathways in the cell. Recent advances in the field of annexins made also with the use of knock-out animal models revealed that these proteins are indeed important constituents of specific signaling pathways. In this review we provide evidence supporting the hypothesis that annexins, as membrane-binding proteins and organizers of the membrane lateral heterogeneity, may participate in lipid mediated signaling pathways by affecting the distribution and activity of lipid metabolizing enzymes (most of the reports point to phospholipase A₂) and of protein kinases regulating activity of these enzymes. Moreover, some experimental data suggest that annexins may directly interact with lipid metabolizing enzymes and, in a calcium-dependent or independent manner, with some of their substrates and products. On the basis of these observations, many investigators suggest that annexins are capable of linking Ca²⁺, redox and lipid signaling to coordinate vital cellular responses to the environmental stimuli.     

Glycerol-3-phosphate Acyltransferase Isoform-4 (GPAT4) Limits Oxidation of Exogenous Fatty Acids in Brown Adipocytes

Glycerol-3-phosphate acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4^−/− mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4^−/− mice fed a 45% fat diet was 12% higher. Core body temperature was 1 ºC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4^−/− mice did not result from increased heat loss, because both cold tolerance and response to a β3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4^−/− BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4^−/− brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of β-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state.     

Lipids at membrane contact sites: cell signaling and ion transport

Communication between organelles is essential to coordinate cellular functions and the cell's response to physiological and pathological stimuli. Organellar communication occurs at membrane contact sites (MCSs), where the endoplasmic reticulum (ER) membrane is tethered to cellular organelle membranes by specific tether proteins and where lipid transfer proteins and cell signaling proteins are located. MCSs have many cellular functions and are the sites of lipid and ion transfer between organelles and generation of second messengers. This review discusses several aspects of MCSs in the context of lipid transfer, formation of lipid domains, generation of Ca²⁺ and cAMP second messengers, and regulation of ion transporters by lipids.     

Nanodiscs: A toolkit for membrane protein science

Membrane proteins are involved in numerous vital biological processes, including transport, signal transduction and the enzymes in a variety of metabolic pathways. Integral membrane proteins account for up to 30% of the human proteome and they make up more than half of all currently marketed therapeutic targets. Unfortunately, membrane proteins are inherently recalcitrant to study using the normal toolkit available to scientists, and one is most often left with the challenge of finding inhibitors, activators and specific antibodies using a denatured or detergent solubilized aggregate. The Nanodisc platform circumvents these challenges by providing a self‐assembled system that renders typically insoluble, yet biologically and pharmacologically significant, targets such as receptors, transporters, enzymes, and viral antigens soluble in aqueous media in a native‐like bilayer environment that maintain a target''s functional activity. By providing a bilayer surface of defined composition and structure, Nanodiscs have found great utility in the study of cellular signaling complexes that assemble on a membrane surface. Nanodiscs provide a nanometer scale vehicle for the in vivo delivery of amphipathic drugs, therapeutic lipids, tethered nucleic acids, imaging agents and active protein complexes. This means for generating nanoscale lipid bilayers has spawned the successful use of numerous other polymer and peptide amphipathic systems. This review, in celebration of the Anfinsen Award, summarizes some recent results and provides an inroad into the current and historical literature.     

Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors

Sec14-like phosphatidylinositol transfer proteins (PITPs) play important biological functions in integrating multiple aspects of intracellular lipid metabolism with phosphatidylinositol-4-phosphate signaling. As such, these proteins offer new opportunities for highly selective chemical interference with specific phosphoinositide pathways in cells. The first and best characterized small molecule inhibitors of the yeast PITP, Sec14, are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and a hallmark feature of NPPMs is their exquisite targeting specificities for Sec14 relative to other closely related Sec14-like PITPs. Our present understanding of Sec14::NPPM binding interactions is based on computational docking and rational loss-of-function approaches. While those approaches have been informative, we still lack an adequate understanding of the basis for the high selectivity of NPPMs among closely related Sec14-like PITPs. Herein, we describe a Sec14 motif, which we term the VV signature, that contributes significantly to the NPPM sensitivity/resistance of Sec14-like phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer proteins. The data not only reveal previously unappreciated determinants that govern Sec14-like PITP sensitivities to NPPMs, but enable predictions of which Sec14-like PtdIns/PtdCho transfer proteins are likely to be NPPM resistant or sensitive based on primary sequence considerations. Finally, the data provide independent evidence in support of previous studies highlighting the importance of Sec14 residue Ser173 in the mechanism by which NPPMs engage and inhibit Sec14-like PITPs.     

Influence of Lipid Mimetics on Gating of Ryanodine Receptor

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Signaling roles of diacylglycerol kinases

Diacylglycerol kinases (DGKs) attenuate diacylglycerol signaling by converting this lipid to phosphatidic acid (PA). The nine mammalian DGKs that have been identified are widely expressed, but each isoform has a unique tissue and subcellular distribution. Their kinase activity is regulated by mechanisms that modify their access to diacylglycerol, directly affect their kinase activity, or alter their ability to bind to other proteins. In many cases, these enzymes regulate the activity of proteins that are modulated by either diacylglycerol or PA. Experiments using cultured cells and model organisms have demonstrated that DGKs have prominent roles in neuronal transmission, lymphocyte signaling, and carcinogenesis.     

Specific SHP-2 partitioning in raft domains triggers integrin-mediated signaling via Rho activation

Cell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts. By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling. Serum-depleted, nonattached cells expressing the raft SHP-2 form, but not non-raft SHP-2, display signaling events resembling those observed after fibronectin attachment, such as beta1 integrin clustering, 397Y-FAK phosphorylation, and ERK activation, and also increases Rho-GTP levels. Expression of the dominant negative N19Rho abrogates raft-SHP-2-induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling. Expression of a catalytic inactive SHP-2 mutant abrogates the adhesion-induced feedback inhibition of Rho activity, suggesting that SHP-2 contributes to adhesion-induced suppression of Rho activity. Because raft recruitment of SHP-2 occurs physiologically after cell attachment, these results provide a mechanism by which SHP-2 may influence cell adhesion and migration by spatially regulating Rho activity.