Louise H. Naylor

Functional coupling of endogenous serotonin (5-HT1B) and calcitonin (C1a) receptors in CHO cells to a cyclic AMP-responsive luciferase reporter gene

Abstract: A cyclic AMP‐responsive reporter cell line has been established through the stable expression of a luciferase reporter plasmid in Chinese hamster ovary (CHO) cells. Reporter cells showed a dose‐dependent expression of luciferase in response to incubation with forskolin. These CHO cells were screened for endogenous G protein‐coupled receptors capable of stimulating or inhibiting adenylyl cyclase, by monitoring changes in luciferase expression. Serotonin (5‐HT) receptor agonist ligands caused an inhibition of forskolin‐stimulated luciferase expression in the rank order 5‐carboxamidotryptamine > 5‐HT > sumatriptan > 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin. The response to 5‐HT was reversed by the 5‐HT1 receptor antagonists cyanopindolol and pindolol, but not the 5‐HT2 receptor antagonist ketanserin. Calcitonin was more potent than calcitonin gene‐related peptide (CGRP) at stimulating luciferase expression in this cell line, and these responses were insensitive to the CGRP receptor antagonist, CGRP (8–37). These results were consistent with the presence of 5‐HT1B‐like and calcitonin (C1a‐like) receptors in CHO cells, with the responses to 5‐HT and CGRP being pertussis and cholera toxin‐sensitive, respectively. This reporter gene assay gave the expected pharmacological profile for these receptors when compared with cyclic AMP accumulation assays, confirming its value as a functional assay for G protein‐coupled receptors linked to adenylyl cyclase.

Investigation of the Role of Conserved Serine Residues in the Long Form of the Rat D2 Dopamine Receptor Using Site‐Directed Mutagenesis

Abstract: Three serine residues (Ser193, Ser194, Ser197) in the fifth transmembrane‐spanning region of the D2 dopamine receptor have been mutated separately to alanine and the effects of the mutations determined in ligand‐binding experiments with [3H]spiperone. For many antagonists the mutations had little effect, showing that the overall conformation of the mutant receptors was similar to that of the native, although there were effects on the binding of certain antagonists. The effect of the mutations on agonist binding to the free receptor (uncoupled from G proteins) was determined in the presence of GTP (100 µM). This showed that there was no single mode of binding of catecholamine agonists to the receptor and that all three serine residues can participate in the binding of some agonists, possibly through hydrogen bonds to the catechol hydroxyl groups. Coupling of the mutant receptors to G proteins was assessed from agonist‐binding curves in the absence of GTP, when higher and lower affinity agonist‐binding sites were seen. Receptor/G protein coupling was generally unaffected by the Ala193 and Ala194 mutations, but the Ala197 mutation eliminated receptor/G protein coupling for some agonists. These data show that the interactions of agonists with the free and coupled forms of the receptor are different.

Evaluation of a CRE-Directed Luciferase Reporter Gene Assay as an Alternative to Measuring cAMP Accumulation

A CHO reporter cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was used to investigate the relationship between cAMP accumulation and cAMP dependent reporter gene expression and therefore, to assess the reporter gene system as an alternative functional assay. Timecourse experiments showed that cAMP accumulation preceded luciferase expression and that longer incubations (>2 h) were required to gain results with the reporter gene system. However, forskolin concentration dose-response studies revealed a 100-fold amplification of the signal measured by luciferase expression compared with direct cAMP accumulation, indicating that the reporter gene system afforded greater sensitivity. EC50 values determined for agonist activation of an inhibitory (5-HTlB-like) G-protein-coupled receptor (GPCR) were the same, and for a stimulatory GPCR (calcitonin Cla-like) were 10-fold lower, using the reporter gene system compared to cAMP accumulation assays, indicating the suitability of the reporter system for measuring the activity of receptors differentially coupled to the cAMP pathway. The phorbol ester, PMA, and the Ca2+ ionophore, A23187, were able to potentiate forskolin-stimulated luciferase expression but not cAMP accumulation, suggesting that the former could also be used to monitor cross-talk between different signal transduction pathways at the level of gene transcription.

Effect of Multiple Serine/Alanine Mutations in the Transmembrane Spanning Region V of the D2 Dopamine Receptor on Ligand Binding

Abstract: Three conserved serine residues (Ser193, Ser194, and Ser197) in transmembrane spanning region (TM) V of the D2 dopamine receptor have been mutated to alanine, individually and in combination, to explore their role in ligand binding and G protein coupling. The multiple Ser → Ala mutations had no effect on the binding of most antagonists tested, including [3H]spiperone, suggesting that the multiple mutations did not affect the overall conformation of the receptor protein. Double or triple mutants containing an Ala197 mutation showed a decrease in affinity for domperidone, whereas Ala193 mutants showed an increased affinity for a substituted benzamide, remoxipride. However, dopamine showed large decreases in affinity (>20‐fold) for each multiple mutant receptor containing the Ser193 Ala mutation, and the high‐affinity (coupled) state of the receptor (in the absence of GTP) could not be detected for any of the multiple mutants. A series of monohydroxylated phenylethylamines and aminotetralins was tested for their binding to the native and multiple mutant D2 dopamine receptors. The results obtained suggest that Ser193 interacts with the hydroxyl of S‐5‐hydroxy‐2‐dipropylaminotetralin (OH‐DPAT) and Ser197 with the hydroxyl of R‐5‐OH‐DPAT. We predict that Ser193 interacts with the hydroxyl of R‐7‐OH‐DPAT and the 3‐hydroxyl (m‐hydroxyl) of dopamine. Therefore, the conserved serine residues in TMV of the D2 dopamine receptor are involved in hydrogen bonding interactions with selected antagonists and most agonists tested and also enable agonists to stabilise receptor‐G protein coupling.

Structural Studies on D2 Dopamine Receptors: Mutation of a Histidine Residue Specifically Affects the Binding of a Subgroup of Substituted Benzamide Drugs

Abstract: A histidine residue (His394) that is likely to be located in the ligand‐binding region of the D2 dopamine receptor has been mutated to a leucine (Leu394), and the properties of the mutant receptor have been determined. For a range of antagonists the mutation has only a minor effect on the affinity of the receptor for the antagonist. The mutation does, however, elicit a structurally specific effect on the affinity with which certain members of the substituted benzamide class of antagonist bind to the receptor. Some of these drugs, e.g., sulpiride, sultopride, and tiapride, bind with reduced affinity to the mutated receptor, whereas others, e.g., clebopride and metoclopramide, bind with increased affinity. However, the Na+/H+ sensitivity of the binding of sulpiride to the receptor is not reduced by the mutation. These findings have been interpreted in terms of the productive or unfavourable interaction of the His394 residue with these compounds.

Functional analysis of the D2L dopamine receptor expressed in a cAMP-responsive luciferase reporter cell line.

A Chinese hamster ovary (CHO) cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was stably transfected with the long form of the rat D2 dopamine receptor. Saturation binding analysis using [3H]spiperone showed that the receptor was expressed at low levels (Bmax = 96.5+/-15.8 fmol/mg), but with an affinity characteristic of the D2 receptor (Kd = 21.5+/-3.7 pM). Luciferase expression in this cell line was modified in a dose dependent manner with dopamine receptor agonists (N-propylapomorphine > apomorphine > quinpirole > dopamine) and antagonists (spiperone > (+)-butaclamol > D0710 > (-)-sulpiride > tiapride > remoxipride), according to their rank order of potency in binding and cAMP accumulation studies. Dopamine-mediated inhibition of forskolin-stimulated luciferase expression was pertussis toxin sensitive. This demonstrated the efficiency of the luciferase reporter gene assay for the functional testing of D2 dopamine receptors, which are negatively coupled to the adenylyl cyclase signaling pathway, when heterogously expressed at low levels in CHO cells.

Transient Gene Expression Levels from Multigene Expression Vectors

Multigene expression vectors are commonly utilized whereby the (over)expression of two or more genes is desired simultaneously and often at supposedly equivalent levels. We have characterized dual‐gene and pEE14.4 RlucMFluc expression plasmids in which the second hCMV‐MIE promoter is replaced with a c‐myc IRES to enable one‐step coordinated expression of multiple genes in eukaryotic cells. The efficacy of these plasmids has been tested in Chinese hamster ovary (CHO) cells using renilla and firefly luciferase reporter genes, with the reporter gene in either position 1 or 2 from the 5′ to 3′ direction, respectively. The dual‐gene constructs gave enhanced second position gene expression levels compared to the first gene during transient transfection experiments (4‐ to 50‐fold increase 24 h post‐transfection). The pEE14.4 RlucMFluc plasmid gave enhanced first cistron expression compared to the second cistron (∼19‐fold increase 24 h post‐transfection). More equivalent transient expression levels were obtained by undertaking co‐transfection of the appropriate single gene plasmids. Reporter gene mRNA levels in the transfected cells were also evaluated by qRT‐PCR and compared to the observed protein expression levels. Analysis of the resulting data showed that transcriptional limitation of the first cistron in the dual‐gene expression system and not translational limitation was the major limiting factor. Taken together these data suggest that relative protein expression levels expected from heterologous gene products in a multigene vector cannot be predicted on copy number alone and it is important to characterize multigene or oligocistronic systems prior to use.

Development of a Generic Dual-Reporter Gene Assay for Screening G-Protein-Coupled Receptors:

Multiple assay formats have been developed for the pharmacological characterization of G-protein-coupled receptors (GPCRs) and for screening orphan receptors. However, the increased pace of target identification and the rapid expansion of compound libraries present the need to develop novel assay formats capable of screeningmultipleGPCRs simultaneously. To address this need, the authors have developed a generic dual-reporter gene assay that can detect ligand activity at 2 GPCRs within the same assay. Two stableHEK293 cell lineswere generated expressing either a firefly (Photinus) luciferase gene under the control ofmultiple cAMP-response elements (CREs) or a Renillaluciferase gene under the control ofmultiple 12-Otetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs). Coseeded reporter cells were used to assess ligandbinding activity at bothGβ s-and Gβ q-coupled receptors. By selectively coexpressing receptors with a chimeric G-protein, agonist activitywas assessed atGβ i/o-coupled receptors in combinationwith eitherGβ s-or Gβ q-coupled receptors. The dual-reporter gene assaywas shown to be capable of simultaneously performing duplexed screens for a variety of agonist and/or antagonist combinations. The data generated from the duplexed reporter assays were pharmacologically relevant, and Zβ factor analysis indicated the suitability of both agonist and antagonist screens for use in high-throughput screening.

The effect of ICER on screening methods involving CRE-mediated reporter gene expression

We describe a mechanism whereby increasing levels of cAMP in Chinese hamster ovary (CHO) and other cell lines lead to a significant repression in cAMP response element (CRE)-mediated luciferase reporter gene expression. This effect was shown to be mediated by a modulatory factor located downstream of cyclic AMP (cAMP), which displayed the temporal regulation pattern of an immediate early gene. The expression of this inducible cAMP early repressor (ICER) was shown to be coincident with the time and concentration dependency of the repression of CRE-mediated luciferase gene expression on the treatment of CHO cells with forskolin. Furthermore, this phenomenon was also observed in JEG and GH3 cell lines (both previously reported to express ICER), but not in COS-7 cells, which do not express ICER. These studies suggest that, in certain cell lines, expression of ICER can be induced at pharmacologically elevated cAMP levels, leading to a potent inhibition of CRE-mediated gene expression. We therefore conclude that screening methodologies employing such CRE-linked reporter genes (particularly in high-throughput screening assays) may produce false functional responses in certain cell lines. Moreover, such effects are likely to be exacerbated in screening assays in which receptors either are overexpressed or high concentrations of potent cAMP-elevating compounds are used.

Partial agonism at serotonin 5-HT1B and dopamine D2L receptors using a luciferase reporter gene assay

We have used a luciferase reporter gene assay to study the functional responses of two G-protein-coupled receptors in Chinese hamster ovary (CHO) cells. The rank order of potency of drugs for the endogenous 5-HT1B receptor was 5-Hydroxytryptamine (5-HT) > zolmitriptan > dihydroergocristine > (-)lisuride (with no response to bromocriptine). However, only 5-HT and (-)lisuride produced a full functional response, with zolmitriptan and dihydroergocristine achieving 69+/-2% and 50+/-1% of the maximal response. In the same cells stably transfected with the rat dopamine D2L receptor, dopamine and bromocriptine produced a full agonist functional response, whilst (-)lisuride produced a biphasic response curve, indicating activity at both the endogenous 5-HT1B and exogenous dopamine D2L receptors. Using the receptor specific antagonists, pindolol and (+)butaclamol, (-)lisuride was shown to produce 52% of the maximal response at the dopamine D2 receptor relative to dopamine. In comparison to a cAMP accumulation assay, the rank orders of potency and intrinsic activity were the same for all compounds used. These results demonstrate that this reporter gene assay is capable of discriminating both potency and efficacy of drugs and can be used to characterise partial agonists at endogenously and heterologously expressed receptors in CHO cells.