The pheromone biosynthesis activating neuropeptide (PBAN) receptor of Heliothis virescens: identification, functional expression, and structure-activity relationships of ligand analogs.

Pheromone biosynthesis activating neuropeptide (PBAN) promotes synthesis and release of sex pheromones in moths. We have identified and functionally expressed a PBAN receptor from Heliothis virescens (HevPBANR) and elucidated structure-activity relationships of PBAN analogs. Screening of a larval CNS cDNA library revealed three putative receptor subtypes and nucleotide sequence comparisons suggest that they are produced through alternative splicing at the 3'-end. RT-PCR amplified preferentially HevPBANR-C from female pheromone glands. CHO cells expressing HevPBANR-C are highly sensitive to PBAN and related analogs, especially those sharing the C-terminal pentapeptide core, FXPRLamide (X=T, S or V). Alanine replacements in the C-terminal hexapeptide (YFTPRLamide) revealed the relative importance of each residue in the active core as follows: R5>L6>F2>>P4>T3>>Y1. This study provides a framework for the rational design of PBANR-specific agonists and/or antagonists that could be exploited for disruption of reproductive function in agriculturally important insect pests.

Functional characterisation of the Anopheles leucokinins and their cognate G-protein coupled receptor.

Identification of the Anopheles gambiae leucokinin gene from the completed A. gambiae genome revealed that this insect species contains three leucokinin peptides, named Anopheles leucokinin I-III. These peptides are similar to those identified in two other mosquito species, Aedes aegypti and Culex salinarius. Additionally, Anopheles leucokinin I displays sequence similarity to Drosophila melanogaster leucokinin. Using a combination of computational and molecular approaches, a full-length cDNA for a candidate leucokinin-like receptor was isolated from A. stephensi, a close relative of A. gambiae. Alignment of the known leucokinin receptors--all G protein-coupled receptors (GPCRs)--with this receptor, identified some key conserved regions within the receptors, notably transmembrane (TM) domains I, II, III, VI and VII. The Anopheles leucokinins and receptor were shown to be a functional receptor-ligand pair. All three Anopheles leucokinins caused a dose-dependent rise in intracellular calcium ([Ca2+]i) when applied to S2 cells co-expressing the receptor and an aequorin transgene, with a potency order of I>II>III. Drosophila leucokinin was also found to activate the Anopheles receptor with a similar EC50 value to Anopheles leucokinin I. However, when the Anopheles peptides were applied to the Drosophila receptor, only Anopheles leucokinin I and II elicited a rise in [Ca2+]i. This suggests that the Anopheles receptor has a broader specificity for leucokinin ligands than the Drosophila receptor. Antisera raised against the Anopheles receptor identified a doublet of approx. 65 and 72 kDa on western blots, consistent with the presence of four N-glycosylation sites within the receptor sequence, and the known glycosylation of the receptor in Drosophila. In Anopheles tubules, as in Drosophila, the receptor was localised to the stellate cells. Thus we provide the first identification of Anopheles mosquito leucokinins (Anopheles leucokinins) and a cognate leucokinin receptor, characterise their interaction and show that Dipteran leucokinin signalling is closely conserved between Drosophila and Anopheles.

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.

Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling.

Luminous proteins include primary light producers, such as aequorin, and secondary photoproteins that in some organisms red-shift light emission for better penetration in space. When expressed in heterologous systems, both types of proteins may act as versatile reporters capable of monitoring phenomena as diverse as calcium homoeostasis, protein sorting, gene expression, and so on. The Ca(2+)-sensitive photoprotein aequorin was targeted to defined intracellular locations (organelles, such as mitochondria, endoplasmic reticulum, sarcoplasmic reticulum, Golgi apparatus and nucleus, and cytoplasmic regions, such as the bulk cytosol and the subplasmalemmal rim), and was used to analyse Ca(2+) homoeostasis at the subcellular level. We will discuss this application, reviewing its advantages and disadvantages and the experimental procedure. The applications of green fluorescent protein (GFP) are even broader. Indeed, the ability to molecularly engineer and recombinantly express a strongly fluorescent probe has provided a powerful tool for investigating a wide variety of biological events in live cells (e.g. tracking of endogenous proteins, labelling of intracellular structures, analysing promoter activity etc.). More recently, the demonstration that, using appropriate mutants and/or fusion proteins, GFP fluorescence can become sensitive to physiological parameters or activities (ion concentration, protease activity, etc.) has further expanded its applications and made GFP the favourite probe of cell biologists. We will here present two applications in the field of cell signalling, i.e. the use of GFP chimaeras for studying the recruitment of protein kinase C isoforms and the activity of intracellular proteases.

Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley.

Transient influx of Ca(2+) constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae-derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca(2+)](cyt)), which peaked at approximately 1 microM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca(2+)](cyt) signature was achieved by elicitor concentrations sufficient to stimulate Ca(2+) influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca(2+)](cyt) but not the rapidly induced [Ca(2+)](cyt) transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca(2+) influx across the plasma membrane or of Ca(2+) release from internal stores suggests that the elicitor-induced sustained increase of [Ca(2+)](cyt) predominantly results from the influx of extracellular Ca(2+). Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca(2+)](cyt), and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca(2+)](cyt) is causally involved in signaling the activation of pathogen defense in parsley.

Cloning and characterization of cDNA encoding a novel human leukotriene B(4) receptor.

By homology screening using BLAST searches of expressed sequence tags (ESTs), we have found a previously unidentified cDNA encoding a putative seven-transmembrane receptor with highest similarity to the leukotriene B(4) receptor, BLTR. Analysis of calcium flow in transfected cells, along with sequence analysis, revealed that the EST encoded a functionally inactive protein, lacking the segment corresponding to the C-terminal part of the putative receptor protein. The missing segment was obtained by PCR amplification of a human leukocyte cDNA library and ligated to the truncated EST cDNA. The novel cDNA encodes a full-length receptor with 39% identity to the previously cloned BLTR. Studies of intracellular calcium flow of transfected HeLa cells exposed to various leukotrienes showed that also the novel BLTR-like receptor can be activated by leukotriene B(4), and it is therefore tentatively named BLTR2.

Rapid determination of gap junction formation using HeLa cells microinjected with cDNAs encoding wild-type and chimeric connexins.

A procedure for rapidly determining the functionality of gap junctions constructed of recombinant connexins in communication-deficient HeLa cells is described. Nuclear microinjection of cDNA encoding wild-type connexins (Cx) 26, 32, 43, and a range of connexin-aequorin (Cx-Aeq) chimerase resulted in generation of gap junction intercellular communication channels. Expression of recombinant protein was detected in > 95% of cells 18-72 h following nuclear microinjection, and the functionality of the channels generated was determined according to their ability to transfer the fluorescent dye tracers Lucifer yellow and propidium iodide. The dye transfer results obtained correlated closely with other published studies using stably transfected cells and yet are obtained as rapidly as 18 h following microinjection of cDNA. Expression of a truncated form of Cx43 (Cx43 delta 244) by this new method indicated diminished intercellular transfer of both dyes and supports a channel-gating mechanism that postulates interaction between the carboxyl tail and the intracellular loop.

Aequorea victoria bioluminescence moves into an exciting new era.

Bioluminescence has revolutionized research into many cellular and molecular-biological processes, ranging from intracellular signalling to gene transcription. This article focuses on the chemistry and biotechnological exploitation of the two proteins involved in bioluminescence of the jellyfish Aequorea victoria--aequorin and green fluorescent protein. Engineered recombinant aequorin has led to a novel technological approach to monitoring calcium signals in organelles and subcellular domains. A new generation of intracellular calcium indicators has been produced in which engineered variants of green fluorescent protein are used to probe their ionic environment using intramolecular fluorescence-resonance-energy transfer.

Reduced Ca2+ uptake by mitochondria in pyruvate dehydrogenase-deficient human diploid fibroblasts.

Physiological and pathological Ca2+ loads are thought to be taken up by mitochondria via a process dependent on aerobic metabolism. We sought to determine whether human diploid fibroblasts from a patient with an inherited defect in pyruvate dehydrogenase (PDH) exhibit a decreased ability to sequester cytosolic Ca2+ into mitochondria. Mobilization of Ca2+ stores with bradykinin (BK) increased the cytosolic Ca2+ concentration ([Ca2+]c) to comparable levels in control and PDH-deficient fibroblasts. In normal fibroblasts transfected with plasmid DNA encoding mitochondrion-targeted apoaequorin, BK elicited an increase in Ca2(+)-dependent aequorin luminescence corresponding to an increase in the mitochondrial Ca2+ concentration ([Ca2+]mt) of 2.0 +/- 0.2 microM. The mitochondrial uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone blocked the BK-induced [Ca2+]mt increase, although it did not affect the [Ca2+]c transient. Basal [Ca2+]c and [Ca2+]mt in control and PDH-deficient cells were similar. However, confocal imaging of the potential-sensitive dye JC-1 indicated that the percentage of highly polarized mitochondria was reduced from 30 +/- 1% in normal cells to 19 +/- 2% in the PDH-deficient fibroblasts. BK-elicited [Ca2+]mt transients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability to take up cytosolic Ca2+. Thus cells with compromised aerobic metabolism have a reduced capacity to sequester Ca2+.

DNA translocation across planar bilayers containing Bacillus subtilis ion channels.

The mechanisms by which genetic material crosses prokaryotic membranes are incompletely understood. We have developed a new methodology to study the translocation of genetic material via pores in a reconstituted system, using techniques from electrophysiology and molecular biology. We report here that planar bilayer membranes become permeable to double-stranded DNA (kilobase range) if Bacillus subtilis membrane vesicles containing high conductance channels have been fused into them. The translocation is an electrophoretic process, since it does not occur if a transmembrane electrical field opposing the movement of DNA, a polyanion, is applied. It is not an aspecific permeation through the phospholipid bilayer, since it does not take place if no proteins have been incorporated into the membrane. The transport is also not due simply to the presence of polypeptides in the membrane, since it does not occur if the latter contains gramicidin A or a eukaryotic, multi-protein vesicle fraction exhibiting 30-picosiemens anion-selective channel activity. The presence of DNA alters the behavior of the bacterial channels, indicating that it interacts with the pores and may travel through their lumen. These results support the idea that DNA translocation may take place through proteic pores and suggest that some of the high conductance bacterial channels observed in electrophysiological experiments may be constituents of the DNA translocating machinery in these organisms.

Possible role for mitochondrial calcium in angiotensin II- and potassium-stimulated steroidogenesis in bovine adrenal glomerulosa cells.

In adrenal zona glomerulosa cells, the action of angiotensin II (Ang II) and of potassium (K+) on aldosterone synthesis is mediated by the Ca2+ messenger system. The major part of the steroidogenic pathway takes place inside the mitochondria, and Ca2+ must enter the mitochondrial matrix to stimulate the steroidogenic cascade. To examine how changes in the cytosolic free calcium concentration ([Ca2+]c) induced by Ang II and K+ are relayed into the mitochondrial matrix, we transfected bovine adrenal zona glomerulosa cells in primary culture with a chimeric complementary DNA encoding for the signal presequence targeting human cytochrome c oxidase subunit VIII to the matrix, linked to a complementary DNA coding for the Ca2+-sensitive photoprotein aequorin. Resting mitochondrial free calcium concentration ([Ca2+]m) amounted to 0.41 +/- 0.18 microM (n = 40). Ang II induced a concentration-dependent (EC50 = 11.3 +/- 6.0 nM), biphasic rise of [Ca2+]m. After a large transient initial peak (5.13 +/- 0.89 microM, n = 28), [Ca2+]m decreased to a plateau that remained higher than basal [Ca2+]m for several minutes in the presence of the hormone. By contrast, studies in cells transfected with cytosolic aequorin indicated that the rise of [Ca2+]c triggered by Ang II was confined to 1.34 +/- 0.26 microM (n = 17). In Ca2+-free medium, a reduced peak [Ca2+]m response to Ang II occurred without a secondary plateau. On readdition of extracellular Ca2+, in the presence of the hormone, the resulting Ca2+ influx was accompanied by small rise of [Ca2+]m. The mitochondrial uncoupler, carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone, prevented the Ang II-induced [Ca2+]m rise but not the [Ca2+]c response, thus demonstrating the mitochondrial location of transfected aequorin. In contrast to Ang II, K+ (13 mM) induced a sustained [Ca2+]c response, which was relayed without amplification into the mitochondrial matrix as a plateau of[Ca2+]m. This plateau of[Ca2+]m was suppressed by the addition of the dihydropyridine, nifedipine (200 nM). The inhibitor of the mitochondrial Na+/Ca2+ exchanger, CGP37157, reduced significantly the rate of decrease of [Ca2+]m following the peak induced by Ang II. In cells whose [Ca2+]c was clamped at various levels (0.05-0.860 microM) with ionomycin, a concentration-dependent stimulation of pregnenolone output was induced by Ca2+. Under these conditions, the output of pregnenolone--the early product of steroidogenesis--was markedly potentiated by CGP37157. These results suggest the existence of microdomains of high [Ca2+]c elicited by Ang II in the proximity of mitochondria. Moreover, our observations are consistent with a mitochondrial site of action for calcium in the activation of the steroidogenic cascade.

Generation of cell transfectants expressing cardiac calcium ion channel and calcium indicator protein aequorin.

Chinese hamster ovary (CHO) cells stably coexpressing cardiac calcium ion channel [L-type calcium channel or ryanodine receptor (RyR)] and the calcium-sensitive bioluminescent protein aequorin were generated by transfecting aequorin cDNA. In a selected clone, C1-17, carrying the L-type calcium channel, depolarization induced by high concentration of K+ produces aequorin luminescence. In another clone, R3-7, carrying RyR, caffeine produces aequorin luminescence. In the presence of selective calcium ion channel blockers, the aequorin luminescence was inhibited in a dose-dependent manner. These results indicate that functionally expressed calcium ion channels in these transformants can be monitored through the activation of endogenous aequorin luminescence following a physiological signal similar to that of native calcium channel. Moreover, the aequorin system compared very well with Fura-2 measurements. Thus, the recombinant cell models, which expressed cloned calcium channel and aequorin, will contribute to the elucidation of Ca2+ movement through the cell surface and intracellular calcium ion channels.

Regeneration and luminescence of aequorin in Chinese hamster ovary cells transformed with cDNA for apoaequorin.

Aequorin, a photoprotein which is regenerated from apoaequorin by incubation with coelenterazine, emits light when it binds Ca2+. The aim of this study was to determine if apoaequorin could be used in adherent mammalian cells for measuring cytosolic Ca2+, and imaging Ca2+, at the single cell level. Chinese hamster ovary (CHO-K1) cells were stably transformed with apoaequorin cDNA and expressed apoaequorin while attached to the culture dishes. Maximal luminescence intensity was obtained when 0.5 x 10(6) cells/ml were grown and incubated with 2.5 microM coelenterazine for 4 hr at 20 degrees C. Ca2+ mobilizing agents (ionomycin and maitotoxin) induced luminescence in CHO-K1 transformed cells. However, imaging of light emission from single cells proved to be unsuccessful. Ca2+ could be readily measured in the adherent CHO-K1 cells, but imaging was not possible at the single cell level.

The moss, Physcomitrella patens, transformed with apoaequorin cDNA responds to cold shock, mechanical perturbation and pH with transient increases in cytoplasmic calcium.

The gene for apoaequorin has been used previously to indicate cytosolic calcium changes in higher plants. Here we report the transformation of the moss Physcomitrella patens with the cDNA for apoaequorin. Stable transformants were obtained in the wild type which reconstitute the calcium-sensitive luminescent protein aequorin in vivo after incubation in coelenterazine, and continue to grow normally. The wild type responds to cold-shock (0-10 degrees C) with increases in cytosolic calcium. Mechanical perturbation, in the form of touch, also induces transient increases in cytosolic calcium. A smaller response to pH, distinct from the touch response and exhibiting different kinetics, can also be detected.

Targeting aequorin and green fluorescent protein to intracellular organelles.

Two proteins of Aequorea victoria were molecularly engineered and produced in mammalian cells, in order to serve as specific reporters of subcellular microenvironments. Aequorin (AEQ), a Ca(2+)-sensitive photoprotein, was successfully targeted to three intracellular locations: cytosol, nucleus and mitochondria. The recombinant apoprotein, reconstituted into active AEQ by the addition of the prosthetic group to the culture medium, allows the direct measurement of [Ca2+] within those compartments, thus directly addressing questions of large biological interest. The same approach was utilized for the green fluorescent protein (GFP) for specific labelling, in vivo, of the various subcellular structures. GFP was targeted to mitochondria: the recombinant protein, strongly fluorescent in a highly reducing environment, provides a powerful tool for visualizing these organelles in living cells, and may represent the prototype of a new family of intracellularly targeted fluorescent probes.

Patterns of free calcium in multicellular stages of Dictyostelium expressing jellyfish apoaequorin.

To examine the patterns of high free cytosolic calcium or [Ca2+]i during Dictyostelium's development, we expressed apoaequorin in D. discoideum, reconstituted aequorin and observed the resultant patterns of calcium-dependent luminescence. Specific, high calcium zones are seen throughout normal multicellular development and are roughly coincident with those regions that later differentiate into stalk or stalk-like cells. A slug, for example, shows a primary high calcium zone within its front quarter and a secondary one around its tail; while a mound shows such a zone around the periphery of its base. Combined with previous evidence, our findings support the hypothesis that high [Ca2+]i feeds back to favor the stalk pathway. We also discovered several high calcium zones within the mound's base that do not coincide with any known prepatterns in D. discoideum. These include two, relatively persistent, antipodal strips along the mound's periphery. These various persistent zones of high calcium are largely made up of frequent, 10 to 30 second long, semiperiodic calcium spikes. Each of these spikes generates a correspondingly short-lived, 200 to 500 microns long, high calcium band which extends along the nearby surface. Similar, but relatively large and infrequent, spikes generate cross bands which extend across migrating slugs and just behind their advancing tips as well as across the peripheries of rotating mounds and midway between their antipodal strips. Moreover, calcium has a doubling time of about a second as various spikes rise. This last observation suggests that the calcium bands seen in Dictyostelium may be generated by so-called fast calcium waves.

Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation.

Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles (Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325-328; Brini, M., Murgia, M., Pasti, L., Picard, D., Pozzan, T., and Rizzuto, R. (1993) EMBO J. 12, 4813-4819; Kendall, J. M., Dormer, R. L., and Campbell, A. K. (1992) Biochem. Biophys. Res. Commun. 189, 1008-1016). Up until now, however, only limited attention has been paid to the use of recombinant photoproteins for measuring, in mammalian cells, the [Ca2+] in the cytoplasm, a compartment for which effective Ca2+ probes are already available. Here we describe this approach in detail, highlighting the advantages, under various experimental conditions, of using recombinant cytosolic aequorin (cytAEQ) instead of classical fluorescent indicators. We demonstrate that cytAEQ is expressed recombinantly at high levels in transiently transfected cell lines and primary cultures as well as in stably transfected clones, and we describe a simple algorithm for converting aequorin luminescence data into [Ca2+] values. We show that although fluorescent indicators at the usual intracellular concentrations (50-100 microM) are associated with a significant buffering of the [Ca2+]c transients, this problem is negligible with recombinantly expressed aequorin. The large dynamic range of the photoprotein also allows an accurate estimate of the large [Ca2+]c increases that are observed in some cell types such as neurons. Finally, cytAEQ appears to be an invaluable tool for measuring [Ca2+]c in cotransfection experiments. In particular, we show that when cotransfected with an alpha 1-adrenergic receptor (coupled to inositol 1,4,5-trisphosphate generation), cytAEQ faithfully monitors the subpopulation of cells expressing the receptor, whereas the signal of fura-2, at the population level, is dominated largely by that of the untransfected cells.

Sequence of the cDNA encoding the Ca(2+)-activated photoprotein obelin from the hydroid polyp Obelia longissima.

A cDNA clone encoding the Ca(2+)-activated photoprotein, obelin (Obl), from Obelia longissima was sequenced. The nucleotide (nt) sequence contained two long overlapping open reading frames (ORFs), one of which encoded apoobelin (apoObl). The deduced amino acid (aa) sequence of apoObl revealed that this 195-aa protein has three EF-hand structures that are characteristic for Ca(2+)-binding domains. Strong aa homology was shown among apoObl, apoaequorin and apoclytin. The second ORF present in the obl cDNA consists of 139 codons and encodes a very basic protein with a calculated pI of 10.56 and a molecular mass of 16,153 Da.

Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin cDNA.

A new method is described for measuring intracellular free calcium concentrations, [(Ca2+)i], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium-specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca2+)i] in developing cells markedly increases at the aggregation stage and again at the culmination stage after a temporary drop at the migration stage. Except for the vegetative stage, the cells at all stages of development exhibit a sharp transient increase in [(Ca2+)i] upon stimulation with a cAMP (50 nM) pulse, high responses being observed at the migration and culmination stages. Separated prestalk cells of migrating slugs contain more than twice as much [(Ca2+)i] and show three times as large a response to cAMP stimulation as prespore cells.

Requirement of the C-terminal proline residue for stability of the Ca(2+)-activated photoprotein aequorin.

cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.