Intracellular Ca2+ signals in Dictyostelium chemotaxis are mediated exclusively by Ca2+ influx.
AuthorsNebl T, Fisher PR
JournalJ Cell Sci
PubMed ID9427292
'We measured folate- and cAMP-induced changes in cytoplasmic free calcium concentration ([Ca2+]i) using recombinant aequorin reconstituted in living Dictyostelium cells with coelenterazine-h. The resulting semi-synthetic protein displayed increased sensitivity to Ca2+ allowing accurate measurement of chemoattractant-induced transients at low resting levels. Both folate- and cAMP-induced Ca2+ responses were developmentally regulated, ... More
Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer.
AuthorsMercier JF, Salahpour A, Angers S, Breit A, Bouvier M
JournalJ Biol Chem
PubMed ID12244098
'Quantitative bioluminescence resonance energy transfer (BRET) analysis was applied to the study of beta(1)- and beta(2)-adrenergic receptor homo- and heterodimerization. To assess the relative affinity between each of the protomers, BRET saturation experiments were carried out in HEK-293T cells. beta(1)- and beta(2)-adrenergic receptors were found to have similar propensity to ... More
The use of Renilla luciferase, Oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate.
AuthorsInouye S, Shimomura O
JournalBiochem Biophys Res Commun
PubMed ID9144537
'To investigate the use of various luciferases as reporter protein, the substrate specificity of recombinant Renilla luciferase, Oplophorus luciferase and recombinant apoaequorin was examined using 23 kinds of coelenterazine analogues as the substrate. The intensity of luminescence was generally highest with Oplophorus luciferase and lowest with apoaequorin, but varied widely ... More
Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals.
AuthorsZhao H, Doyle TC, Wong RJ, Cao Y, Stevenson DK, Piwnica-Worms D, Contag CH
JournalMol Imaging
PubMed ID15142411
'In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which ... More
Imaging [Ca2+]i with aequorin using a photon imaging detector.
AuthorsMiller AL, Karplus E, Jaffe LF
JournalMethods Cell Biol
PubMed ID8201982
Preparation and handling of aequorin solutions for the measurement of cellular Ca2+.
AuthorsShimomura O
JournalCell Calcium
PubMed ID1959127
Main characteristics of the various types of aequorin presently available for measuring cellular Ca2+, i.e. heterogeneous aequorin, isoaequorins, recombinant aequorin, fluorescein-labeled aequorin and semi-synthetic aequorins, are summarized. Basic techniques of preparing and handling the solutions of those aequorins for measuring Ca2+, including such techniques as concentrating aequorin solutions, freeze-drying, changing ... More
Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium.
AuthorsShimomura O, Musicki B, Kishi Y, Inouye S
JournalCell Calcium
PubMed ID8519061
15 kinds of recombinant semi-synthetic aequorins and a recombinant fluorescein-conjugated aequorin were prepared and their properties in Ca(2+)-triggered luminescence were studied. The semi-synthetic aequorins showed a wide range of Ca(2+)-sensitivity. The luminescence intensity of a high-sensitivity type (hcp-aequorin) was greater than 10(4)-times that of a low-sensitivity type (n-aequorin) at pCa ... More
Membrane permeability of coelenterazine analogues measured with fish eggs.
AuthorsShimomura O
JournalBiochem J
PubMed ID9291095
To determine the suitability of various coelenterazine analogues for the regeneration of aequorin in living cells, the membrane permeabilities of 11 analogues were measured using the eggs of the killifish Fundulus grandis by soaking the eggs in solutions containing the analogues. The results indicated that e-coelenterazine, which has an exceptionally ... More
The relative rate of aequorin regeneration from apoaequorin and coelenterazine analogues.
AuthorsShimomura O, Kishi Y, Inouye S
JournalBiochem J
PubMed ID8280050
The regeneration of an active semi-synthetic aequorin, from apoaequorin produced in cells and a coelenterazine analogue, is a key step in measuring Ca2+ in the cells. The relative rates of the regeneration of semi-synthetic aequorins from apoaequorin and 28 synthetic coelenterazine analogues were compared. The results indicated that the rate ... More
Semi-synthetic aequorins with improved sensitivity to Ca2+ ions.
AuthorsShimomura O, Musicki B, Kishi Y
JournalBiochem J
PubMed ID2803250
Thirty-seven coelenterazine analogues were synthesized and incorporated into apo-aequorin, yielding 30 semi-synthetic aequorins that have the capacity to emit a significant amount of light in the presence of Ca2+. The properties of resultant photoproteins were investigated. The most prominent feature of those photoproteins was the wide range in their sensitivities ... More
Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions.
AuthorsPfleger KD, Seeber RM, Eidne KA
JournalNat Protoc
PubMed ID17406254
A substantial range of protein-protein interactions can be readily monitored in real time using bioluminescence resonance energy transfer (BRET). The procedure involves heterologous coexpression of fusion proteins, which link proteins of interest to a bioluminescent donor enzyme or acceptor fluorophore. Energy transfer between these proteins is then detected. This protocol ... More
Imaging reversal of multidrug resistance in living mice with bioluminescence: MDR1 P-glycoprotein transports coelenterazine.
AuthorsPichler A, Prior JL, Piwnica-Worms D
JournalProc Natl Acad Sci U S A
PubMed ID14755051
Coelenterazine is widely distributed among marine organisms, producing bioluminescence by calcium-insensitive oxidation mediated by Renilla luciferase (Rluc) and calcium-dependent oxidation mediated by the photoprotein aequorin. Despite its abundance in nature and wide use of both proteins as reporters of gene expression and signal transduction, little is known about mechanisms of ... More
The TXP motif in the second transmembrane helix of CCR5. A structural determinant of chemokine-induced activation.
CCR5 is a G-protein-coupled receptor activated by the chemokines RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein 1alpha and 1beta, and monocyte chemotactic protein 2 and is the main co-receptor for the macrophage-tropic human immunodeficiency virus strains. We have identified a sequence motif (TXP) in ... More
Mechanisms of cell-surface rerouting of an endoplasmic reticulum-retained mutant of the vasopressin V1b/V3 receptor by a pharmacological chaperone.
AuthorsRobert J, Auzan C, Ventura MA, Clauser E
JournalJ Biol Chem
PubMed ID16210325
Cell-surface expression and biological functions of several intracellular-retained G protein-coupled receptors are restored by membrane-permeable ligands called pharmacological chaperones. We have previously demonstrated that a mutation of the hydrophobic motif 341FNX2LLX3L350 in the C terminus of the human pituitary vasopressin V3 receptor (MUT V3R) led to it being retained in ... More
Identification of natural ligands for the orphan G protein-coupled receptors GPR7 and GPR8.
AuthorsBrezillon S, Lannoy V, Franssen JD, Le Poul E, Dupriez V, Lucchetti J, Detheux M, Parmentier M
JournalJ Biol Chem
PubMed ID12401809
GPR7 and GPR8 are two structurally related orphan G protein-coupled receptors, presenting high similarities with opioid and somatostatin receptors. Two peptides, L8 and L8C, derived from a larger precursor, were recently described as natural ligands for GPR8 (Mori, M., Shimomura, Y., Harada, M., Kurihara, M., Kitada, C., Asami, T., Matsumoto, ... More
Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer.
AuthorsKroeger KM, Hanyaloglu AC, Seeber RM, Miles LE, Eidne KA
JournalJ Biol Chem
PubMed ID11278883
The ability of G-protein-coupled receptors (GPCRs) to interact to form new functional structures, either forming oligomers with themselves or forming associations with other intracellular proteins, has important implications for the regulation of cellular events; however, little is known about how this occurs. Here, we have employed a newly emerging technology, ... More
Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?
AuthorsCréton R, Steele ME, Jaffe LF
JournalCell Calcium
PubMed ID9502193
Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe ... More
Intracellular free calcium oscillations in normal and cleavage-blocked embryos and artificially activated eggs of Xenopus laevis.
AuthorsKeating TJ, Cork RJ, Robinson KR
JournalJ Cell Sci
PubMed ID7983182
We have measured levels of intracellular free calcium ([Ca2+]i) in albino Xenopus laevis embryos using recombinant aequorin and a photon-counting system. We observed sinusoidal oscillations in [Ca2+]i that had the same frequency as cleavage, with cleavage occurring when [Ca2+]i was lowest. An increase in calcium was seen to precede first ... More
Cause of spectral variation in the luminescence of semisynthetic aequorins.
AuthorsShimomura O
JournalBiochem J
PubMed ID7887908
Aequorin emits light in the presence of Ca2+, decomposing into apoaequorin, coelenteramide and CO2. Semisynthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with analogues of coelenterazine, showed widely different sensitivities to Ca2+ as well as certain spectral variations. A group of semisynthetic aequorins, e-type aequorins, showed bimodal luminescence, ... More
Multiple charged and aromatic residues in CCR5 amino-terminal domain are involved in high affinity binding of both chemokines and HIV-1 Env protein.
AuthorsBlanpain C, Doranz BJ, Vakili J, Rucker J, Govaerts C, Baik SS, Lorthioir O, Migeotte I, Libert F, Baleux F, Vassart G, Doms RW, Parmentier M
JournalJ Biol Chem
PubMed ID10574939
CCR5 is a functional receptor for MIP-1alpha, MIP-1beta, RANTES (regulated on activation normal T cell expressed), MCP-2, and MCP-4 and constitutes the main coreceptor for macrophage tropic human and simian immunodeficiency viruses. By using CCR5-CCR2b chimeras, we have shown previously that the second extracellular loop of CCR5 is the major ... More
Posttranslational modification impact on the mechanism by which amyloid-β induces synaptic dysfunction.
Authors
JournalEMBO Rep
PubMed ID28420656
Molecular and functional characterization of somatostatin-type signalling in a deuterostome invertebrate.
Authors
JournalOpen Biol
PubMed ID32898470
Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis.
Authors
JournalCell
PubMed ID34048700
Influenza A Virus Infection Activates NLRP3 Inflammasome through Trans-Golgi Network Dispersion.
Authors
JournalViruses
PubMed ID35062292
Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome.
Authors
JournalSci Rep
PubMed ID29740074
D2-like dopamine and β-adrenergic receptors form a signaling complex that integrates Gs- and Gi-mediated regulation of adenylyl cyclase.