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doi: 10.1371/journal.pone.0027073. Epub 2011 Oct 31.

Differential modulation of Beta-adrenergic receptor signaling by trace amine-associated receptor 1 agonists

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Differential modulation of Beta-adrenergic receptor signaling by trace amine-associated receptor 1 agonists

Gunnar Kleinau et al. PLoS One.2011.

Abstract

Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Molecular structures of ADRB1/2 and TAAR1 ligands.
The trace amines TYR, PEA and OA are agonists of TAAR1. In contrast, TYR and PEA are antagonists on ADRB1/2, most likely due to less hydroxyl groups compared OA or already known agonistic beta-adrenergic ligands (grey circles). OA has an additional hydroxyl group (grey circle), but yet a hydroxyl group less (dashed grey circle) compared to full ADRB1/2 agonists isoprenaline, adrenaline and noradrenaline.
Figure 2
Figure 2. Functional characterization of hTAAR1 interacting with different trace amines.
HEK293 cells transiently transfected with hTAAR1 and were incubated for 45 minutes with 10 mM of PEA, OA and TYR. Basal signaling activity as well as activation of the Gs protein/adenylyl cyclase pathway was determined by AlphaScreen technology. Data represent mean ± SEM of cAMP accumulation after stimulation from 4–5 independent experiments performed in triplicates. TAAR1 showed an elevated ligand independent basal activity. PEA was the most potent agonist (p<0.01), followed by TYR (p<0.05) and OA (p<0.05). Data were analyzed using a paired one-tailed t-test.
Figure 3
Figure 3. Characterization of hADRB1 and hADRB2 after trace amine challenge.
HEK293 cells were transiently transfected with hADRB1 and hADRB2, respectively. Cells were pre-incubated with increasing concentrations of the trace amines TYR, PEA or OA (6.7 nM to 6700 nM) for 15 minutes. For competition studies ISOP with increasing concentrations (10000 nM to 1 nM) was added and incubated for additional 30 minutes. As controls hADRB1 and hADRB2 were incubated with each substance alone with the same concentration for 45 minutes. Three to four independent experiments measured in duplex or tripletts mean ± SEM are depicted here. Fold over mock was calculated and expressed as percentage of ISOP stimulation (100%). Maximal values were statistically analyzed using paired one-tail t-test compared to ISOP maximal stimulation.A-B) hADRB1 and hADRB2 were stimulated with ISOP and TYR alone and with both substances simultaneously. TYR shows no agonistic effect on hADRB1 (p<0.05) and hADRB2 (p<0.01), but acts as an allosteric antagonist on hADRB1/2, indicated by a decreased maximum of stimulation (p<0.05) with comparable EC50 values.C-D) PEA shows no agonistic effects on both hADRB1 (p<0.01) and hADRB2 (p<0.05) but leads to a decreased maximum of stimulation by ISOP when pre-incubated with PEA (p<0.05). The EC50 value is similar to stimulation with ISOP alone.E) OA acts as an agonist on hADRB1 but with a decreased efficacy. Simultaneous incubation with ISOP and OA reveals no antagonistic effect of OA on hADRB1 (table 1).F) OA is an orthosteric antagonist on hADRB2, indicated by a right shift of the EC50 value. OA however showed no agonistic effect on hADRB2 (p<0.05).
Figure 4
Figure 4. Defining the ligand binding region of aminergic receptors.
A) The pocket-like ligand binding region (inner crevice surface) of the human β2-adrenergic receptor (pdb entry code 2RH1) is surrounded by amino acids (lilac sticks, labeled) which are also known from mutagenesis studies to be important for ligand binding and signal transduction.B) The inverse agonist carazolol is embedded tightly in this pocket of the β2-adrenergic receptor (top-view) and interacts with residues of TMH 3, 5, and 7 by hydrogen bonds . Differences in binding and effects on receptor conformation compared to agonists were found to be relatively small, mainly manifested in the interaction pattern to TMH5 or induced side chain rotamer conformations at TMH5 (amino acids at positions 5.41, 5.42 and 5.46) .C) The crystal structure of dopamine-3 receptor (pdb entry code 3PBL) with the antagonist eticlopride shows a similar localization between the transmembrane helices compared to carazolol in the adrenergic receptor (B). An aspartate (red stick) at position 3.32 in helix 3 is well known to function as an anchor point for binding of ligands at aminergic receptors.
Figure 5
Figure 5. Amino acids covering the ligand binding region of β-adrenergic receptors and TAARs.
Amino acids covering the ligand binding regions of β-adrenergic receptors 1 and 2 are compared with corresponding residues of human TAAR subtypes, dopamine-3 receptor and invertebrate tyramine (Apis) (TAR) or octopamine receptor (Bombyx) (OAR) (see also amino acid sequence alignment figure S2). This comparison reveals potential overlapping binding determinants which are predestinated to interact with shared ligands. The amino acids of ADRB1 and ADRB2 ligand binding region are identified by analyzing solved crystal structures complexed with different ligands (figure 4). Residues which are directly involved in ligand binding at adrenergic receptors are marked by a star-symbol (*). The numbering is provided by theBallesteros and Weinstein numbering scheme and consecutively to the entire amino acid sequence. Especially TAAR1 shows similar or even identical side chains (bold) with the adrenergic receptors, dopamine-3 receptor and OAR or TAR. Highly conserved amino acids between all receptors are marked by a gray background. Conserved residues within hTAAR subtypes are in italic with a partial gray background. These five residues are located at TMH 2, 3, 6 and 7 and likely encode TAAR specificities compared to other aminergic receptors.
Figure 6
Figure 6. TAAR1 shows similarity in the ligand binding region compared with β-adrenergic receptors.
A) Superimposition of the hTAAR1 homology model (extracellular top-side view, green backbone) and the hADRB2 crystal structure (lilac sticks hADRB2, backbone not shown) reveals similarities of residues which are known to be important for ligand binding and signal transduction in adrenergic receptors (figure 4 and figure 5). These identical amino acids (sticks) should be involved in binding of shared ligands like isoprenaline. Additional side chains of hTAAR1 covering the putative ligand binding region (figure 5) are represented as green lines. Cysteine bridges (yellow) between loops 1 and 2 or loop 2 and TMH3 are highlighted and labeled.B) The recently published crystal structure of the turkey β1-adrenergic receptor co-crystallized with the agonist isoprenaline (pdb entry code 2Y03 [80]) shows main key players for intermolecular hydrogen bonding (yellow dotted lines) at aminergic receptors like side chains at positions D3.32 (TMH3) (red stick); S5.42, S5.46 (TMH5); and N7.39 (TMH7) (lilac sticks). Interestingly, the inner-pocket surface (translucent) between the extracellular ends of the helices and ECL2 shows an unoccupied volume which might form a second binding site for small molecules.
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