Receptor crosstalk: haloperidol treatment enhances A(2A) adenosine receptor functioning in a transfected cell model.

UNLABELLED: A(2A) adenosine receptors are considered an excellent target for drug development in several neurological and psychiatric disorders. It is noteworthy that the responses evoked by A(2A) adenosine receptors are regulated by D(2) dopamine receptor ligands. These two receptors are co-expressed at the level of the basal ganglia and interact to form functional heterodimers. In this context, possible changes in A(2A) adenosine receptor functional responses caused by the chronic blockade/activation of D(2) dopamine receptors should be considered to optimise the therapeutic effectiveness of dopaminergic agents and to reduce any possible side effects. In the present paper, we investigated the regulation of A(2A) adenosine receptors induced by antipsychotic drugs, commonly acting as D(2) dopamine receptor antagonists, in a cellular model co-expressing both A(2A) and D(2) receptors. Our data suggest that the treatment of cells with the classical antipsychotic haloperidol increased both the affinity and responsiveness of the A(2A) receptor and also affected the degree of A(2A)-D(2) receptor heterodimerisation. In contrast, an atypical antipsychotic, clozapine, had no effect on A(2A) adenosine receptor parameters, suggesting that the two classes of drugs have different effects on adenosine-dopamine receptor interaction. Modifications to A(2A) adenosine receptors may play a significant role in determining cerebral adenosine effects during the chronic administration of antipsychotics in psychiatric diseases and may account for the efficacy of A(2A) adenosine receptor ligands in pathologies associated with dopaminergic system dysfunction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-010-9201-z) contains supplementary material, which is available to authorized users.

Partial agonist actions at dopamine D2L receptors are modified by co-transfection of D3 receptors: potential role of heterodimer formation.

The effects of aripiprazole, S33592, bifeprunox, N-desmethylclozapine and preclamol acting as partial agonists on recombinant D(2L) and D(3) receptors expressed both separately and concomitantly in COS-7 cells are evaluated here. Aripiprazole, S33592, bifeprunox, N-desmethylclozapine and preclamol behave as partial agonists on D(2L) receptors coupled with adenylyl cyclase, but they behave as antagonists on co-expression of D(3) with D(2L) receptors. These data raise the intriguing hypothesis that antipsychotic actions of "partial agonists" such as aripiprazole may not reflect inefficient stimulation of D(2) and/or D(3) receptors but, by analogy with other antipsychotics, may instead represent a blockade of D(2)/D(3) heterodimers (and/or D(3) receptors) that are "weakly" coupled to transduction mechanisms postsynaptically of the dopaminergic pathway.

S33138 [N-[4-[2-[(3aS,9bR)-8-cyano-1,3a,4,9b-tetrahydro[1]-benzopyrano[3,4-c]pyrrol-2(3H)-yl)-ethyl]phenylacetamide], a preferential dopamine D3 versus D2 receptor antagonist and potential antipsychotic agent: I. Receptor-binding profile and functional actions at G-protein-coupled receptors.

The novel, potential antipsychotic, S33138 (N-[4-[2-[(3aS,9bR)-8-cyano-1,3a,4,9b-tetrahydro[1]benzopyrano[3,4-c]pyrrol-2(3H)-yl)-ethyl]phenylacetamide), displayed approximately 25-fold higher affinity at human (h) dopamine D(3) versus hD(2L) (long isoform) and hD(2S) (short isoform) receptors (pK(i) values, 8.7, 7.1, and 7.3, respectively). Conversely, haloperidol, clozapine, olanzapine, and risperidone displayed similar affinities for hD(3), hD(2L), and hD(2S) sites. In guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]-GTPgammaS) filtration assays, S33138 showed potent, pure, and competitive antagonist properties at hD(3) receptors, displaying pK(B) and pA(2) values of 8.9 and 8.7, respectively. Higher concentrations were required to block hD(2L) and hD(2S) receptors. Preferential antagonist properties of S33138 at hD(3) versus hD(2L) receptors were underpinned in antibody capture/scintillation proximity assays (SPAs) of Galpha(i3) recruitment and in measures of extracellular-regulated kinase phosphorylation. In addition, in cells cotransfected with hD(3) and hD(2L) receptors that assemble into heterodimers, S33138 blocked (pK(B), 8.5) the inhibitory influence of quinpirole upon forskolin-stimulated cAMP formation. S33138 had low affinity for hD(4) receptors (<5.0) but revealed weak antagonist activity at hD(1) receptors (Galphas/SPA, pK(B), 6.3) and hD(5) sites (adenylyl cyclase, 6.5). Modest antagonist properties were also seen at human serotonin (5-HT)(2A) receptors (Galpha(q)/SPA, pK(B), 6.8, and inositol formation, 6.9) and at 5-HT(7) receptors (adenylyl cyclase, pK(B), 7.1). In addition, S33138 antagonized halpha(2C) adrenoceptors ([(35)S]GTPgammaS, 7.2; Galpha(i3)/SPA, 6.9; Galpha(o)/SPA, 7.3, and extracellular-regulated-kinase, 7.1) but not halpha(2A) or halpha(2B) adrenoceptors (<5.0). Finally, in contrast to haloperidol, clozapine, olanzapine, and risperidone, S33138 displayed negligible affinities for multiple subtypes of alpha(1)-adrenoceptor, muscarinic, and histamine receptor. In conclusion, S33138 possesses a distinctive receptor-binding profile and behaves, in contrast to clinically available antipsychotics, as a preferential antagonist at hD(3) versus hD(2) receptors.

Partial agonist actions of aripiprazole and the candidate antipsychotics S33592, bifeprunox, N-desmethylclozapine and preclamol at dopamine D(2L) receptors are modified by co-transfection of D(3) receptors: potential role of heterodimer formation.

Aripiprazole and the candidate antipsychotics, S33592, bifeprunox, N-desmethylclozapine (NDMC) and preclamol, are partial agonists at D(2) receptors. Herein, we examined their actions at D(2L) and D(3) receptors expressed separately or together in COS-7 cells. In D(2L) receptor-expressing cells co-transfected with (D(3) receptor-insensitive) chimeric adenylate cyclase-V/VI, drugs reduced forskolin-stimulated cAMP production by approximately 20% versus quinpirole (48%). Further, quinpirole-induced inhibition was blunted by aripiprazole and S33592, confirming partial agonist properties. In cells co-transfected with equal amounts of D(2L)and D(3) receptors (1 : 1), efficacies of aripiprazole and S33592 were attenuated. Further, in cells co-transfected with D(2L) and an excess of D(3) receptors (1 : 3), aripiprazole and S33592 were completely inactive, and they abolished the actions of quinpirole. Likewise, bifeprunox, NDMC and preclamol lost agonist properties in cells co-transfected with D(2L)and D(3) receptors. Accordingly, at split D(2trunk)/D(3tail) and D(3trunk)/D(2tail) chimeras, agonist actions of quinpirole were blocked by aripiprazole and S33592 that, like bifeprunox, NDMC and preclamol, were inactive alone. Conversely, when a 12 amino acid sequence in the third intracellular loop of D(3) receptors was replaced by the homologous sequence of D(2L) receptors, aripiprazole, S33592, bifeprunox, NDMC and preclamol inhibited cAMP formation by approximately 20% versus quinpirole (42%). Moreover, at D(2L) receptor-expressing cells co-transfected with modified D(3i3(D2)) receptors, drugs behaved as partial agonists. To summarize, low efficacy agonist actions of aripiprazole, S33592, bifeprunox, NDMC and preclamol at D(2L) receptors are abrogated upon co-expression of D(3) receptors, probably due to physical association and weakened coupling efficacy. These findings have implications for the functional profiles of antipsychotics.

The impact of G-protein-coupled receptor hetero-oligomerization on function and pharmacology.

Although highly controversial just a few years ago, the idea that G-protein-coupled receptors (GPCRs) may undergo homo-oligomerization or hetero-oligomerization has recently gained considerable attention. The recognition that GPCRs may exhibit either dimeric or oligomeric structures is based on a number of different biochemical and biophysical approaches. Although much effort has been spent to demonstrate the mechanism(s) by which GPCRs interact with each other, the physiological relevance of this phenomenon remains elusive. An additional source of uncertainty stems from the realization that homo-oligomerization and hetero-oligomerization of GPCRs may affect receptor binding and activity in different ways, depending on the type of interacting receptors. In this brief review, the functional and pharmacological effects of the hetero-oligomerization of GPCR on binding and cell signaling are critically analyzed.

Paired activation of two components within muscarinic M3 receptor dimers is required for recruitment of beta-arrestin-1 to the plasma membrane.

beta-Arrestins regulate the functioning of G protein-coupled receptors in a variety of cellular processes including receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled receptor-mediated recruitment of beta-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via beta-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit beta-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated muscarinic M(3) receptor, which is deleted in most of the third intracellular loop (M(3)-short). Although still capable of activating phospholipase C, this receptor loses almost completely the ability to recruit beta-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M(3)-short was co-expressed with the M(3) receptor. Under these conditions, the M(3)/M(3)-short heterodimer could not recruit beta-arrestin-1 to the plasma membrane, even though the control M(3)/M(3) homodimer could. We next tested the ability of chimeric adrenergic muscarinic alpha(2)/M(3) and M(3)/alpha(2) heterodimeric receptors to co-immunoprecipitate with beta-arrestin-1 following stimulation with adrenergic and muscarinic agonists. beta-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each receptor may exert on the M(2)/M(3) heterodimer to recruit beta-arrestin-1. Remarkably, we observed that M(2)/M(3) heterodimers recruit significantly greater amounts of beta-arrestin-1 than their respective M(3)/M(3) or M(2)/M(2) homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of beta-arrestin-1 to muscarinic M(3) receptors requires paired stimulation of two receptor components within the same receptor dimer.

The paired activation of the two components of the muscarinic M3 receptor dimer is required for induction of ERK1/2 phosphorylation.

Muscarinic M(3) receptors stimulate ERK1/2, the mitogen-activated protein kinase pathway. A mutant of the muscarinic M(3) receptor in which most of the third intracellular (i3) loop had been deleted (M(3)-short) completely lost the ability to stimulate the ERK1/2 phosphorylation in COS-7 cells. This loss was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. In co-transfected cells, M(3)-short greatly reduced the ability of M(3) to activate ERK1/2. In another set of experiments we tested the ability of a mutant M(3)/M(2)(16aa) receptor, in which the first 16 amino acids of the i3 loop of the M(3) receptor were replaced with the corresponding segment of the muscarinic M(2) receptor to stimulate ERK1/2 phosphorylation. This mutant is not coupled to Galpha(q), but it is weakly coupled to Galpha(i). Despite its coupling modification this receptor was able to stimulate ERK1/2 phosphorylation. Again, M(3)-short greatly reduced the ability of M(3)/M(2)(16aa) to activate ERK1/2 in co-transfected cells. Similar results were obtained in stable-transfected Chinese hamster ovary (CHO) cells lines. In CHO M(3) cells carbachol induced a biphasic increase of ERK1/2 phosphorylation; a first increase at doses as low as 0.1 microm and a second increase starting from 10 microm. In CHO M(3)-short and in double-transfected CHO M(3)/M(3)-short cells we observed only the lower doses increase of ERK1/2 phosphorylation; no further increase was observed up to 1 mm carbachol. This suggests that in double-transfected CHO cells M(3)-short prevents the effect of the higher doses of carbachol on the M(3) receptor. In a final experiment we tested the ability of co-transfected chimeric alpha(2)/M(3) and M(3)/alpha(2) receptors to activate the ERK1/2 pathway. When given alone, carbachol and, to a lesser extent, clonidine, stimulated the coupling of the co-transfected chimeric receptors to the phospholipase C second messenger pathway, but they were unable to stimulate ERK1/2 phosphorylation. On the contrary, a strong stimulation of ERK1/2 phosphorylation was observed when the two agonists were given together despite the fact that the overall increase in phosphatidylinositol hydrolysis was not dissimilar from that observed in cells treated with carbachol alone. Our data suggest that the activation of the ERK1/2 pathway requires the coincident activation of the two components of a receptor dimer.

Potent activation of dopamine D3/D2 heterodimers by the antiparkinsonian agents, S32504, pramipexole and ropinirole.

Recombinant, human dopamine D3 and D2 receptors form functional heterodimers upon co-expression in COS-7 cells. Herein, actions of the antiparkinsonian agents, S32504, ropinirole and pramipexole, at D3/D2L heterodimers were compared to their effects at the respective monomers and at split, chimeric D3trunk/D2tail and D2trunk/D3tail receptors: the trunk incorporated transmembrane domains (TDs) I-V and the tail TDs VI and VII. In binding assays with the antagonist [3H]nemonapride, all agonists were potent ligands of D3 receptors showing, respectively, 100-, 18- and 56-fold lower affinity at D2L receptors, mimicking the selective D3 receptor antagonist, S33084 (100-fold). At D3trunk/D2tail receptors, except for ropinirole, all drugs showed lower affinities than at D3 sites, whereas for D2trunk/D3tail receptors, affinities of all drugs were higher than at D2L sites. The proportion of high affinity binding sites recognized by S32504, pramipexole and ropinirole in membranes derived from cells co-expressing D3 and D2L sites was higher than in an equivalent mixture of membranes from cells expressing D3 or D2L sites, consistent with the promotion of heterodimer formation. In contrast, the percentage of high and low affinity sites (biphasic isotherms) recognized by S33084 was identical. Functional actions were determined by co-transfection of a chimeric adenylyl cyclase (AC)-V/VI insensitive to D3 receptors. Accordingly, D3 receptor-transfected cells were irresponsive whereas, in D2L receptor-transfected cells, agonists suppressed forskolin-stimulated cAMP production with modest potencies. In cells co-transfected with D3 and D2L receptors, S32504, ropinirole and pramipexole potently suppressed AC-V/VI with EC50s 33-, 19- and 11-fold lower than at D2L receptors, respectively. S32504 also suppressed AC-V/VI activity at split D3trunk/D2tail and D2trunk/D3tail chimeras transfected into COS-7 cells. In conclusion, antiparkinson agents behave as potent agonists at D3/D2'heterodimers', though any role in their actions in vivo remains to be demonstrated.

Functional rescue of the inactive splice variant of the dopamine D3 receptor D3nf.

We previously found that the dopamine D3 receptor can be split at the third cytoplasmic loop into two fragments (D3trunk and D3tail), and that the mixture of the two fragments retains the binding and functional activity of the wild type receptor. The dopamine D3 receptor gene gives rise to several inactive receptor splice variants, one of which is the D3nf. Since this gene variant very closely resembles our D3trunk fragment, in this study we investigated if the transfection of D3nf with D3tail could result in the rescue of a functional dopamine receptor. Our experiments showed that D3tail can indeed rescue the activity of D3nf, and that the pharmacological profile of this split D3nf/D3tail receptor is identical to that of the wild type D3 receptor.

An unusual form of the association binding kinetics of N-[3H]methylscopolamine to the split muscarinic M2trunk/M2tail receptor.

The muscarinic M(2) receptor was split at the third cytoplasmic loop into two fragments: the one containing the first five transmembrane regions and the N-terminal part of the third cytoplasmic loop was named M(2trunk), while the other, which contained the last two transmembrane regions and the C-terminal part of the third cytoplasmic loop, was named M(2tail). As seen in many other G protein-coupled receptors, when these two fragments were transfected together in COS-7 cells they rescued the pharmacological profile and the functional activity of the wild-type M(2) receptor. Conversely, N-[(3)H]methylscopolamine ([(3)H]NMS) association binding experiments showed a substantial difference between the wild-type M(2) and the split M(2trunk)/M(2tail) receptors. The progression of the association binding kinetic of the M(2trunk)/M(2tail) receptor was strictly dependent upon the amount of the fragment DNA transfected. When the amount of transfected DNA was 4 microg/plate and the B(max) of [(3)H]NMS at equilibrium was around 200 fmol/mg protein the form of the association was that of classical saturation, but when the amount of transfected DNA was lower the [(3)H]NMS association reached a maximum binding point and then declined to a lower equilibrium binding level. The form of the association was temperature-dependent: as the temperature was lowered, the maximum binding point tended to be higher. We suggest that this peculiar form of the [(3)H]NMS association binding to the muscarinic M(2trunk)/M(2tail) receptor is attributable to a less stable interaction between the trunk and the tail fragments of the split receptor.

Deoxamuscaroneoxime derivatives as useful muscarinic agonists to explore the muscarinic subsite: demox, a modulator of orthosteric and allosteric sites at cardiac muscarinic M2 receptors.

A series of muscarinic agonists, straight chained, branched, cyclic alkyl and aromatic derivatives of the oxime 1 (demox) was designed with the aim of investigating their activity on muscarinic receptor subtypes. Effects on M1 receptor were assessed functionally by a microphysiometer apparatus, while M2, M3, and M4 receptor potency and affinity were studied on isolated preparations of guinea pig heart, ileum, and lung, respectively. The results suggest that the substitution of a hydrogen with a long side-chain or bulky group generally induces a decrease in potency at M1 and M3 subtypes, while a general increase in this parameter is obtained at M2 subtype. Among the agonists 2-18, compound 4 behaves as a full agonist with a preference for M3 subtype. Moreover, compound 12 is inactive at M1 and M4 receptors while it displays a full agonist activity at M2 and M3 subtypes. Since demox displays a variable response on cardiac M2 receptors regulating heart force, an in-depth inquiry of the functional behaviour of this compound was carried out at M2 receptors. In presence of 10(-11) and 10(-10) M demox, the binding of [3H]-NMS was increased by approximately 30% as a consequence of an increase of the association of [3H]-NMS to membranes; this effect was not observed in presence of a higher concentration of [3H]-NMS. Higher concentrations of demox decreased the binding of [3H]-NMS to heart atrial membranes but significantly retarded the dissociation of this radioligand. Our results suggest that demox may interact with orthosteric and allosteric sites of atrial M2 muscarinic receptor.