Aequorin functional assay for characterization of G-protein-coupled receptors: implementation with cryopreserved transiently transfected cells.

Assay technologies that measure intracellular Ca(2+) release are among the predominant methods for evaluation of GPCR function. These measurements have historically been performed using cell-permeable fluorescent dyes, although the use of the recombinant photoprotein aequorin (AEQ) as a Ca(2+) sensor has gained popularity with recent advances in instrumentation. The requirement of the AEQ system for cells expressing both the photoprotein and the GPCR target of interest has necessitated the labor-intensive development of cell lines stably expressing both proteins. With the goal of streamlining this process, transient transfections were used to either (1) introduce AEQ into cells stably expressing the GPCR of interest or (2) introduce the GPCR into cells stably expressing the AEQ protein, employing the human muscarinic M(1) receptor as a model system. Robust results were obtained from cryopreserved cells prepared by both strategies, yielding agonist and antagonist pharmacology in good agreement with literature values. Good reproducibility was observed between multiple transient transfection events. These results indicate that transient transfection is a viable and efficient method for production of cellular reagents for use in AEQ assays.

Identification of ETA and ETB binding domains using ET-derived photoprobes.

Using the structure of ET-1 as a template, a series of photosensitive analogs were developed to investigate the binding domain of ETA and ETB receptors. Accordingly, a p-benzoyl-l-phenylalanine (Bpa) residue was introduced into the peptide chain following a pattern aiming at scanning N- to C-terminal portions of the molecule. Among the analogs, those containing a Bpa amino acid in position 7 ([L-Bpa7, Tyr(125I)13]hET-1) or 12 ([Nle7, L-Bpa12, Tyr(125I)13]hET-1) exhibited the capacity to activate both receptors, thus showing that residues Met-7 and Val-12 of ET-1 do not play a key role in the activation process. The binding capacity of the probes was also evaluated on transfected CHO cells overexpressing either ETA or ETB receptors. Subsequently, these photoprobes were used to label ETA and ETB receptors overexpressed in transfected CHO cells. Enzymatic digestions and chemical cleavages were then performed on ligand-receptor complexes and fragments produced by the lysis were analyzed to point out putative interaction areas on the receptors. Results showed that Phe147-Lys166, covering the second segment of EC I and the top part of TM III, contains a contact point for [Nle7, L-Bpa12, Tyr(125I)13]hET-1 on ETA receptors whereas Ile292-Trp319, spanning from the second half of the intracellular loop III up to the middle turns of TM VI, includes a residue that can interact with [L-Bpa7, Tyr(125I)13]hET-1. Moreover, upon binding of [Nle7, L-Bpa12, Tyr(125I)13]hET-1, it was observed that Thr263-Met266 (EC II) of the ETB receptor would come close with the ligand.

[D-1-Nal4]endomorphin-2 is a potent micro-opioid receptor antagonist in the aequorin luminescence-based calcium assay.

A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca(2+) levels, was used to examine relative potency and efficacy of the micro-opioid receptor antagonists. A series of position 3- and 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) analogues containing D-3-(1-naphthyl)-alanine (D-1-Nal) or D-3-(2-naphthyl)-alanine (D-2-Nal), which were previously shown to reverse antinociception induced by endomorphin-2 in the in vivo hot-plate test in mice, was tested in the aequorin luminescence-based calcium assay to examine their micro-opioid antagonist potency in vitro. A recombinant mammalian cell line expressing the micro-opioid receptor together with a luminescent reporter protein, apoaequorin, was used in the study. The results obtained in this functional assay indicated that analogues with D-1-Nal or D-2-Nal substitutions in position 4 of endomorphin-2 are strong micro-opioid receptor antagonists, while those substituted in position 3 are partial agonists. Exceptional antagonist potency in the calcium assay was observed for [D-1-Nal(4)]endomorphin-2. The pA(2) value for this analogue was 7.95, compared to the value of 8.68 obtained for the universal, non-selective opioid antagonist of the alkaloid structure, naloxone. The obtained results were compared with the data from the hot-plate test in mice. In that in vivo assay [D-1-Nal(4)]endomorphin-2 was also the most potent analogue of the series.

Functional characterization of opioid receptor ligands by aequorin luminescence-based calcium assay.

A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in intracellular calcium levels, was used to examine relative potency and efficacy of the mu-opioid agonists endomorphin-1, endomorphin-2, morphiceptin, and their position 3-substituted analogs, as well as the delta-agonist deltorphin-II. The results of the aequorin assay, performed on recombinant cell lines, were compared with those obtained in the functional assay on isolated tissue preparations (guinea pig ileum and mouse vas deferens). A range of nine opioid peptide ligands produced a similar rank order of potency for the mu- and delta-opioid receptor agonists in both functional assays. The highest potency at the mu-receptor was observed for endomorphin-1, endomorphin-2, and [D-1-Nal3]morphiceptin, whereas deltorphin-II was the most potent delta-receptor agonist. In the aequorin assay, the mu- and delta-agonist-triggered luminescence was inhibited by the opioid antagonists naloxone and naltrindole, respectively. We can conclude that the use of the aequorin assay for new mu- and delta-receptor-selective opioid analogs gives pharmacologically relevant data and allows high-throughput compound screening, which does not involve radioactivity or animal tissues. This is the first study that validates the application of this assay in the screening of opioid analogs.

Desensitization of the human motilin receptor by motilides.

Tachyphylaxis may have contributed to the failure of the motilide ABT-229 [N-ethyl, N-methyl 4'' deoxy erythromycin (EM)-B enolether] in clinical trials. We compared the desensitizing potency of structurally related motilides [EM-A, EM-A enolether (ME4), N-ethyl, N-methyl EM-A (ME36), EM-B enolether (ME67), N-ethyl, N-methyl EM-A enolether (EM523), ABT-229 and 4'' deoxy EM-A enolether (KOS1326)] in a Chinese hamster ovary (CHO)-K1 cell line expressing the human motilin receptor (MTLR) and in rabbit duodenal segments. CHO-MTLR cells were preincubated with motilides prior to stimulation with motilin. The negative logarithm of the preincubation concentration reducing the maximal motilin-induced Ca(2+) flux to 50% was calculated (pDC(50)). Internalization was visualized in CHO-K1 cells containing an enhanced green fluorescent protein (EGFP)-tagged MTLR and quantified in binding experiments. The contractile response of repeated stimulations was measured in duodenal segments. In CHO-MTLR cells, the pDC(50) was ABT-229 (8.78) > motilin (7.77) > EM-A (4.78), different from their order of potency to induce Ca(2+) release (pEC(50)): motilin (9.39) > ABT-229 (8.46) > EM-A (7.11). In cells with the EGFP-tagged MTLR, ABT-229 decreased membrane fluorescence by 25 +/- 2% compared with 16 +/- 2% for motilin and 8 +/- 2% for EM-A. Binding studies confirmed that EM-A did not induce MTLR internalization (residual binding 96 +/- 4% compared with motilin, 31 +/- 3% and ABT-229, 21 +/- 1%). Comparison of the pDC(50) and pEC(50) values of the other motilides ME4 (5.90; 8.08), ME67 (6.03; 8.12), ME36 (3.32; 6.62), EM-523 (6.02; 8.22), and KOS1326 (7.32; 8.14) suggested that the strong desensitizing properties of ABT-229 are mostly related to the removal of the 4''-OH of the cladinose sugar. The decline of the contractile response in duodenal segments correlated with the pDC(50). The ability to desensitize and internalize the MTLR is not only determined by potency. This may be an important criterion for the development of a clinically useful compound.

Activation of GPR54 promotes cell cycle arrest and apoptosis of human tumor cells through a specific transcriptional program not shared by other Gq-coupled receptors.

GPR54 is a receptor for peptides derived from the metastasis suppressor gene KiSS-1. To investigate the intracellular mechanisms involved in the reduction of the metastatic potential of MDA-MB-435S cells expressing GPR54, a time course stimulation by kisspeptin-10 over a period of 25 h was performed using cDNA microarrays. Comparison with the bradykinin B(2) receptor revealed a distinct pattern of gene regulation despite a common coupling to the G(q/11) class of G-proteins. Inhibitors of PLC and PK-C abolished the transcriptional regulation of all tested genes, while an inhibitor of p42/44 affected a subset of genes controlled both by GPR54 and B(2). Among the genes specifically up-regulated by GPR54, we found several proapoptotic genes. Stimulation of GPR54 promoted apoptosis while no significant change was observed after B(2) receptor activation. Our results suggest that the metastasis suppressor properties of GPR54 are mediated in part by cell cycle arrest and induction of apoptosis in malignant cells.

Adaptation of aequorin functional assay to high throughput screening.

AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.

Aequorin-based functional assays for G-protein-coupled receptors, ion channels, and tyrosine kinase receptors.

Aequorin is a photoprotein originating from jellyfish, whose luminescent activity is dependent on the concentration of calcium ions. Due to the high sensitivity and low background linked to luminescent assays, as well as to its absence of toxicity and its large linear dynamic range, aequorin has been used as an intracellular calcium indicator since its discovery in the early 1960s. The first applications of aequorin involved its microinjection in cells. The cloning of its gene in 1985 opened the way to the stable expression of aequorin in cell lines or even entire organisms. Here we present the validation of aequorin as a functional assay for the screening of G-protein-coupled receptors, ion channels, and tyrosine kinase receptors, as well as for their pharmacological characterization in agonist and antagonist detection assays. We optimized our cell suspension-based assay and determined that the most sensitive assay was performed at room temperature, with mitochondrially expressed aequorin and using coelenterazine derivative h for reconstitution of aequorin. The robustness of the assay and the current availability of luminometers with integrated injectors allow aequorin to fit perfectly with high throughput functional assays requirements.