Ligand modulation of [35S]GTPγS binding at human α2A, α2B and α2C adrenoceptors

Audinot, Valérie; Fabry, Nelly; Nicolas, Jean-Paul; Beauverger, Philippe; Newman‐Tancredi, Adrian; Millan, Mark J.; Try, Anne; Bornancin, Frédéric; Canet, Emmanuel; Boutin, Jean A.

doi:10.1016/s0898-6568(02)00030-x

Cited by 27 publications

(23 citation statements)

References 36 publications

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“…Figure 2 shows that all constructs induced [ 35 S]GTP␥S binding well above the level of nontransfected HEK cells. These assays were done with the full agonist norepinephrine, the strong partial agonists dopamine and clonidine (a structurally independent compound), and two weak partial agonists, norphenephrine and octopamine (Audinot et al, 2002;Peltonen et al, 2003). With all five agonists, wild-type and mutant receptors were indistinguishable in the rates and in the amplitudes of stimulated [ 35 S]GTP␥S binding.…”

Section: Resultsmentioning

confidence: 99%

Fluorescence Resonance Energy Transfer Analysis of α_2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes

Zürn¹,

Zabel²,

Vilardaga³

et al. 2008

Mol Pharmacol

100

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Several lines of evidence suggest that G-protein-coupled receptors can adopt different active conformations, but their direct demonstration in intact cells is still missing. Using a fluorescence resonance energy transfer (FRET)-based approach we studied conformational changes in ␣ 2A -adrenergic receptors in intact cells. The receptors were C-terminally labeled with cyan fluorescent protein and with fluorescein arsenical hairpin binder at different sites in the third intracellular loop: N-terminally close to transmembrane domain V (I3-N), in the middle of the loop (I3-M), or C-terminally close to transmembrane domain VI (I3-C). All constructs retained normal ligand binding and signaling properties. Changes in FRET between the labels were determined in intact cells in response to different agonists. The full agonist norepinephrine evoked similar FRET changes for all three constructs. The strong partial agonists clonidine and dopamine induced partial FRET changes for all constructs. However, the weak partial agonists octopamine and norphenephrine only induced detectable changes in the construct I3-C but no change in I3-M and I3-N. Dopamineinduced FRET-signals were Ϸ1.5-fold slower than those for norepinephrine in I3-C and I3-M but Ͼ3-fold slower in I3-N. Our data indicate that the different ligands induced conformational changes in the receptor that were sensed differently in different positions of the third intracellular loop. This agrees with X-ray receptor structures indicating larger agonist-induced movements at the cytoplasmic ends of transmembrane domain VI than V and suggests that partial agonism is linked to distinct conformational changes within a G-protein-coupled receptor.Stimulation of G-protein-coupled receptors (GPCRs) by an agonist leads to a conformational change and to a transition of the receptor into an active conformation, which can then couple to its G-protein. Conformational changes have been well established to occur within the transmembrane domains (TMs) III and VI (Gether, 2000;Hubbell et al., 2003). These changes are believed to be transmitted into the third intracellular loop. This loop seems to contain the key domains for coupling to G-proteins, particularly in its C terminus (adjacent to TMVI) but also in its N terminus (adjacent to TMV) regions (Wess, 1998).Whereas classic theory assumed that receptors simply switch between "off" and "on" states, more recent data indicate that agonists of different efficacy might induce different changes in receptor conformations (Kobilka and Deupi, 2007). To accommodate the growing body of evidence for multiple conformational states into theoretical considerations, different models have been proposed. These models propose either that each agonist might promote its own specific active receptor conformation, thus leading to an almost unlimited number of receptor conformations R n *, or suggest that there might be a limited number of active conformations into which different agonists might switch a receptor (Kenakin, 1995). The accumulating evid...

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Section: Resultsmentioning

confidence: 99%

Fluorescence Resonance Energy Transfer Analysis of α_2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes

Zürn¹,

Zabel²,

Vilardaga³

et al. 2008

Mol Pharmacol

100

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“…The functional status of MOP receptor can be measured in concentration-effect curves for agonist-stimulated [ 35 S]GTP␥S binding (Audinot et al, 2002). We performed a set of experiments to determine this initial stage of G protein activation to reveal possible changes in MOP receptor-mediated G protein-coupling of DRG in animals with and without FCA inflammation.…”

Section: Fig 2 Stimulation Of [ 35 S]gtp␥s Binding To Ht (A) Sc (B)mentioning

confidence: 99%

Characterization of μ Opioid Receptor Binding and G Protein Coupling in Rat Hypothalamus, Spinal Cord, and Primary Afferent Neurons during Inflammatory Pain

Shaqura

Zöllner²,

Mousa³

et al. 2003

J Pharmacol Exp Ther

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“…An intriguing finding differentiating S33138 from other antipsychotics was its interaction with h␣ 2C -ARs despite low affinities for their h␣ 2A and h␣ 2B counterparts. All h␣ 2 -AR subtypes couple via G i /G o to downstream pathways converging on ERK 1/2 (Hieble et al, 1995), and, by analogy to other ␣ 2 -AR antagonists, S33138 abolished activation of h␣ 2C -ARs in procedures of G-protein activation, recruitment of G␣ i3 and G␣ o , and ERK 1/2 phosphorylation (Alblas et al, 1993;Jasper et al, 1998;Audinot et al, 2002). Few ligands possessing a marked preference for ␣ 2C -versus ␣ 2A /␣ 2B -ARs have been described previously (Hieble et al, 1995), so S33138 may serve as a useful template for construction of chemically novel selective ␣ 2C -AR antagonists.…”

mentioning

confidence: 99%

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 D₃versus D₂Receptor Antagonist and Potential Antipsychotic Agent: I. Receptor-Binding Profile and Functional Actions at G-Protein-Coupled Receptors

Millan

Cour²,

Novi³

et al. 2007

J Pharmacol Exp Ther

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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-

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Ligand modulation of [35S]GTPγS binding at human α2A, α2B and α2C adrenoceptors

Cited by 27 publications

References 36 publications

Fluorescence Resonance Energy Transfer Analysis of α_2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes

Fluorescence Resonance Energy Transfer Analysis of α_2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes

Characterization of μ Opioid Receptor Binding and G Protein Coupling in Rat Hypothalamus, Spinal Cord, and Primary Afferent Neurons during Inflammatory Pain

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 D₃versus D₂Receptor Antagonist and Potential Antipsychotic Agent: I. Receptor-Binding Profile and Functional Actions at G-Protein-Coupled Receptors

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Ligand modulation of [35S]GTPγS binding at human α2A, α2B and α2C adrenoceptors

Cited by 27 publications

References 36 publications

Fluorescence Resonance Energy Transfer Analysis of α2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes

Fluorescence Resonance Energy Transfer Analysis of α2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes

Characterization of μ Opioid Receptor Binding and G Protein Coupling in Rat Hypothalamus, Spinal Cord, and Primary Afferent Neurons during Inflammatory Pain

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 D3versus D2Receptor Antagonist and Potential Antipsychotic Agent: I. Receptor-Binding Profile and Functional Actions at G-Protein-Coupled Receptors

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Fluorescence Resonance Energy Transfer Analysis of α_2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes

Fluorescence Resonance Energy Transfer Analysis of α_2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes

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 D₃versus D₂Receptor Antagonist and Potential Antipsychotic Agent: I. Receptor-Binding Profile and Functional Actions at G-Protein-Coupled Receptors