Traditionally, the interaction of G protein-coupled receptors has been described by models that assume that the receptor exists as a monomer coupled to G protein in a 1:1 stoichiometry. However, these classical models of receptor/G protein coupling may be oversimplified. It has now been shown that many G protein-coupled receptors can form dimers or higher order oligomers and that this phenomenon has relevance to receptor function (for a review, see Ref. 6). Dopamine receptors have also been shown to form dimers and higher order oligomers. Evidence has been provided for D 1 , D 2 , and D 3 homodimers in transfected cell lines (7-9), and D 2 receptors have been shown to exist as dimers in human and rat brain tissues (10). Moreover, Rocheville et al. (11) have recently shown that the dopamine D 2 receptors not only form homodimers but also form heterodimers with somatostatin SSTR 5 receptors. In addition, Gines et al. (12) have shown that the dopamine D 1 receptor forms hetero-oligomers with the adenosine A 1 receptor.As the issue of G protein-coupled receptor homo-and heterodimerization is becoming more and more important, it is crucial to define the mechanism(s) of receptor-receptor interactions in order to predict which receptors can interact with one another. The results obtained until now suggest that more than one mechanism exists and that one receptor can interact with another in more than one way.One of the mechanisms that have been proposed to explain receptor dimerization is the phenomenon of domain swapping

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.

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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 ϳ25-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-5Ј-O-(3-[35 S]thio)-triphosphate ([ 35 S]-GTP␥S) 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 G␣ 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 (Ͻ5.0) but revealed weak antagonist activity at hD 1 receptors (G␣s/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 (G␣ 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 h␣ 2C adrenoceptors ([ 35 S]GTP␥S, 7.2; G␣ i3 /SPA, 6.9; G␣ o /SPA, 7.3, and extracellular-regulated-kinase, 7.1) but not h␣ 2A or h␣ 2B adrenoceptors (Ͻ5.0). Finally, in contrast to haloperidol, clozapine, olanzapine, and risperidone, S33138 displayed negligible affinities for multiple subtypes of ␣ 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.Schizophrenia is a complex, progressive, and debilitating disorder of early onset that afflicts approximately 1% of the population. It is characterized by disorganized thought and a constellation of symptoms generally classified as positive (delusions and hallucinations), negative (social withdrawal, blunted affect, and mutism), and cognitive (deficits in attention, working and verbal memory, social cognition, and execArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.126706.ABBREVIATIONS: EPS, extrapyramidal motor symptom; AR, adrenoceptor; H 1 , histamine; DA, dopamine;3a,4, R(ϩ)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-

␤-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 ␤-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via ␤-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit ␤-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 ␤-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 ␤-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 ␣ 2 /M 3 and M 3 /␣ 2 heterodimeric receptors to co-immunoprecipitate with ␤-arrestin-1 following stimulation with adrenergic and muscarinic agonists. ␤-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 ␤-arrestin-1. Remarkably, we observed that M 2 /M 3 heterodimers recruit significantly greater amounts of ␤-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 ␤-arrestin-1 to muscarinic M 3 receptors requires paired stimulation of two receptor components within the same receptor dimer.Over the past several years, receptor dimerization has become an established concept in the field of G protein-coupled receptors (1, 2). Although the mechanism(s) by which receptors may undergo dimerization has yet to be elucidated in detail, it is becoming ever more clear that the phenomenon is playing a key role in receptor maturation, G protein coupling, and downstream signaling besides regulating such processes as internalization and desensitization. One of the most critical issues in receptor homodimerization and heterodimerization is whether signal transduction may require pair activation of receptor dimers. Many of the recent reports on G protein coupling consider separate stimulation of receptor dimers to be sufficient for activating G proteins in co-transfected cells (3-7). Accordingly, heterodimerization would not necessarily interfere with G protein coupling and the receptor monomer should be sufficient by itself to ac...

The recommended laboratory diagnostic approach for Lyme disease is a standard two-tiered testing (STTT) algorithm where the first tier is typically an enzyme immunoassay (EIA) that if positive or equivocal is reflexed to Western immunoblotting as the second tier. bioMérieux manufactures one of the most commonly used first-tier EIAs in the United States, the combined IgM/IgG Vidas test (LYT). Recently, bioMérieux launched its dissociated first-tier tests, the Vidas Lyme IgM II (LYM) and IgG II (LYG) EIAs, which use purified recombinant test antigens and a different algorithm than STTT. The dissociated LYM/LYG EIAs were evaluated against the combined LYT EIA using samples from 471 well-characterized Lyme patients and controls. Statistical analyses were conducted to assess the performance of these EIAs as first-tier tests and when used in two-tiered algorithms, including a modified twotiered testing (MTTT) approach where the second-tier test was a C6 EIA. Similar sensitivities and specificities were obtained for the two testing strategies (LYT versus LYM/LYG) when used as first-tier tests (sensitivity, 83 to 85%; specificity, 85 to 88%) with an observed agreement of 80%. Sensitivities of 68 to 69% and 76 to 77% and specificities of 97% and 98 to 99% resulted when the two EIA strategies were followed by Western immunoblotting and when used in an MTTT, respectively. The MTTT approach resulted in significantly higher sensitivities than did STTT. Overall, the LYM/LYG EIAs performed equivalently to the LYT EIA in test-to-test comparisons or as first-tier assays in STTT or MTTT with few exceptions.

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