C2 domain membrane penetration by group IVA cytosolic phospholipase A2 induces membrane curvature changes

Group IVA cytosolic phospholipase A₂ (cPLA₂α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA₂α binds zwitterionic lipids such as phosphatidylcholine in a Ca²⁺-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA₂α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA₂α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA₂α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA₂α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA₂α.     

Lactosylceramide Interacts with and Activates Cytosolic Phospholipase A2α

Lactosylceramide (LacCer) is a member of the glycosphingolipid family and is known to be a bioactive lipid in various cell physiological processes. However, the direct targets of LacCer and cellular events mediated by LacCer are largely unknown. In this study, we examined the effect of LacCer on the release of arachidonic acid (AA) and the activity of cytosolic phospholipase A₂α (cPLA₂α). In CHO-W11A cells, treatment with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthase, reduced the glycosphingolipid level, and the release of AA induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or platelet-activating factor was inhibited. The addition of LacCer reversed the PPMP effect on the stimulus-induced AA release. Exogenous LacCer stimulated the release of AA, which was decreased by treatment with an inhibitor of cPLA₂α or silencing of the enzyme. Treatment of CHO-W11A cells with LacCer induced the translocation of full-length cPLA₂α and its C2 domain from the cytosol to the Golgi apparatus. LacCer also induced the translocation of the D43N mutant of cPLA₂α. Treatment of L929 cells with TNF-α induced LacCer generation and mediated the translocation of cPLA₂α and AA release, which was attenuated by treatment with PPMP. In vitro studies were then conducted to test whether LacCer interacts directly with cPLA₂α. Phosphatidylcholine vesicles containing LacCer increased cPLA₂α activity. LacCer bound to cPLA₂α and its C2 domain in a Ca²⁺-independent manner. Thus, we propose that LacCer is a direct activator of cPLA₂α.     

Chain length specificity for activation of cPLA2�� by C1P: use of the dodecane delivery system to determine lipid-specific effects

Previously, our laboratory demonstrated that ceramide-1-phosphate (C1P) specifically activated group IVA cytosolic phospholipase A₂ (cPLA₂α) in vitro. In this study, we investigated the chain length specificity of this interaction. C1P with an acyl-chain of ≥6 carbons efficiently activated cPLA₂α in vitro, whereas C₂-C1P, was unable to do so. Delivery of C1P to cells via the newly characterized ethanol/dodecane system demonstrated a lipid-specific activation of cPLA₂α, AA release, and PGE₂ synthesis (EC₅₀ = 400 nM) when compared to structurally similar lipids. C1P delivered as vesicles in water also induced a lipid-specific increase in AA release. Mass spectrometric analysis demonstrated that C1P delivered via ethanol/dodecane induced a 3-fold increase in endogenous C1P with little metabolism to ceramide. C1P was also more efficiently delivered (>3-fold) to internal membranes by ethanol/dodecane as compared to vesiculated C1P. Using this now established delivery method for lipids, C₂-C1P was shown to be ineffective in the induction of AA release as compared with C₆-C1P, C₁₆-C1P, and C_18:1 C1P. Here, we demonstrate that C1P requires ≥6 carbon acyl-chain to activate cPLA₂α. Thus, published reports on the biological activity of C₂-C1P are not via eicosanoid synthesis. Furthermore, this study demonstrates that the alcohol/dodecane system can be used to efficiently deliver exogenous phospholipids to cells for the examination of specific biological effects.—Wijesinghe, D. S., P. Subramanian, N. F. Lamour, L. B. Gentile, M. H. Granado, A. Bielawska, Z. Szulc, A. Gomez-Munoz, and C. E. Chalfant. Chain length specificity for activation of cPLA₂α by C1P: use of the dodecane delivery system to determine lipid-specific effects.     

Annular Anionic Lipids Stabilize the Integrin αIIbβ3 Transmembrane Complex

Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbβ3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg⁹⁹⁵) contacts with β3(Asp⁷²³) but paradoxically do not diminish the contribution of αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbβ3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact.     

Ceramide 1-Phosphate Is Required for the Translocation of Group IVA Cytosolic Phospholipase A2 and Prostaglandin Synthesis

Little is known about the regulation of eicosanoid synthesis proximal to the activation of cytosolic phospholipase A₂α (cPLA₂α), the initial rate-limiting step. The current view is that cPLA₂α associates with intracellular/phosphatidylcholine-rich membranes strictly via hydrophobic interactions in response to an increase of intracellular calcium. In opposition to this accepted mechanism of two decades, ceramide 1-phosphate (C1P) has been shown to increase the membrane association of cPLA₂α in vitro via a novel site in the cationic β-groove of the C2 domain (Stahelin, R. V., Subramanian, P., Vora, M., Cho, W., and Chalfant, C. E. (2007) J. Biol. Chem. 282, 20467–204741). In this study we demonstrate that C1P is a proximal and required bioactive lipid for the translocation of cPLA₂α to intracellular membranes in response to inflammatory agonists (e.g. calcium ionophore and ATP). Last, the absolute requirement of the C1P/cPLA₂α interaction was demonstrated for the production of eicosanoids using murine embryonic fibroblasts (cPLA₂α^−/−) coupled to “rescue” studies. Therefore, this study provides a paradigm shift in how cPLA₂α is activated during inflammation.Eicosanoids are a class of bioactive lipids derived from the 20-carbon fatty acid, arachidonic acid (AA),² including prostaglandins, prostacyclins, thromboxanes, and leukotrienes. The production of AA is the initial rate-limiting step in the production of eicosanoids, and the major phospholipase that regulates eicosanoids synthesis in response to agonists is group IVA cytosolic phospholipase A₂ (cPLA₂α) (2, 3). Activation of cPLA₂ in cells requires the association of the enzyme with intracellular membranes in a Ca²⁺-dependent manner. This translocation of cPLA₂α from the cytosol to intracellular membranes is mediated by a Ca²⁺-dependent lipid binding domain (CaLB domain) located at the N terminus of the enzyme (4–7). The CaLB domain is ∼60 amino acids and binds phosphatidylcholine (PC) in a Ca²⁺-dependent manner (3, 8–10). However, it is not known if physiologic calcium is sufficient to activate and translocate cPLA₂α to membranes in cells or if activation also requires the generation of other activating lipids, such as the focus of this study, ceramide 1-phosphate (C1P).One possible activating lipid, phosphatidylinositol 4,5-diphosphate, was ruled out by Balboa and co-workers (11) as a lipid co-factor required for the translocation of the enzyme. This group showed that the interaction with this lipid (via its catalytic domain) was required for full activity of cPLA₂α after the enzyme translocated to the membrane (11). Another recent report by Leslie and co-workers (12) confirmed these findings, and a recent study by our laboratory corroborated these findings utilizing biophysical approaches (1). Specifically, we showed that C1P induced a dramatic increase of cPLA₂α activity strictly by increasing the residence time of cPLA₂α to membranes, whereas phosphatidylinositol 4,5-diphosphate enhanced the enzymes catalytic activity and membrane penetration (13, 14).Recent studies from our laboratory have also demonstrated that C1P enhances the association of cPLA₂α with membranes in vitro via a novel interactions site adjacent to the calcium binding region II of the C2 domain. Mutations of specific amino acids of this region significantly reduced the affinity for C1P (>65%) without an effect on basal enzyme activity, calcium-dependent PC affinity (supplemental Table 1), and phosphatidylinositol 4,5-diphosphate activation/affinity (1, 14). The identification and characterization of the C1P interaction site in cPLA₂α allowed our laboratory to determine whether C1P played a role in regulating cPLA₂α translocation and, thus, eicosanoid synthesis in response to inflammatory agonists.     

Interfacial Kinetic and Binding Properties of Mammalian Group IVB Phospholipase A2 (cPLA2β) and Comparison with the Other cPLA2 Isoforms

The cytosolic (group IV) phospholipase A₂ (cPLA₂s) family contains six members. We have prepared recombinant proteins for human α, mouse β, human γ, human δ, human ϵ, and mouse ζ cPLA₂s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA₂β action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca²⁺ only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca²⁺ on the interfacial binding of mouse cPLA₂β and its C2 domain to vesicles. Ca²⁺-dependent binding of mouse cPLA₂β to cardiolipin-containing vesicles requires a patch of basic residues near the Ca²⁺-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA₂δ also displays Ca²⁺- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A₁, and phospholipase A₂ activities of the full set of mammalian cPLA₂s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA₂δ stands out as having relatively high phospholipase A₁ activity. We also tested the susceptibility of all cPLA₂ family members to a panel of previously reported inhibitors of human cPLA₂α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA₂β. These in vitro studies help determine the regulation and function of the cPLA₂ family members.     

Simultaneous activation of p38 and JNK by arachidonic acid stimulates the cytosolic phospholipase A2-dependent synthesis of lipid droplets in human monocytes

Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LDs are formed by two different routes: (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling, leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60–70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A₂α (cPLA₂α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA₂α activation by phosphorylation. Collectively, these results suggest that concomitant activation of p38 and JNK by AA cooperate to activate cPLA₂α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis.     

A Sequential Mechanism for Exosite-mediated Factor IX Activation by Factor XIa

During blood coagulation, the protease factor XIa (fXIa) activates factor IX (fIX). We describe a new mechanism for this process. FIX is cleaved initially after Arg¹⁴⁵ to form fIXα, and then after Arg¹⁸⁰ to form the protease fIXaβ. FIXα is released from fXIa, and must rebind for cleavage after Arg¹⁸⁰ to occur. Catalytic efficiency of cleavage after Arg¹⁸⁰ is 7-fold greater than for cleavage after Arg¹⁴⁵, limiting fIXα accumulation. FXIa contains four apple domains (A1–A4) and a catalytic domain. Exosite(s) on fXIa are required for fIX binding, however, there is lack of consensus on their location(s), with sites on the A2, A3, and catalytic domains described. Replacing the A3 domain with the prekallikrein A3 domain increases K_m for fIX cleavage after Arg¹⁴⁵ and Arg¹⁸⁰ 25- and ≥90-fold, respectively, and markedly decreases k_cat for cleavage after Arg¹⁸⁰. Similar results were obtained with the isolated fXIa catalytic domain, or fXIa in the absence of Ca²⁺. Forms of fXIa lacking the A3 domain exhibit 15-fold lower catalytic efficiency for cleavage after Arg¹⁸⁰ than for cleavage after Arg¹⁴⁵, resulting in fIXα accumulation. Replacing the A2 domain does not affect fIX activation. The results demonstrate that fXIa activates fIX by an exosite- and Ca²⁺-mediated release-rebind mechanism in which efficiency of the second cleavage is enhanced by conformational changes resulting from the first cleavage. Initial binding of fIX and fIXα requires an exosite on the fXIa A3 domain, but not the A2 or catalytic domain.     

JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A₂ (cPLA₂α). This work dissects the pathway leading to cPLA₂α activation and LD biogenesis. Both processes were Ca²⁺-independent, as they took place after pharmacological blockade of Ca²⁺ transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA₂α, which abrogates its Ca²⁺ binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca²⁺ but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA₂α at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA₂α at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA₂α and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.     

Hyperforin induces Ca-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A2 activation through select phospholipid interactions

Here, we investigated the modulation of cytosolic phospholipase A₂ (cPLA₂)-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10 µM) versus unstimulated cells, which was blocked by cPLA₂α-inhibition, and induced translocation of cPLA₂ to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca²⁺ by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA₂ at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA₂ phosphorylation. However, neither AA release nor translocation of cPLA₂ was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA₂ in the presence of Ca²⁺. However, when Ca²⁺ was omitted, hyperforin caused a prominent increase in cPLA₂ activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional ¹H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA₂ activation by enabling its access towards select lipid membranes independent of Ca²⁺ ions. Such Ca²⁺- and phosphorylation-independent mechanism of cPLA₂ activation may apply also to other membrane-interfering molecules.     

Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages

Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A₂ group IVA (cPLA₂α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA₂α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA₂α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E₂ production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA₂α inhibitor blocked HA-induced AA release and PGE₂ production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA₂α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE₂ production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE₂ production and COX2 expression were blocked in Tlr4^−/− and Myd88^−/− mice, but not in Cd44^−/− mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA₂α/COX2^high and COX1/ALOX15/ALOX5/LTA4H^low gene and PGE₂/PGD₂/15-HETE^high and LXA₄^low eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.     

Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of α1PDX (α1-Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras

α₁-Protease inhibitor Portland (α₁PDX) is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α₁PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α₁PDX with those of serpin B8 demonstrated that both the P4–P1 RXXR recognition sequence as well as the P1′–P5′ sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin(298–300) loop but not the other loops differentially affected furin reactivity with serpin B8 and α₁PDX in a manner that depended on the serpin RCL-primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298–300 loop whose substitution in α₁PDX differentially affected furin reactivity depending on the furin loop and serpin RCL-primed sequences. These studies demonstrate that RCL-primed residues, strand 3C exosites, and the furin(298–300) loop are critical determinants of serpin reactivity with furin, which may be exploited in the design of specific and selective α₁PDX inhibitors of PCs.     

Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A2

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca²⁺ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca²⁺. Addition of exogenous AA to cells spreading in the absence of extracellular Ca²⁺ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A₂ (cPLA₂) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA₂ phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca²⁺, all cPLA₂ became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca²⁺, the time for complete cPLA₂ phosphorylation was lengthened to <4 min. Maximal translocation of cPLA₂ from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca²⁺ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA₂ translocated to the membrane in the presence of extracellular Ca²⁺ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca²⁺ only 55–65% of the total cPLA₂ was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca²⁺. Although translocation of cPLA₂ from cytosol to membrane was Ca²⁺ dependent, phosphorylation of cPLA₂ was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA₂, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca²⁺. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA₂ phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA₂ and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA₂ translocation and phosphorylation remained unaffected. In conclusion, although cPLA₂-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA₂ phosphorylation and the release of AA.     

Molecular and in vivo phenotyping of missense variants of the human glucagon receptor

Naturally occurring missense variants of G protein–coupled receptors with loss of function have been linked to metabolic disease in case studies and in animal experiments. The glucagon receptor, one such G protein–coupled receptor, is involved in maintaining blood glucose and amino acid homeostasis; however, loss-of-function mutations of this receptor have not been systematically characterized. Here, we observed fewer glucagon receptor missense variants than expected, as well as lower allele diversity and fewer variants with trait associations as compared with other class B1 receptors. We performed molecular pharmacological phenotyping of 38 missense variants located in the receptor extracellular domain, at the glucagon interface, or with previously suggested clinical implications. These variants were characterized in terms of cAMP accumulation to assess glucagon-induced Gα_s coupling, and of recruitment of β-arrestin-1/2. Fifteen variants were impaired in at least one of these downstream functions, with six variants affected in both cAMP accumulation and β-arrestin-1/2 recruitment. For the eight variants with decreased Gα_s signaling (D63^ECDN, P86^ECDS, V96^ECDE, G125^ECDC, R225^3.30H, R308^5.40W, V368^6.59M, and R378^7.35C) binding experiments revealed preserved glucagon affinity, although with significantly reduced binding capacity. Finally, using the UK Biobank, we found that variants with wildtype-like Gα_s signaling did not associate with metabolic phenotypes, whereas carriers of cAMP accumulation-impairing variants displayed a tendency toward increased risk of obesity and increased body mass and blood pressure. These observations are in line with the essential role of the glucagon system in metabolism and support that Gα_s is the main signaling pathway effecting the physiological roles of the glucagon receptor.     

Study of the Role of Cytosolic Phospholipase A2 Alpha in Eicosanoid Generation and Thymocyte Maturation in the Thymus

The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A₂ (cPLA₂α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA₂α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA₂α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA₂α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA₂α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA₂α.     

Localization and function of cytosolic phospholipase A2α at the Golgi

Cytosolic phospholipase A₂α (cPLA₂α, Group IVA phospholipase A₂) is a central mediator of arachidonate release from cellular phospholipids for the biosynthesis of eicosanoids. cPLA₂α translocates to intracellular membranes including the Golgi in response to a rise in intracellular calcium level. The enzyme’s calcium-dependent phospholipid-binding C2 domain provides the targeting specificity for cPLA₂α translocation to the Golgi. However, other features of cPLA₂α regulation are incompletely understood such as the role of phosphorylation of serine residues in the catalytic domain and the function of basic residues in the cPLA₂α C2 and catalytic domains that are proposed to interact with anionic phospholipids in the membrane to which cPLA₂α is targeted. Increasing evidence strongly suggests that cPLA₂α plays a role in regulating Golgi structure, tubule formation and intra-Golgi transport. For example, recent data suggests that cPLA₂α regulates the transport of tight junction and adherens junction proteins through the Golgi to cell–cell contacts in confluent endothelial cells. However, there are now examples where data based on knockdown using siRNA or pharmacological inhibition of enzymatic activity of cPLA₂α affects fundamental cellular processes yet these phenotypes are not observed in cells from cPLA₂α deficient mice. These results suggest that in some cases there may be compensation for the lack of cPLA₂α. Thus, there is continued need for studies employing highly specific cPLA₂α antagonists in addition to genetic deletion of cPLA₂α in mice.     

Phosphatidylinositol-4,5-Bisphosphate Enhances Anionic Lipid Demixing by the C2 Domain of PKCα

The C2 domain of PKCα (C2α) induces fluorescence self-quenching of NBD-PS in the presence of Ca²⁺, which is interpreted as the demixing of phosphatidylserine from a mixture of this phospholipid with phosphatidylcholine. Self-quenching of NBD-PS was considerably increased when phosphatidylinositol-4,5-bisphosphate (PIP₂) was present in the membrane. When PIP₂ was the labeled phospholipid, in the form of TopFluor-PIP₂, fluorescence self-quenching induced by the C2 domain was also observed, but this was dependent on the presence of phosphatidylserine. An independent indication of the phospholipid demixing effect given by the C2α domain was obtained by using ²H-NMR, since a shift of the transition temperature of deuterated phosphatidylcholine was observed as a consequence of the addition of the C2α domain, but only in the presence of PIP₂. The demixing induced by the C2α domain may have a physiological significance since it means that the binding of PKCα to membranes is accompanied by the formation of domains enriched in activating lipids, like phosphatidylserine and PIP₂. The formation of these domains may enhance the activation of the enzyme when it binds to membranes containing phosphatidylserine and PIP₂.     

Interleukin-1β induces the synthesis and activity of cytosolic phospholipase A2 and the release of prostaglandin E2 in human amnion-derived WISH cells

The objective of this study was to examine the expression and activity of cytosolic phospholipase A₂ (cPLA₂) in relation to prostaglandin E₂ (PGE₂) synthesis in human amnion-derived WISH cells in response to stimulation by interleukin-1β (IL-1β). cPLA₂ activity was characterized by sensitivity to heat and acid treatment, stability to dithiothreitol, and inhibition by the specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF₃). Treatment of WISH cells with IL-1β (0.01–1 ng/mL) for up to 24 h resulted in a significant increase in PGE₂ release in a concentration- and time-dependent manner accompanied by increases both in total cellular cPLA₂ activity and in cPLA₂ protein levels detected by Western blot analysis. The parallel increase in total cellular cPLA₂ activity and cPLA₂ protein level indicates that IL-1β may induce the synthesis of CPLA₂. Incubation of the cells with 10 μM AACOCF₃ for 24 h significantly inhibited IL-1β-induced PGE₂ production strongly suggesting that cPLA₂ mediates IL-1β-induced PGE₂ formation. In unstimulated cells, there is appreciable total cellular cPLA₂ activity and protein, but these cells produce low amounts of PGE₂ until stimulated by IL-1β, suggesting that cPLA₂ translocation from cytosol to the membrane is necessary for its bioactivity. In contrast to IL-1β, treatment with phorbol ester (12-O-tetradecanoyl phorbol-13-acetate, TPA, 10⁻¹⁰−10⁻⁶ M) for 24 h significantly inhibited total cellular cPLA₂ activity in a concentration-dependent manner. The amount of total cellular cPLA₂ protein seen on Western blot remained unchanged following TPA treatment. These data suggest that in WISH cells, IL-1β induces both translocation to the membrane and de novo synthesis of cPLA₂ protein to sustain prostaglandin (PG) synthesis. In contrast, TPA may only cause cPLA₂ translocation but no increase in cPLA₂ protein synthesis, resulting in limited PFG synthesis. Our results provide a mechanism for the effect of IL-1β on prostaglandin synthesis in human amnion cells and provide support for a role of cPLA₂ in the mechanism initiating human parturition.