Some G-protein-coupled receptors display 'constitutive activity', that is, spontaneous activity in the absence of agonist. This means that a proportion of the receptor population spontaneously undergoes an allosteric transition, leading to a conformation that can bind G proteins. The process has been shown to occur with recombinant receptors expressed at high density, and/or mutated, but also non-mutated recombinant receptors expressed at physiological concentrations. Transgenic mice that express a constitutively active mutant of the beta2-adrenergic receptor display cardiac anomalies; and spontaneous receptor mutations leading to constitutive activity are at the origin of some human diseases. Nevertheless, this process has not previously been found to occur in animals expressing normal levels of receptor. Here we show that two isoforms of the recombinant rat H3 receptor display high constitutive activity. Using drugs that abrogate this activity ('inverse agonists') and a drug that opposes both agonists and inverse agonists ('neutral antagonist'), we show that constitutive activity of native H3 receptors is present in rodent brain and that it controls histaminergic neuron activity in vivo. Inverse agonists may therefore find therapeutic applications, even in the case of diseases involving non-mutated receptors expressed at normal levels.

Histamine H 3 receptor inverse agonists are known to enhance the activity of histaminergic neurons in brain and thereby promote vigilance and cognition. 1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride (BF2.649) is a novel, potent, and selective nonimidazole inverse agonist at the recombinant human H 3 receptor. On the stimulation of guanosine 5Ј-O-(3-[35 S]thio)triphosphate binding to this receptor, BF2.649 behaved as a competitive antagonist with a K i value of 0.16 nM and as an inverse agonist with an EC 50 value of 1.5 nM and an intrinsic activity ϳ50% higher than that of ciproxifan. Its in vitro potency was ϳ6 times lower at the rodent receptor. In mice, the oral bioavailability coefficient, i.e., the ratio of plasma areas under the curve after oral and i.v. administrations, respectively, was 84%. BF2.649 dose dependently enhanced tele-methylhistamine levels in mouse brain, an index of histaminergic neuron activity, with an ED 50 value of 1.6 mg/kg p.o., a response that persisted after repeated administrations for 17 days. In rats, the drug enhanced dopamine and acetylcholine levels in microdialysates of the prefrontal cortex. In cats, it markedly enhanced wakefulness at the expense of sleep states and also enhanced fast cortical rhythms of the electroencephalogram, known to be associated with improved vigilance. On the two-trial object recognition test in mice, a promnesiant effect was shown regarding either scopolamine-induced or natural forgetting. These preclinical data suggest that BF2.649 is a valuable drug candidate to be developed in wakefulness or memory deficits and other cognitive disorders.The cerebral histaminergic neurons seem to play a critical role in the maintenance of wakefulness and higher cerebral functions, e.g., attention or learning (for review, see Schwartz et al., 1991;Haas and Panula, 2003). Hence, druginduced activation of histaminergic neurotransmission in the central nervous system represents a promising therapeutic target in a large variety of neuropsychiatric disorders in which these functions are compromised and for which available therapeutic opportunities are limited in this respect .Stimulation of postsynaptic H 1 and/or H 2 receptors by agonists is, however, not acceptable due to unavoidable and detrimental actions of these drugs at peripheral, i.e., mainly cardiovascular and gastric targets. In contrast, presynaptic H 3 receptors are almost exclusively expressed in the central nervous system, and their blockade by drugs such as thioperamide markedly enhances the activity of histaminergic neurons, as shown namely by the increases in histamine (HA) release and turnover in rodent brain (Arrang et al., Article, publication date, and citation information can be found at

The formation of G protein-coupled receptor (GPCR) heteromers elicits signaling diversification and holds great promise for improved drug selectivity. Most studies have been conducted in heterologous expression systems; however, in vivo validation is missing from most cases thus questioning the physiological significance of GPCR heteromerization. Melatonin MT1 and MT2 receptors have been shown to exist as homo- and heteromers in vitro. We show here that the effect of melatonin on rod photoreceptor light sensitivity is mediated by melatonin MT1/MT2 receptor heteromers. This effect involves activation of the heteromer-specific PLC/PKC pathway and is abolished in MT1−/− and MT2−/− mice as well as in mice overexpressing a non-functional MT2 receptor mutant that competes with the formation of functional MT1/MT2 heteromers in photoreceptor cells. This study establishes the essential role of melatonin receptor heteromers in retinal function and supports the physiological importance of GPCR heteromerization. Finally, our work may have important therapeutic implications, as the heteromer complex may provide a unique pharmacological target to improve photoreceptor functioning and to extend the viability of photoreceptors during aging.

Background: There is cross-talk between serotonin and melatonin hormones. Results: There is evidence for unidirectional transactivation and a heteromer-specific signaling profile for formation of functional melatonin MT 2 and serotonin 5-HT 2C receptor heteromers. Conclusion: A new potential target of the antidepressant agomelatine is identified. Significance: The importance of binding of multitarget drugs to GPCR heteromers in psychiatric disorders is demonstrated.

G protein-coupled receptors (GPCRs) are allosteric proteins that adopt inactive (R) and active (R*) conformations in equilibrium. R* is promoted by agonists or occurs spontaneously, leading to constitutive activity of the receptor. Conversely, inverse agonists promote R and decrease constitutive activity. The existence of another pharmacological entity, referred to as ''protean'' agonists (after Proteus, the Greek god who could change shape), was assumed on theoretical grounds. It was predicted from the existence of constitutive activity that a same ligand of this class could act either as an agonist or an inverse agonist at the same GPCR. Here, we show that proxyfan, a high-affinity histamine H 3-receptor ligand, acts as a protean agonist at recombinant H 3 receptors expressed in the same Chinese hamster ovary cells. In support of the physiological relevance of the process, we show that proxyfan also behaves as a protean agonist at native H 3 receptors known to display constitutive activity. On neurochemical and behavioral responses in rodents and cats, proxyfan displays a spectrum of activity ranging from full agonism to full inverse agonism. Thus, protean agonism demonstrates the existence of ligand-directed active states LR* different from, and competing with, constitutively active states R* of GPCRs, and defines a pharmacological entity with important therapeutic implications.

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1 Constitutive activity of the recombinant and native rat and human H 3 receptors (H 3 Rs) was studied using H 3 R-mediated [ 35 cells. Both e ects were correlated to receptor density and revealed that constitutive activity of the hH 3 R, although lower than that of the rH 3 R in this assay, was again observed at physiological densities (5500 fmol mg 71 protein). Ciproxifan was less potent at the human than the rat receptor, not only as an antagonist (K i =45 nM), but also as an inverse agonist (EC 50 =15 nM). 4 Constitutive activity of the hH 3 R was also evidenced using inhibition of [ 35 S]GTPg[S] binding by unlabelled GTPgS. The expression of the hH 3 R generated a high a nity binding for GTPgS which was increased by imetit, but partially decreased by ciproxifan, therefore acting as a partial inverse agonist. [ 3S]GTPg[S]binding to rat brain membranes was decreased in several regions by thioperamide, ciproxifan and FUB 465, three inverse agonists at the H 3 R, whose e ects were blocked by proxyfan, a neutral antagonist. [ 35 S]GTPg[S] binding was also decreased by an A 1 -adenosine receptor inverse agonist, but remained unchanged in the presence of inverse agonists at D 2 /D 3 dopamine, H 1 and H 2 histamine, a 2 -adrenergic and d opioid receptors. 6 In conclusion, the present study shows that the recombinant rat and human H 3 receptors expressed at physiological densities display constitutive activity and suggests that constitutive activity of native H 3 Rs is one of the highest among G-protein-coupled receptors present in rat brain.

Starting from the sequence of the human histamine H 3 receptor (hH 3 R) cDNA, we have cloned the corresponding rat cDNA. Whereas the two deduced proteins show 93.5% overall homology and di er only by ®ve amino acid residues at the level of the transmembrane domains (TMs), some ligands displayed distinct a nities. Thioperamide and ciproxifan were about 10 fold more potent at the rat than at the human receptor, whereas FUB 349 displayed a reverse preference. Histamine, (R)a-methylhistamine, proxyfan or clobenpropit were nearly equipotent at H 3 receptors of both species. The inverse discrimination patterns of ciproxifan and FUB 349 were partially changed by mutation of one amino acid (V122A), and fully abolished by mutation of two amino acids (A119T and V122A), in TM3 of the rH 3 R located in the vicinity of Asp 114 purported to salt-link the ammonium group of histamine. Therefore, these two residues appear to be responsible for the distinct pharmacology of the H 3 R in the two species. British Journal of Pharmacology (2000) Introduction Whereas the histamine H 3 receptor (H 3 R) was initially identi®ed in the rat brain (Arrang et al., 1983;1987), its presence in the human brain was con®rmed a few years later (Arrang et al., 1988). In both cases a functional test, the inhibition of [ 3 H]-histamine release from depolarized brain slices, was used, but the pharmacological characterization of the human H 3 R has remained preliminary since the availability of fresh brain tissues obtained during neurosurgery is limited. Nevertheless there were some indications that the pharmacology of the human and the rat H 3 R may slightly di er (Arrang et al., 1988; West et al., 1999 and X. Ligneau, unpublished observation).With the recent cloning of the human H 3 R (hH 3 R) (Lovenberg et al., 1999), it became feasible to determine with greater precision the apparent a nity of ligands at this receptor and assess the existence of species di erences. Namely, for this purpose we have cloned the rat H 3 R (rH 3 R) starting from the published sequence of the hH 3 R and established permanent cell lines expressing the hH 3 R or rH 3 R. This allowed us to identify ligands displaying distinct apparent a nities at the H 3 R of the two species. Then we tried to identify the amino acid residues responsible for such discrimination using site-directed mutagenesis of the rH 3 R.