Regulation of membrane raft recruitment of the bradykinin B2 receptor by close association with the ATP/UTP receptor P2Y2.

Several G protein-coupled receptors are present in lipid rafts. We have shown that most of the P2Y2 receptor (P2Y2R) protein is fractionated into lipid rafts in COS 7 cells. In the same cells, about 25-30% of the bradykinin B2 receptor (B2R) protein is also fractionated into lipid rafts. When both P2Y2R and B2R are co-expressed, the distribution of P2Y2R remained unchanged, but more B2R shifted into the raft fraction. This indicates that the interaction between both receptors recruited B2R into the lipid rafts. After 15 min of UTP stimulation, both receptors almost completely disappeared from the cell surface by endocytosis as observed with a confocal fluorescence microscope. Furthermore, with bradykinin stimulation for 15 min, portions of both receptors disappeared from the cell surface and were endocytosed. As we reported previously with both CHO-K1 cells and HEK 293 cells, continuous stimulation of COS7 cells with GT1b and CSC resulted in the disappearance of both P2Y2R and B2R from the cell membrane surface. Thus, both P2Y2R and B2R migrate into membrane rafts and are endocytosed in parallel with signal crosstalk, clearly indicating that both closely interact on membrane rafts. The P2Y2R N-glycosylation deficient mutant does not migrate to the cell surface. It remains predominantly in the endoplasmic reticulum and is fractionated into raft fractions. In the presence of this glycosylation mutant, most of B2R remains in the endoplasmic reticulum, and is fractionated into the raft fraction. These findings demonstrate that in the membrane rafts of the endoplasmic reticulum, both receptors are already closely associated, and B2R shifts into the rafts by affinity with P2Y2R.

N-glycan-dependent cell-surface expression of the P2Y2 receptor and N-glycan-independent distribution to lipid rafts.

P2Y2 receptor (P2Y2R) is a G-protein-coupled receptor (GPCR) that couples with Gαq/11 and is stimulated by ATP and UTP. P2Y2R is involved in pain, proinflammatory changes, and blood pressure control. Some GPCRs are localized in lipid rafts for interaction with other signaling molecules. In this study, we prepared N-glycan-deficient mutants by mutating the two consensus Asn residues for N-glycosylation to Gln to examine intracellular localization and association with lipid rafts. Western blotting of the wild type (WT) protein and mutants (N9Q, N13Q, N9Q/N13Q) in COS-7 cells showed that both Asn residues were glycosylated in the WT. Fluorescent microscopy analysis showed that WT, N9Q and N13Q were expressed in the endoplasmic reticulum (ER), Golgi body, and cell membrane, but N9Q/N13Q was only found in the ER. WT, N9Q and N13Q moved from the cell surface to endosomes within 15 min after UTP stimulation. WT and the N9Q/N13Q glycosylation-deficient mutant appeared in the detergent insoluble membrane fraction, lipid raft. These findings suggest that P2Y2R is localized in lipid rafts in the ER during biosynthesis, and that N-glycosylation is required for subsequent expression in the cell membrane. In the presence of epoxomicin, a proteasome inhibitor, there was a significant increase in the level of N9Q/N13Q, which suggests that N-glycan-deficient P2Y2R undergoes proteasomal degradation.

Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase from oyster hepatopancreas†.

We have previously reported that oyster hepatopancreas contained three unusual α-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase (α-Kdo-ase), (ii) a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid α-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the α-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we found that instead of expressing Kdn-sialidase, our cloned gene expressed α-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant α-Kdo-ase (R-α-Kdo-ase) in yeast and CHO-S cells, but not in various Escherichia coli strains. The deduced amino acid sequence contains two Asp boxes (S(277)PDDGKTW and S(328)TDQGKTW) commonly found in sialidases, but is devoid of the signature FRIP-motif of sialidase. The R-α-Kdo-ase effectively hydrolyzed the Kdo in the core-oligosaccharide of the structurally defined lipopolysaccharide (LPS), Re-LPS (Kdo(2)-Lipid A) from Salmonella minnesota R595 and E. coli D31m4. However, Rd-LPS from S. minnesota R7 that contained an extra outer core phosphorylated heptose was only slowly hydrolyzed. The complex type LPS from Neisseria meningitides A1 and M992 that contained extra 5-6 sugar units at the outer core were refractory to R-α-Kdo-ase. This R-α-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans.

Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica.

A novel sphingomyelin-binding protein (clamlysin) was purified from the foot muscle of a brackishwater clam, Corbicula japonica. The purified 24.8-kDa protein lysed sheep, horse and rabbit erythrocytes and the hemolytic activity was inhibited by sphingomyelin, but not other phospholipids or glycosphingolipids. The open reading frame of the clamlysin gene encoded a putative 26.9-kDa protein (clamlysin B) which showed high sequence similarity with the actinoporin family. A surface plasmon resonance assay confirmed that clamlysin B specifically bound to sphingomyelin. Furthermore, two cDNA variants of clamlysin, encoding putative 31.4 kDa (clamlysin A) and 11 kDa (clamlysin C) proteins, were isolated. Only the 31.4-kDa variant was found to exhibit sphingomyelin-binding activity. Clamlysin A and B, but not C, shared a sequence (domain II) conserved in all known sphingomyelin-binding proteins. Domain II fused with a glutathione S-transferase bound to sphingomyelin. Horse erythrocytes, mouse melanoma B16 and GM95 cells, and Chinese hamster ovary CHO-K1 cells, but not the same cells treated with bacterial sphingomyelinase, were immunostained with clamlysin B. These results indicate that clamlysin B binds to the sphingomyelin of living cells and thus would be useful as a molecular probe to detect sphingomyelin.

Gangliosides and chondroitin sulfate desensitize and internalize B2 bradykinin receptors.

Prolonged or repeated agonist activation of G-protein-coupled receptors (GPCRs) initiates their desensitization and internalization, rendering them unresponsive to agonist activation. We analyzed how gangliosides and chondroitin sulfate affect B2 bradykinin (BK) receptors (B2Rs). Gangliosides and chondroitin sulfate did not stimulate intracellular Ca(2+) release from B2R-expressing CHO-K1 cells, but repeated exposure desensitized B2Rs to BK stimulation. Microscopic observation of DsRed-fused B2Rs revealed that several gangliosides and chondroitin sulfate C (CSC) effectively internalized B2Rs. Ganglioside-CSC treatment of B2R mutant-expressing cells failed to desensitize and internalize the mutant receptors. As this mutant lacks the first extracellular domain and cannot activate GPCR kinase (GRK), gangliosides and CSC likely initiate B2R desensitization and endocytosis through GRK-mediated B2R phosphorylation.

The mucin box and signal/anchor sequence of rat neutral ceramidase recruit bacterial sphingomyelinase to the plasma membrane.

Sphingolipid metabolites act as lipid mediators in various cellular events. We found that the mucin box and signal/anchor sequence of a rat neutral ceramidase recruit bacterial sphingomyelinase to the plasma membranes of mammalian cells. The mucin box-fused sphingomyelinase hydrolyzed cellular sphingomyelin efficiently to generate ceramide.

Homodimer formation by the ATP/UTP receptor P2Y2 via disulfide bridges.

Many class C G-protein coupled receptors (GPCRs) function as homo- or heterodimers and several class A GPCRs have also been shown to form a homodimer. We expressed human P2Y2 receptor (P2Y2R) in cultured cells and compared SDS-PAGE patterns under reducing and non-reducing conditions. Under non-reducing conditions, approximately half of the P2Y2Rs were electrophoresed as a dimer. We then produced Cys to Ser mutants at four sites (Cys25, Cys106, Cys183 and Cys278) in the extracellular domains of P2Y2R and examined the effect on dimer formation and receptor activity. All single mutants formed dimers similarly to the wild-type protein, but C25S, C106S and C183S P2Y2R lost activity, while C278S P2Y2R maintained weak activity. Coexpression with wild-type P2Y2R recovered the activity of the C25S mutant. These results show that Cys106 and Cys183 are required for monomer or homodimer activity; Cys25 is required for monomer activity, but it is not needed in one protomer for homodimer activity; and Cys278 can be replaced in the monomer and homodimer. Approximately, half of C25S/C278S double mutants were electrophoresed as a dimer, similarly to the wild-type and single mutants, and dimers with the wild-type protein were active. These results suggest involvement of Cys106 and Cys183 in disulfide bonding between protomers in homodimer formation.

Close association of B2 bradykinin receptors with P2Y2 ATP receptors.

Two G-protein-coupled receptors (GPCRs) that couple with Gαq/11, B2 bradykinin (BK) receptor (B2R) and ATP/UTP receptor P2Y2 (P2Y2R), are ubiquitously expressed and responsible for vascular tone, inflammation, and pain. We analysed the cellular signalling of P2Y2Rs in cells that express B2Rs. B2R desensitization induced by BK or B2R internalization-inducing glycans cross-desensitized the P2Y2R response to ATP/UTP. Fluorescence resonance energy transfer from P2Y2R-AcGFP to B2R-DsRed was detected in the cells and on the cell surfaces, showing the close association of these GPCRs. BK- and ATP-induced cross-internalization of P2Y2R and B2R, respectively, was shown in a ß-galactosidase complementation assay using P2Y2R or B2R fused to the H31R substituted α donor peptide of a ß-galactosidase reporter enzyme (P2Y2R-α or B2R-α) with coexpression of the FYVE domain of endofin, an early endosome protein, fused to the M15 acceptor deletion mutant of ß-galactosidase (the ω peptide, FYVE-ω). Arrestin recruitment to the GPCRs by cross-activation was also shown with the similar way. Coimmunoprecipitation showed that B2R and P2Y2R were closely associated in the cotransfected cells. These results indicate that B2R couples with P2Y2R and that these GPCRs act together to fine-tune cellular responsiveness. The collaboration between these receptors may permit rapid onset and turning off of biological events.

Gangliosides stimulate bradykinin B2 receptors to promote calmodulin kinase II-mediated neuronal differentiation.

Gangliosides mediate neuronal differentiation and maturation and are indispensable for the maintenance of brain function and survival. As part of our ongoing efforts to understand signaling pathways related to ganglioside function, we recently demonstrated that neuronal cells react to exogenous gangliosides GT1b and GD1b. Both of these gangliosides are enriched in the synapse-forming area of the brain and induce Ca(2+) release from intracellular stores, activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and activation of cdc42 to promote reorganization of cytoskeletal actin and dendritic differentiation. Here, we show that bradykinin B2 receptors transduce these reactions as a mediator for ganglioside glycan signals. The B2 antagonist Hoe140 inhibited ganglioside-induced CaMKII activation, actin reorganization and early development of axon- and dendrite-like processes of primary cultured hippocampal neurons. Furthermore, we confirmed by yeast reporter assay that major b-series gangliosides, GT1b, GD1b and GD3, stimulated B2 bradykinin receptors. We hypothesize that this B2 receptor-mediated ganglioside signal transduction pathway is one mechanism that modulates neuronal differentiation and maturation.

C18:3-GM1a induces apoptosis in Neuro2a cells: enzymatic remodeling of fatty acyl chains of glycosphingolipids.

GM1a [Gal beta1-3GalNAc beta1-4(NeuAc alpha2-3)Gal beta1-4Glc beta1-1Cer] is known to support and protect neuronal functions. However, we report that alpha-linolenic acid-containing GM1a (C18:3-GM1a), which was prepared using the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase, induced apoptosis in neuronal cells. Intranucleosomal DNA fragmentation, chromatin condensation, and caspase activation, all typical features of apoptosis, were observed when mouse neuroblastoma Neuro2a cells were cultured with C18:3-GM1a but not GM1a containing stearic acid (C18:0) or oleic acid (C18:1). The phenotype of Neuro2a cells induced by C18:3-GM1a was similar to that evoked by lyso-GM1a. However, lyso-GM1a caused a complete disruption of lipid microdomains of Neuro2a cells and hemolysis of sheep erythrocytes, whereas C18:3-GM1a did neither. C18:3-GM1a, but not lyso-GM1a, was found to be abundant in lipid microdomains after the removal of loosely bound GM1a by BSA. The activation of stress-activated protein kinase/c-Jun N-terminal kinase in Neuro2a cells was observed with lyso-GM1a but not C18:3-GM1a. These results indicate that the mechanism of apoptosis induced by C18:3-GM1a is distinct from that caused by lyso-GM1a. This study also clearly shows that fatty acid composition of gangliosides significantly affected their pharmacological activities when added to the cell cultures and suggests why naturally occurring gangliosides do not possess polyunsaturated fatty acids as a major constituent.

Subcellular localization of human neutral ceramidase expressed in HEK293 cells.

We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence.

Application of RNA interference to chicken embryos using small interfering RNA.

Gene silencing using small interfering RNA (siRNA) is not established in avian species. The present study was performed to evaluate RNA interference (RNAi) in the chicken embryo by using a dual fluorescence reporter assay, a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting the GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells and whole embryos by lipofection and microelectroporation, respectively. GFP- and RFP-expressed cells and embryos were observed under fluorescent microscopy and analyzed by flow cytometer, and their mRNAs were analyzed by reverse transcription-PCR (RT-PCR). The strong fluorescence was observed by introducing both plasmids into cells. The intensity of the green fluorescence generated by GFP was greatly suppressed by introducing GFP-siRNA. RT-PCR analysis showed that introducing GFP-siRNA also decreased GFP mRNA levels. In contrast to GFP, the intensity of the red fluorescence generated by RFP and the RFP mRNA levels remained unchanged. In whole embryos, also, introducing GFP-siRNA specifically suppressed GFP expression, and the suppression was maintained for at least 72 h. Consequently, it was concluded that the gene silencing using siRNA is applicable to analyzing the function of genes of interest during avian embryogenesis.