Inverse Agonism at Dopamine D2 Receptors: a Receptor Recalcitrant to High Levels of Constitutive Activation* Thierry Wurch*, Elisa A

International Congress Series 1249 (2003) 163-183 Inverse agonism at dopamine D2 receptors: a receptor recalcitrant to high levels of constitutive activation* Thierry Wurch*, Elisa A. Boutet-Robinet, Petras J. Pauwels Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, 17, Avenue Jean Moulin, 81106 Castres Cédex, France Received 16 April 2003; accepted 17 April 2003 Abstract Neuroleptic drags have been suggested to act as inverse agonists at the dopamine D2 receptor. Nevertheless, the capacity with which inverse agonism at this receptor subtype can be resolved is limited. Modulation of the constitutive activation of the D2 receptor was investigated in different 35 343 cellular systems by monitoring either [ S]GTP-yS binding responses at mutant Thr Ser D2short receptor or inositol phosphates formation mediated by a chimeric D2/a)B 3ICL receptor. A weak (about — 20% vs. basal [35S]GTP"yS binding response) inverse agonist activity of putative dopamine antagonists (i.e., nemonapride, haloperidol or (+)-butaclamol) was observed with digitonin- 343 permeabilized Chinese hamster ovary (CHO)-Kl cells stably expressing a mutant Thr Ser D2short receptor only if a high (150 mM) KC1 concentration was present in the binding buffer. No ligand- mediated decrease in basal [35S]GTP"/S binding was observed on membrane preparations of the same cells. Markedly increased inverse agonist responses were obtained with a series of dopamine antagonists by exchange of the D2sj10rt receptor's 3ICL by that of the a]B-adrenoceptor and incorporation of an activating mutation (Ala279Glu) in the distal BBXXB motif of its 3ICL and by co- expression with a Gan protein. This chimeric D2/aiB receptor construct displayed a ligand binding profile comparable to that of the wt D2short receptor and an effector activation profile close to that of the wt a1B-adrenoceptor. Most of the putative dopamine antagonists attenuated by — 54% to — 59% basal inositol phosphates (IP) formation, thus clearly acting as inverse agonists. Ziprasidone behaved virtually as a silent antagonist (+5% vs. basal IP level) and antagonised both dopamine (pK#: 7.61)- Abbreviations: AR, adrenoceptor; CHO, Chinese hamster ovary; DA, dopamine; ICL, intracellular loop; IP, inositol phosphates; NPA, norpropylapomorphine; PLC, phospholipase C; (+)-UH 232, c/s-(+)-5-methoxy-l- methyl-2-(di~n-propylammotetralin); S 14066, 3-(l-(benzocyclobutan-l-yhnethyl)piperidin-4-yl)-6-fluoro-l,2- benzisoxazole; TEA, triethanol amine; TM, trans-membrane domain. ^ Some data presented in this paper were taken from Wurch et al. [1]. * Corresponding author. Tel.: +33-5-63-71-43-62; fax: +33-5-63-71-43-63. E-mail address: [email protected] (T. Wurch). 0531-5131/03 © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00679-4 164 T. Wiirch et al. /International Congress Series 1249 (2003) 163-183 and tropapride (pK& 8.52)-mediated IP responses. Clozapine, olanzapine and raclopride displayed partial inverse agonist properties ( — 31%, —67% and —71% vs. tropapride 1 LiM, respectively), whereas bromerguride (+63%) and (+)-UH 232 (+88%) demonstrated positive agonism. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Inverse agonism; [35S]GTP-/S binding assay; Chimeric receptor; Inositol phosphates response; Intrinsic activity; Dopamine antagonist 1. Introduction Determination of the intrinsic activity of receptor ligands remains a major issue in the pharmacological characterization of the activity profile of a compound. The resolving capacity by which compounds with various degrees of intrinsic activity can be differ- entiated is co-determined by the receptor/G protein coupling state. Fine-tuning of the assay system is often necessary to measure the entire window of pharmacological activities ranging from efficacious agonism via neutral antagonism to inverse agonism. Detection of inverse agonism implies that a receptor is constitutively active, a condition which is often achieved either at high receptor expression levels and/or upon introduction of activating receptor mutations [2,3]. Several of such mutations have been described like Asp142Thr, 143 293 Arg Lys in the second intracellular loop (2ICL) and Ala Glu in the 3ICL of the ct1B- 373 272 adrenoceptor (a1B AR) [4-6]; Thr Lys in the 3ICL of the a2A AR [7,8]; and Leu Ala in the 3ICL of the p>2 AR [9]. The dopamine D2 receptor is of particular interest because it represents the major receptor target for neuroleptic drugs [10]. These compounds cover a broad spectrum of chemical structures classified as antagonists at D2_i¡ke receptors and which act as anti- schizophrenic agents [10]. Former studies suggested haloperidol to act as an inverse agonist by increasing prolactin release from GH4Ci pituitary cells expressing a D2short receptor [11,12] or by decreasing basal arachidonic acid release from a CHO pro"5 cell line stably expressing a D2iong receptor isoform [13]. More recent reports also suggested that most of these antagonists may act as inverse agonists at either wt or mutant D2 receptors [14—16]. Surprisingly, the amplitude of inverse agonism by these compounds seems to be comparable. The putative absence of a difference in the ligands' amplitude of inverse agonism may perhaps be due to the low resolving capacity of the investigated assay systems. Thus, it is of interest to develop more sensitive assay systems that would be able to identify putative differences in the magnitude of inverse agonism at D2 receptors. In the present study, [35S]GTP·yS binding experiments were performed in a first attempt 343 on CHO-K1 cells stably expressing a wild-type (wt) or mutant Thr Ser D2short receptor. Either a membrane preparation or digitonin-permeabilized cells were evaluated in different buffer formulations (i.e., in the presence of either Na+ or K+ ions) since the nature of these ions in the assay buffer has been shown to modulate [35S]GTP-yS binding responses for 8- 343 op'ioid receptor and a2A AR [17,18]. Detection of inverse agonism at the mutant Thr Ser D2short receptor was achieved with nemonapride, haloperidol and (+)-butaclamol. Never- theless, the amplitude of the inverse agonist response was weak (about 20%) and apparently similar for these compounds. In order to enhance the magnitude of the inverse T. Wurch et al /International Congress Series 1249 (2003) 163-183 165 agonist response, a chimeric receptor obtained by exchange of the 3ICL of the D2 receptor 279 for that of an a1B AR and containing an activating mutation (Ala Glu equivalent to the 293 279 Ala position in ot1B AR and noted D2/a1B Ala Glu 3ICL) in its 3ICL was constructed. This construct displayed a large amplitude (about 60%) of inverse agonism in the presence of a Gall protein. The intrinsic activity of different agonists and putative antagonists was 279 evaluated at the chimeric D2/a1B Ala Glu 3ICL receptor construct using both kinetic Ca2 + and inositol phosphates (IP) responses. 2. Materials and methods 2.1. Cell culture and transfection procedures Cos-7 and Chinese hamster ovary (CHO)-Kl cell lines were cultured, respectively, in Dulbecco's Modified Eagle Medium and Ham's F12 medium, each one supplemented with 10% heat-inactivated fetal calf serum. Transfection was performed by electroporation [Bio-Rad Gene pulser apparatus (250 V, 250 uF)] using 10 \ig of indicated receptor plasmid in either the presence or absence of 10 jig of recombinant Ga protein plasmid. The culture was prolonged in complete culture medium containing 1% dimethylsulfoxide under conditions dependent on the monitored response. CHO-K1 cell lines stably 343 expressing either a wt or mutant Thr Ser D2short receptor were generated upon dilution of transfected cells (10- to 1000-fold) and selection in complete Ham's F12 medium 343 containing 1.25 mg geneticin/ml. The selected wt and mutant Thr Ser D2Sh0rt receptor cell lines showed 8.31 ± 0.05 and 5.01 ± 0.04 pmol/mg protein, respectively, of specific [3H] nemonapride binding sites on their cellular membranes. 2.2. Construction of chimeric dopamine D2 receptor constructs The short splice form of the human wt dopamine D2 receptor (R.C. 2.1 .DA.02, GenBank accession number: S69899) was modified by exchanging its 3ICL by the equivalent portion of the human a1B-adrenoceptor (a1B AR, R.C. 2.1.ADR.A1B, GenBank accession number: U03865). A modified overlap extension PCR approach was applied by using overlapping complementary oligonucleotide primers allowing the junction of the D2 receptor and a1B AR portions without addition of restriction sites [19]. Incorporation of the Ala279Glu mutation was performed by using a QuickChange site-directed mutagenesis kit as described by the supplier (Stratagene, La Jolla, USA). The chimeric D2/a1B 3ICL receptor constructs were inserted into a pCR3.1 mammalian expression vector and fully sequenced on an ABI Prism 310 Genetic analyzer using a Big Dye Terminator Cycle Sequencing Ready Reaction kit, confirming the respective sequences. 2.3. [3sS]GTPyS binding assay to cellular membrane preparations 343 CHO-K1 cells stably expressing a wt or mutant Thr Ser D2short receptor were grown in complete Ham's F12 medium containing 1.25 mg/ml geneticin. Cells were scraped mechanically in 10 mM Tris-HCl, 0.1 M EDTA (pH 7.5) and centrifuged for 10 min at 45 000 x g. The pellet was homogenised in the same buffer and centrifuged under similar 166 T Winch et al / Intel national Congiess Series 1249 (2003) 163-183 conditions. Membrane preparations were diluted in different types of buffers as indicated. Buffer formulations consisted in 20 mM HEPES, 100 mM of either NaCl or KC1, 3 mM MgCl2, 0.2 mM ascorbic acid, 30 uM GDP (pH 7.4) or in 50 mM triethanolamine (TEA)- HC1, 1 mM MgCl2, 150 mM KC1, 30 uM GDP (pH 7.4). Incubation was performed at 25 °C for 30 min, followed by the addition of 0.5 nM [35S]GTP-yS either in the absence or presence of compound.

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