Adenosine A2A-Dopamine D2 Receptor-Receptor Heteromerization: QUALITATIVE AND QUANTITATIVE ASSESSMENT BY FLUORESCENCE AND BIOLUMINESCENCE ENERGY TRANSFER (original) (raw)
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Molecular determinants of A2AR-D2R allosterism: role of the intracellular loop 3 of the D2R
Journal of neurochemistry, 2012
In the CNS, an antagonistic interaction has been shown between adenosine A 2A and dopamine D 2 receptors (A 2A Rs and D 2 Rs) that may be relevant both in normal and pathological conditions (i.e., Parkinson's disease). Thus, the molecular determinants mediating this receptor-receptor interaction have recently been explored, as the fine tuning of this target (namely the A 2A R/D 2 R oligomer) could possibly improve the treatment of certain CNS diseases. Here, we used a fluorescence resonance energy transfer-based approach to examine the allosteric modulation of the D 2 R within the A 2A R/D 2 R oligomer and the dependence of this receptor-receptor interaction on two regions rich in positive charges on intracellular loop 3 of the D 2 R. Interestingly, we observed a negative allosteric effect of the D 2 R agonist quinpirole on A 2A R ligand binding and activation. However, these allosteric effects were abolished upon mutation of specific arginine residues (217-222 and 267-269) on intracellular loop 3 of the D 2 R, thus demonstrating a major role of these positively charged residues in mediating the observed receptor-receptor interaction. Overall, these results provide structural insights to better understand the functioning of the A 2A R/D 2 R oligomer in living cells. resonance energy transfer; HEK, Human embryonic kidney; IL3, third intracellular loop 3; RIPA, radio immunoprecipitation assay .
Analytical Chemistry, 2004
Previous results from FRET and BRET experiments and computational analysis (docking simulations) have suggested that a portion of the third intracellular loop (I3) of the human dopamine D 2 receptor (D 2 R) and the C-tail from the human adenosine A 2A receptor (A 2A R) are involved in A 2A R-D 2 R heteromerization. The results of the present studies, using pull-down and mass spectrometry experiments, suggest that A 2A R-D 2 R heteromerization depends on an electrostatic interaction between an Argrich epitope from the I3 of the D 2 R (217 RRRRKR 222) and two adjacent Asp residues (DD 401-402) or a phosphorylated Ser (S 374) residue in the C-tail of the A 2A R. A GSTfusion protein containing the C-terminal domain of the A 2A R (GST-A2A CT) was able to pull down the whole D 2 R solubilized from D 2 R-tranfected HEK-293 cells. Second, a peptide corresponding to the Arg-rich I3 region of the D 2 R (215 VLRRRRKRVN 224) and bound to Sepharose was able to pull down both GST-A2A CT and the whole A 2A R solubilized from A 2A R-tranfected HEK-293 cells. Finally, mass spectometry and pull-down data showed that the Arg-rich D 2 R epitope binds to two different epitopes from the C-terminal part of the A 2A R, containing the two adjacent Asp residues or the phosphorylated Ser residue (388 HELKGVCPEPPGLDDPLAQDGAVGS 412 and 370 SAQ-EpSQGNT 378). The present results are the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization, a new, expanding area of protein-protein interactions.
Neurochemistry International, 2013
The molecular interaction between adenosine A 2A and dopamine D 2 receptors (A 2A Rs and D 2 Rs, respectively) within an oligomeric complex has been postulated to play a pivotal role in the adenosine-dopamine interplay in the central nervous system, in both normal and pathological conditions (e.g. Parkinson's disease). While the effects of A 2A R challenge on D 2 R functioning have been largely studied, the reverse condition is still unexplored, a fact that might have impact in therapeutics. Here, we aimed to examine in a real-time mode the D 2 R-mediated allosteric modulation of A 2A R binding when an A 2A R/D 2 R oligomer is established. Thus, we synthesized fluorescent A 2A R agonists and evaluated, by means of a flow cytometry homogeneous no-wash assay and a real-time fluorescence resonance energy transfer (FRET)-based approach, the effects on A 2A R binding of distinct antiparkinsonian drugs in current clinical use (i.e. pramipexole, rotigotine and apomorphine). Our results provided evidence for the existence of a differential D 2 R-mediated negative allosteric modulation on A 2A R agonist binding that was oligomerformation dependent, and with apomorphine being the best antiparkinsonian drug attenuating A 2A R agonist binding. Overall, the here-developed methods were found valid to prospect the ability of drugs acting on D 2 Rs to modulate A 2A R binding, thus featuring as possible helpful tools for the preliminary selection of D 2 R-like candidate drugs in the management of Parkinson's disease.
Journal of Neurochemistry, 2003
The results presented in this paper show that adenosine A2A receptor (A2AR) form homodimers and that homodimers but not monomers are the functional species at the cell surface. Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques have been used to demonstrate in transfected HEK293 cells homodimerization of A2AR, which are heptaspanning membrane receptors with enriched expression in striatum. The existence of homodimers at the cell surface was demonstrated by time-resolved FRET. Although agonist activation of the receptor leads to the formation of receptor clusters, it did not affect the degree of A2AR–A2AR dimerization. Both monomers and dimers were detected by immunoblotting in cell extracts. However, cell surface biotinylation of proteins has made evident that more than 90% of the cell surface receptor is in its dimeric form. Thus, it seems that homodimers are the functional form of the receptor present on the plasma membrane. A deletion mutant version of the A2A receptor, lacking its C-terminal domain, was also able to form both monomeric and dimeric species when cell extracts from transfected cells were analyzed by immunoblotting. This suggests that the C-terminal tail does not participate in the dimerization. This is relevant as the C-terminal tail of A2AR is involved in heteromers formed by A2AR and dopamine D2 receptors. BRET ratios corresponding to A2AR–A2AR homodimers were higher than those encountered for heterodimers formed by A2AR and dopamine D2 receptors. As A2AR and dopamine D2 receptors do indeed interact, these results indicate that A2AR homodimers are the functional species at the cell surface and that they coexist with A2AR/D2 receptor heterodimers.
Journal of …, 2003
The results presented in this paper show that adenosine A 2A receptor (A 2A R) form homodimers and that homodimers but not monomers are the functional species at the cell surface. Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques have been used to demonstrate in transfected HEK293 cells homodimerization of A 2A R, which are heptaspanning membrane receptors with enriched expression in striatum. The existence of homodimers at the cell surface was demonstrated by time-resolved FRET. Although agonist activation of the receptor leads to the formation of receptor clusters, it did not affect the degree of A 2A R-A 2A R dimerization. Both monomers and dimers were detected by immunoblotting in cell extracts. However, cell surface biotinylation of proteins has made evident that more than 90% of the cell surface receptor is in its dimeric form. Thus, it seems that homodimers are the functional form of the receptor present on the plasma membrane.
Journal of medicinal …, 2006
Molecular modeling methods have been applied to construct three-dimensional models for dopaminergic ligand complexes with D2 and D4 receptor subtypes (D2DAR and D4DAR), using the bovine rhodopsin crystal structure as a template for the modeling study. Different dopaminergic ligands, in particular the N-n-propyl-substituted 3-aryl-and 3-cyclohexylpiperidines, were docked into the D2DAR and the D4DAR, to evaluate the agreement between theoretical and experimental results as regards their D2/D4 selectivity. The different position of an aromatic region in the two receptors might explain the structural basis of this biological property.
Adenosine A 2A and dopamine D 2 heteromeric receptor complexes and their function
Journal of Molecular Neuroscience, 2005
The existence of A2A-D2 heteromeric complexes is based on coimmunoprecipitation studies and on fluorescence resonance energy transfer and bioluminescence resonance energy transfer analyses. It has now become possible to show that A2A and D2 receptors also coimmunoprecipitate in striatal tissue, giving evidence for the existence of A2A-D2 heteromeric receptor complexes also in rat striatal tissue. The analysis gives evidence that these heteromers are constitutive, as they are observed in the absence of A2A and D2 agonists. The A2A-D2 heteromers could either be A2A-D2 heterodimers and/or higher-order A2A-D2 hetero-oligomers. In striatal neurons there are probably A2A-D2 heteromeric complexes, together with A2A-D2 homomeric complexes in the neuronal surface membrane. Their stoichiometry in various microdomains will have a major role in determining A2A and D2 signaling in the striatopallidal GABA neurons. Through the use of D2/D1 chimeras, evidence has been obtained that the fifth transmembrane (TM) domain and/or the 13 of the D2 receptor are part of the A2A-D2 receptor interface, where electrostatic epitope-epitope interactions involving the N-terminal part of 13 of the D2 receptor (arginine-rich epitope) play a major role, interacting with the carboxyl terminus of the A2A receptor. Computerized modeling of A2A-D2 heteromers are in line with these findings. It seems likely that A2A receptor-induced reduction of D2 receptor recognition, G protein coupling, and signaling, as well as the existence of A2A-D2 co-trafficking, are the consequence of the existence of an A2A-D2 receptor heteromer. The relevance of A2A-D2 heteromeric receptor complexes for Parkinson’s disease and schizophrenia is emphasized as well as for the treatment of these diseases. Finally, recent evidence for the existence of antagonistic A2A-D3 heteromeric receptor complexes in cotransfected cell lines has been summarized.
Adenosine A 2A-dopamine D 2 receptor–receptor heteromers. Targets for neuro-psychiatric disorders
Parkinsonism & Related Disorders, 2004
BRET competition experiments were performed using a chimeric D 2 R-D 1 R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D 2 R were replaced by those of the dopamine D 1 receptor (D 1 R). Although the wild type D 2 R was able to decrease the BRET signal, the chimera failed to achieve any effect. This suggests that the helix 5-I3-helix 6-E3 portion of D 2 R holds the site(s) for interaction with A 2A R.
Biochemical and biophysical research communications, 2010
Evidence exists that the adenosine receptor A2AR and the dopamine receptor D2R form constitutive heteromers in living cells. Mass spectrometry and pull-down data showed that an arginine-rich domain of the D2R third intracellular loop binds via electrostatic interactions to a specific motif of the A2AR C-terminal tail. It has been indicated that the phosphorylated serine 374 might represent an important residue in this motif. In the present study, it was found that a point mutation of serine 374 to alanine reduced the A2AR ability to interact with D2R. Also, this point mutation abolished the A2AR-mediated inhibition of both the D2R high affinity agonist binding and signaling. These results point to a key role of serine 374 in the A2AR–D2R interface. All together these results indicate that by targeting A2AR serine 374 it will be possible to allosterically modulate A2AR–D2R function, thus representing a new approach for therapeutically modulate D2R function.