The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila

The neuropeptide Pigment Dispersing Factor (PDF) is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R) is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells—the PDF-R(+) LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s) is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into “circadian signalosomes,” whose compositions differ between E and M pacemaker cell types.     

The divergent TGF-beta ligand Dawdle utilizes an activin pathway to influence axon guidance in Drosophila

Axon guidance is regulated by intrinsic factors and extrinsic cues provided by other neurons, glia and target muscles. Dawdle (Daw), a divergent TGF-beta superfamily ligand expressed in glia and mesoderm, is required for embryonic motoneuron pathfinding in Drosophila. In daw mutants, ISNb and SNa axons fail to extend completely and are unable to innervate their targets. We find that Daw initiates an activin signaling pathway via the receptors Punt and Baboon (Babo) and the signal-transducer Smad2. Furthermore, mutations in these signaling components display similar axon guidance defects. Cell-autonomous disruption of receptor signaling suggests that Babo is required in motoneurons rather than in muscles or glia. Ectopic ligand expression can rescue the daw phenotype, but has no deleterious effects. Our results indicate that Daw functions in a permissive manner to modulate or enable the growth cone response to other restricted guidance cues, and support a novel role for activin signaling in axon guidance.     

The role of maternal Activin-like signals in zebrafish embryos

Maternal Activin-like proteins, a subgroup of the TGF-beta superfamily, play a key role in establishing the body axes in many vertebrates, but their role in teleosts is unclear. At least two maternal Activin-like proteins are expressed in zebrafish, including the Vg1 orthologue, zDVR-1, and the nodal-related gene, Squint. Our analysis of embryos lacking both maternal and zygotic squint function revealed that maternal squint is required in some genetic backgrounds for the formation of dorsal and anterior tissues. Conditional inactivation of the ALK4, 5 and 7 receptors by SB-505124 treatment during the cleavage stages ruled out a role for maternal Squint, zDVR-1, or other Activin-like ligands before the mid-blastula transition, when the dorsal axis is established. Furthermore, we show that maternal Squint and zDVR-1 are not required during the cleavage stages to induce zygotic nodal-related gene expression. nodal-related gene expression decreases when receptor inhibition continues past the mid-blastula transition, resulting in a progressive loss of mesoderm and endoderm. We conclude that maternally expressed Activin-like signals do not act before the mid-blastula transition in zebrafish, but do have a variably penetrant role in the later stages of axis formation. This contrasts with the early role for these signals during Xenopus development.     

Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor

Ligand binding to ecdysone receptor (EcR) is an autonomous function of the ligand binding domain (LBD) and is not modified by other receptor domains or tags fused to the LBD. Association and dissociation velocity of hormone to EcR was studied in the absence and presence of its main dimerization partner Ultraspiracle (USP). Mutational analysis of the EcR(LBD) revealed that ligand entry and exit is affected differently by the same point mutation, indicating that different pathways are used for association and dissociation of the ligand. Heterodimerization with wild type USP(LBD) increases ligand association to EcR(LBD) about fivefold and reduces dissociation 18-fold. Opposite effects of the same mutation (N626K) on dissociation velocity of ligand in EcR and EcR/USP indicate that not only hormone binding itself, but also the kinetic behaviour of ligand binding is modified by the dimerization partner. A general effect of the point mutations on the 3D architecture seems unlikely due to the highly selective effects on the kinetics of hormone binding.     

The fastest contracting muscles of nonmammalian vertebrates express only one isoform of the ryanodine receptor.

The skeletal muscles of chickens, frogs, and fish have been reported to express two isoforms (alpha and beta) of the sarcoplasmic reticulum calcium release channel (ryanodine receptor or RYR), while mammals express only one. We have studied patterns of RYR isoform expression in skeletal muscles from a variety of fish, reptiles, and birds with immunological techniques. Immunoblot analysis with a monoclonal antibody that recognizes both nonmammalian RYR isoforms and a polyclonal antibody specific to the alpha isoform show two key results: (a) two reptilian orders share with mammals the pattern of expressing only the alpha (skeletal) RYR isoform in skeletal muscle; and (b) certain functionally specialized muscles of fish and birds express only the alpha RYR isoforms. While both isoforms are expressed in the body musculature of fish and birds, the alpha isoform is expressed alone in extraocular muscles and swimbladder muscles. The appearance of the alpha RYR isoform alone in the extraocular muscles and a fast-contracting sonic muscle in fish (toadfish swimbladder muscle) provides evidence that this isoform is selectively expressed when rapid contraction is required. The functional and phylogenetic implications of expression of the alpha isoform alone are discussed in the context of the mechanism and evolution of excitation-contraction coupling.     

The murine TRAIL receptor signals caspase-independent cell death through ceramide

Death receptors such as the 55 kDa tumor necrosis factor (TNF) receptor (TNF-R55) or Fas can initiate both apoptotic (caspase-dependent) and caspase-independent routes to programmed cell death (PCD). Here, we demonstrate for the first time that the single murine receptor for (TNF)-related apoptosis-inducing ligand (mTRAIL-R2) can induce a caspase-independent form of PCD with necrosis-like features in addition to apoptosis. Analysis of morphological and cellular features of caspase-independent PCD in response to TRAIL and TNF suggests that mTRAIL-R2 and TNF-R55 elicit caspase-independent PCD through similar pathways, although without participation of cathepsins. Cells overexpressing acid ceramidase (AC), an enzyme that metabolizes the sphingolipid ceramide, show enhanced survival from TRAIL-induced caspase-independent PCD but not from apoptosis, implicating a function of ceramide as a key mediator in caspase-independent PCD (but not apoptosis) induced by mTRAIL-R2. In concert with the enhanced resistance of AC-overexpressing cells against caspase-independent PCD induced by TNF, our results suggest that ceramide acts as a common mediator of caspase-independent PCD caused by death receptors such as mTRAIL-R2 and TNF-R55.     

Structure and ligand specificity of the Drosophila melanogaster insulin receptor.

The insulin-binding and protein tyrosine kinase subunits of the Drosophila melanogaster insulin receptor homolog have been identified and characterized by using antipeptide antibodies elicited to the deduced amino acid sequence of the alpha and beta subunits of the human insulin receptor. In D. melanogaster embryos and cell lines, the insulin receptor contains insulin-binding alpha subunits of 110 or 120 kilodaltons (kDa), a 95-kDa beta subunit that is phosphorylated on tyrosine in response to insulin in intact cells and in vitro, and a 170-kDa protein that may be an incompletely processed receptor. All of the components are synthesized from a proreceptor, joined by disulfide bonds, and exposed on the cell surface. The beta subunit is recognized by an antipeptide antibody elicited to amino acids 1142 to 1162 of the human insulin proreceptor, and the alpha subunit is recognized by an antipeptide antibody elicited to amino acids 702 to 723 of the human proreceptor. Of the polypeptide ligands tested, only insulin reacts with the D. melanogaster receptor. Insulinlike growth factors type I and II, epidermal growth factor, and the silkworm insulinlike prothoracicotropic hormone are unable to stimulate autophosphorylation. Thus despite the evolutionary divergence of vertebrates and invertebrates, the essential features of the structure and intrinsic functions of the insulin receptor have been remarkably conserved.     

The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria

There are three isoforms of the inositol 1,4,5- trisphosphate receptor (InsP(3)R), each of which has a distinct effect on Ca(2+) signaling. However, it is not known whether each isoform similarly plays a distinct role in the activation of Ca(2+)-mediated events. To investigate this question, we examined the effects of each InsP(3)R isoform on transmission of Ca(2+) signals to mitochondria and induction of apoptosis. Each isoform was selectively silenced using isoform-specific small interfering RNA in Chinese hamster ovary cells, which express all three InsP(3)R isoforms. ATP-induced cytosolic Ca(2+) signaling patterns were altered, regardless of which isoform was silenced, but in a different fashion depending on the isoform. ATP also induced Ca(2+) signals in mitochondria, which were inhibited more effectively by silencing the type III InsP(3)R than by silencing either the type I or type II isoform. The type III isoform also co-localized most strongly with mitochondria. When apoptosis was induced by activation of either the extrinsic or intrinsic apoptotic pathway, induction was reduced most effectively by silencing the type III InsP(3)R. These findings provide evidence that the type III isoform of the InsP(3)R plays a special role in induction of apoptosis by preferentially transmitting Ca(2+) signals into mitochondria.     

Epstein-Barr virus latent membrane protein 2A preferentially signals through the Src family kinase Lyn

Latent membrane protein 2A (LMP2A) is a viral protein expressed during Epstein-Barr virus (EBV) latency in EBV-infected B cells both in cell culture and in vivo. LMP2A has important roles in modulating B-cell receptor signal transduction and provides survival and developmental signals to B cells in vivo. Although Lyn has been shown to be important in mediating LMP2A signaling, it is still unclear if Lyn is used preferentially or if LMP2A associates promiscuously with other Src family kinase (SFK) members. To investigate the role of various SFKs in LMP2A signaling, we crossed LMP2A transgenic mice (TgE) with Lyn^−/−, Fyn^−/−, or Blk^−/− mice. TgE Lyn^−/− mice had a larger immunoglobulin M (IgM)-positive B-cell population than TgE mice, suggesting that the absence of Lyn prevents LMP2A from delivering survival and developmental signals to the B cells. Both TgE Fyn^−/− and TgE Blk^−/− mice have an IgM-negative population of splenic B cells, similar to the TgE mice. LMP2A was also transiently transfected into the human EBV-negative B-cell line BJAB to determine which SFK members associate with LMP2A. Lyn was detected in LMP2A immunoprecipitates, whereas Fyn was not. Both Lyn and Fyn were able to bind to an LMP2A mutant which contained a sequence shown previously to bind tightly to the SH2 domain of multiple SFK members. From these results, we conclude that LMP2A preferentially associates with and signals through Lyn compared to its association with other SFKs. This preferential association is due in part to the SH2 domain of Lyn associating with LMP2A.     

Inhibition of serotonin 5-hydroxytryptamine2c receptor function through heterodimerization: receptor dimers bind two molecules of ligand and one G-protein

Although dimerization appears to be a common property of G-protein-coupled receptors (GPCRs), it remains unclear whether a GPCR dimer binds one or two molecules of ligand and whether ligand binding results in activation of one or two G-proteins when measured using functional assays in intact living cells. Previously, we demonstrated that serotonin 5-hydroxytryptamine2C (5-HT2C) receptors form homodimers (Herrick-Davis, K., Grinde, E., and Mazurkiewicz, J. (2004) Biochemistry 43, 13963-13971). In the present study, an inactive 5-HT(2C) receptor was created and coexpressed with wild-type 5-HT2C receptors to determine whether dimerization regulates receptor function and to determine the ligand/dimer/G-protein stoichiometry in living cells. Mutagenesis of Ser138 to Arg (S138R) produced a 5-HT2C receptor incapable of binding ligand or stimulating inositol phosphate (IP) signaling. Confocal fluorescence imaging revealed plasma membrane expression of yellow fluorescent protein-tagged S138R receptors. Expression of wild-type 5-HT2C receptors in an S138R-expressing stable cell line had no effect on ligand binding to wild-type 5-HT2C receptors, but inhibited basal and 5-HT-stimulated IP signaling as well as constitutive and 5-HT-stimulated endocytosis of wild-type 5-HT2C receptors. M1 muscarinic receptor activation of IP production was normal in the S138R-expressing cells. Heterodimerization of S138R with wild-type 5-HT2C receptors was visualized in living cells using confocal fluorescence resonance energy transfer (FRET). FRET was dependent on the donor/acceptor ratio and independent of the receptor expression level. Therefore, inactive 5-HT2C receptors inhibit wild-type 5-HT2C receptor function by forming nonfunctional heterodimers expressed on the plasma membrane. These results are consistent with a model in which one GPCR dimer binds two molecules of ligand and one G-protein and indicate that dimerization is essential for 5-HT receptor function.     

Multiple signals regulate axon regeneration through the nogo receptor complex

Several myelin-derived proteins have been identified as components of central nervous system (CNS) myelin, which prevents axonal regeneration in the adult vertebrate CNS. The discovery of the receptor for these proteins was a major step toward understanding the failure of axon regeneration. The receptor complex consists of at least three elements: the p75 receptor (p75^NTR), the Nogo receptor and LINGO-1. Downstream from the receptor complex, RhoA activation has been shown to be a key element of the signaling mechanism of these proteins. Rho activation arrests axon growth, and blocking Rho activation promotes axon regeneration in vivo. Recent studies have identified conventional protein kinase C as an additional necessary component for axon growth inhibition. Possible crosstalk downstream of these signals should be explored to clarify all the inhibitory signals and may provide an efficient molecular target against injuries to the CNS.     

Adenosine 2b receptor (A2bR) signals through adenylate cyclase (AC) 6 isoform in the intestinal epithelial cells

Adenosine 2b receptor (A2bR), a G-protein coupled receptor positively coupled to adenylate cyclase, mediates key events such as chloride, IL-6 and fibronectin secretion in intestinal epithelial cells and is upregulated during intestinal inflammation. In order to gain insight into the overall mechanism of A2bR activation, in this study, we sought to characterize the AC isoform associated with A2bR signaling. The colonic epithelial cell line T84, expressing only the A2b subtype of adenosine receptor, and Chinese hamster ovary (CHO) cells, were used in these studies. cAMP was measured by luminometric assay and AC isoform expression was determined by Western blot, RT-PCR, isoform-specific stealth RNAi and Quantigene. T84 and CHO cells express all nine known AC isoforms. In order to characterize which AC isoform(s) are associated with A2bR, we used the differential inhibition of specific AC isoforms by calcium and nitric oxide. Pretreatment of cells with carbachol or nitric oxide donors such as S-Nitroso-N-acetylpencillamine (SNAP) and PAPANANOATE inhibited A2bR mediated increase in cAMP. Further, overexpression of AC-5 or AC-6 potentiated A2bR-mediated increases in cAMP levels. Finally, transfection with AC isoform-specific RNAi demonstrated that AC-6 but not AC-5 RNAi inhibited adenosine-induced cAMP levels. Taken together, these results suggest that A2bR mediates signaling through AC-6 isoform. Since pro-inflammatory cytokines such as interferon-gamma (IFN-gamma) modulate the expression of specific AC isoforms in the intestinal epithelia, our observation may have therapeutic implications for intestinal inflammation or diarrhea wherein aA2bR is upregulated.     

Interleukin-11 signals through the formation of a hexameric receptor complex

Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. IL-11 has been shown to induce gp130-dependent signaling through the formation of a high affinity complex with the IL-11 receptor (IL-11R) and gp130. Site-directed mutagenesis studies have identified three distinct receptor binding sites of IL-11, which enable it to form this high affinity receptor complex. Here we present data from immunoprecipitation experiments, using differentially tagged forms of ligand and soluble receptor components, which show that multiple copies of IL-11, IL-11R, and gp130 are present in the receptor complex. Furthermore, it is demonstrated that sites II and III of IL-11 are independent gp130 binding epitopes and that both are essential for gp130 dimerization. We also show that a stable high affinity complex of IL-11, IL-11R, and gp130 can be resolved by nondenaturing polyacrylamide gel electrophoresis, and its composition verified by second dimension denaturing polyacrylamide gel electrophoresis. Results indicate that the three receptor binding sites of IL-11 and the Ig-like domain of gp130 are all essential for this stable receptor complex to be formed. We therefore propose that IL-11 forms a hexameric receptor complex composed of two molecules each of IL-11, IL-11R, and gp130.     

An insertion within a variably spliced Drosophila tropomyosin gene blocks accumulation of only one encoded isoform

We have characterized an aberrant allele of a variably spliced Drosophila tropomyosin gene. The allele was recovered from the flightless Ifm(3)3 strain, which has been shown to have structurally and functionally abnormal indirect flight muscles. The transcribed portion of the mutant gene is interrupted by an 8,8 kb insertion of middle repetitive DNA having a structure typical of copia-like Drosophila mobile elements. The insertion is positioned so as to interrupt an exon sequence in one splicing mode and, simultaneously, an intron in the alternate mode. As a consequence of the insertion the allele fails to direct synthesis of the flight muscle-specific tropomyosin isoform, but remains capable of specifying a second isoform, which accumulates in nonfibrillar Drosophila muscles.     

The elastin receptor complex transduces signals through the catalytic activity of its Neu-1 subunit

The binding of elastin peptides on the elastin receptor complex leads to the formation of intracellular signals but how this is achieved remains totally unknown. Using pharmacological inhibitors of the enzymatic activities of its subunits, we show here that the elastin peptide-driven ERK1/2 activation and subsequent pro-MMP-1 production, observed in skin fibroblasts when they are cultured in the presence of these peptides, rely on a membrane-bound sialidase activity. As lactose blocked this effect, the elastin receptor sialidase subunit, Neu-1, seemed to be involved. The use of a catalytically inactive form of Neu-1 and the small interfering RNA-mediated decrease of Neu-1 expression strongly support this view. Finally, we report that N-acetyl neuraminic acid can reproduce the effects of elastin peptides on both ERK1/2 activation and pro-MMP-1 production. Altogether, our results indicate that the enzymatic activity of the Neu-1 subunit of the elastin receptor complex is responsible for its signal transduction, presumably through sialic acid generation from undetermined substrates.     

The valence for ligand of the human mononuclear phagocyte 72 kD high-affinity IgG Fc receptor is one

The valence for ligand of the 72 kD high-affinity IgG FcR present on human mononuclear phagocytes was evaluated. Lysates of U937 cells whose high-affinity FcR had been saturated with equivalent quantities of 125I-IgG1 kappa and unlabeled IgG1 lambda or with 125I-IgG1 lambda and unlabeled IgG1 kappa were incubated with Sepharose-anti-kappa. Eighty-nine percent of the applied 125I-IgG1 kappa was bound, whereas 0.35% of the applied 125I-IgG1 lambda bound (mean of two experiments), indicating that if the receptors are occupied with ligand, the receptors bind only one ligand molecule at a time. Two experiments were performed to show that the receptors were ligand-occupied. First, a monoclonal antibody directed against the 72 kD FcR (FcRmab32) was added to lysates of U937 cells saturated with equal quantities of 125I-IgG1 lambda and IgG1 kappa. This anti-FcR antibody caused a dose-dependent sevenfold increase in the amount of 125I-IgG1 lambda bound to the anti-kappa immunoadsorbent (presumably by cross-linking receptors bearing 125I-IgG1 lambda with receptors bearing IgG1 kappa), whereas monoclonal antibodies (MMA and IV3) directed against two other determinants on U937 caused no such increase. In the second experiment, Sepharose-FcRmab32 adsorbed 60% of the 125I-IgG1 kappa and 46% of the 125I-IgG1 lambda applied in a U937 lysate (bearing high-affinity FcR), whereas only 3% of 125I-IgG1 kappa and 6% of 125I-IgG1 lambda applied in a K562 lysate (bearing no high-affinity FcR) were adsorbed. We interpret these data to indicate that in detergent solution the valency of the high-affinity FcR on U937 cells is one.     

Expression of the chicken hepatic glycoprotein receptor in Xenopus oocytes: conservation of ligand and receptor targeting signals.

We have obtained expression of the beta-N-acetylglucosamine-binding receptor from chicken hepatocytes in Xenopus oocytes by injecting mRNA synthesized in vitro from a full length cDNA cloned into an expression vector (Mellow et al: J. Biol Chem 263: 5468-5473, 1988). Immunoprecipitation of the receptor after labeling of oocytes with [35S]-methionine for times ranging from 6 to 72 h revealed 4-5 closely spaced bands of 25-30 kDa after SDS-PAGE. Although these bands were largely resistant to endoglycosidase H cleavage, endoglycosidase F reduced the size of all bands to a single species at 23-24 kDa, indicating that they resulted from heterogeneity in glycosylation of a single polypeptide. Incubation of oocytes expressing this receptor with [125I]-GlcNAc-BSA resulted in 1.8 to 10 x higher levels of cell-associated ligand in mRNA-injected vs. water-injected control oocytes, 2-35% of cell-associated counts was removed by EGTA rinse at 20 degrees C, suggesting that most ligand was inaccessible (presumably intracellular). Immunoprecipitation of sucrose gradient fractions detected receptor molecules predominantly in a light organelle at 1.09-1.12 g/cc (the density of early endosomes and plasma membrane vesicles), with no evidence of the receptor in much heavier yolk platelet fractions even in the presence of ligand. In contrast, internalized [125I]-GlcNAc-BSA was found either at the top of the gradients or in organelles at 1.09-1.17 g/cc and in yolk platelets. TCA precipitation indicated that much intracellular ligand was degraded to acid-soluble fragments. Addition of vitellogenin (the yolk protein precursor) to the medium together with the [125I]-GlcNAc-BSA shifted much of the ligand into yolk platelets. These data indicate that the chicken glycoprotein receptor expressed in oocytes mediates binding and internalization of this ligand into an organelle in which ligand-receptor dissociation occurs, allowing for separation of these two molecules into different compartments. The behavior of ligand in Xenopus oocytes expressing the chicken receptor closely resembles its behavior in hepatocytes.