Homodimerization of adenosine A2A receptors: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer (original) (raw)
Related papers
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.
Adenosine A2A and Dopamine D2 Heteromeric Receptor Complexes and Their Function
Journal of Molecular Neuroscience, 2005
The existence of A 2A -D 2 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 A 2A and D 2 receptors also coimmunoprecipitate in striatal tissue, giving evidence for the existence of A 2A -D 2 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 A 2A and D 2 agonists. The A 2A -D 2 heteromers could either be A 2A -D 2 heterodimers and/or higher-order A 2A -D 2 hetero-oligomers. In striatal neurons there are probably A 2A -D 2 heteromeric complexes, together with A 2A -D 2 homomeric complexes in the neuronal surface membrane. Their stoichiometry in various microdomains will have a major role in determining A 2A and D 2 signaling in the striatopallidal GABA neurons. Through the use of D 2 /D 1 chimeras, evidence has been obtained that the fifth transmembrane (TM) domain and/or the I3 of the D 2 receptor are part of the A 2A -D 2 receptor interface, where electrostatic epitope-epitope interactions involving the N-terminal part of I3 of the D 2 receptor (arginine-rich epitope) play a major role, interacting with the carboxyl terminus of the A 2A receptor. Computerized modeling of A 2A -D 2 heteromers are in line with these findings. It seems likely that A 2A receptor-induced reduction of D 2 receptor recognition, G protein coupling, and signaling, as well as the existence of A 2A -D 2 co-trafficking, are the consequence of the existence of an A 2A -D 2 receptor heteromer. The relevance of A 2A -D 2 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 A 2A -D 3 heteromeric receptor complexes in cotransfected cell lines has been summarized.
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.
Adenosine A2A-Dopamine D2 Receptor-Receptor Heteromerization
Journal of Biological Chemistry, 2003
The abbreviations used are: HSMR, heptaspanning membrane receptor; GPCR, G protein-coupled receptor; YFP, yellow fluorescent protein; BRET, bioluminescence resonance energy transfer; FRET, fluorescence resonance energy transfer; E n , extracellular loop n; I n , intracellular loop n; Mes, 4-morpholineethanesulfonic acid; ANOVA, analysis of variance; Rluc, Renilla luciferase; EYFP enhanced yellow fluorescent protein; CHO, Chinese hamster ovary; GFP, green fluorescent protein; PDB, Protein Data Bank; GABA, ␥-aminobutyric acid; PBS, phosphate-buffered saline; CH, channel; A 2A R, adenosine A 2A receptor; D n R, dopamine D n receptor.
Journal of Biological Chemistry, 2003
There is evidence for strong functional antagonistic interactions between adenosine A2A receptors (A2ARs) and dopamine D2 receptors (D2Rs). Although a close physical interaction between both receptors has recently been shown using co-immunoprecipitation and co-localization assays, the existence of a A2AR-D2R protein-protein interaction still had to be demonstrated in intact living cells. In the present work, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques were used to confirm the occurrence of A2AR-D2R interactions in co-transfected cells. The degree of A2AR-D2R heteromerization, measured by BRET, did not vary after receptor activation with selective agonists, alone or in combination. BRET competition experiments were performed using a chimeric D2R-D1R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D2R were replaced by those of the dopamine D1 receptor (D1R). Although the wild type D2R 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 D2R holds the site(s) for interaction with A2AR. Modeling of A2AR and D2R using a modified rhodopsin template followed by molecular dynamics and docking simulations gave essentially two different possible modes of interaction between D2R and A2AR. In the most probable one, helix 5 and/or helix 6 and the N-terminal portion of I3 from D2R approached helix 4 and the C-terminal portion of the C-tail from the A2AR, respectively.
Trafficking of Adenosine A2A and Dopamine D2 Receptors
Journal of Molecular Neuroscience, 2005
An interaction between adenosine A 2A and dopamine D 2 receptors has been demonstrated previously. It is generally found that agonist treatment internalizes receptors, including A 2A and D 2 , whereas less is known of the long-term effects involved in the agonist-mediated trafficking of A 2A and D 2 receptors. Furthermore, the possible influence of the antagonists on receptor trafficking is still undefined. The present studies focus on the long-term effects of A 2A and D 2 agonist and D 2 antagonist treatments on both A 2A and D 2 receptor trafficking studied at three different time intervals-3, 15, and 24 h. In addition, with the fluorescence resonance energy transfer technique, formation of heteromeric A 2A and D 2 receptor complexes was shown in the cotransfected CHO cell line. Confocal microscopy analysis showed that a 3-h treatment with the D 2 agonist induced coaggregation of A 2A /D 2 receptors. These A 2A /D 2 receptor coaggregates internalized after 15 h with a recruitment of the receptors back to the cell membrane after 24 h. In contrast to the effects of the agonist treatment, a 3-h treatment with the D 2 -like antagonist raclopride increased both A 2A and D 2 immunoreactivity, indicating that the D 2 antagonist stabilizes the D 2 receptor and thereby reduces the internalization of both of the A 2A and D 2 receptors. Taken together, an activation of either A 2A and D 2 receptor or blockade of D 2 receptors will cause long-lasting changes in A 2A and D 2 receptor trafficking.
Journal of Biological Chemistry, 2002
Antagonistic and reciprocal interactions are known to exist between adenosine and dopamine receptors in the striatum. In the present study, double immunofluorescence experiments with confocal laser microscopy showed a high degree of colocalization of adenosine A 2A receptors (A 2A R) and dopamine D 2 receptors (D 2 R) in cell membranes of SH-SY5Y human neuroblastoma cells stably transfected with human D 2 R and in cultured striatal cells. A 2A R/D 2 R heteromeric complexes were demonstrated in coimmunoprecipitation experiments in membrane preparations from D 2 R-transfected SH-SY5Y cells and from mouse fibroblast Ltk ؊ cells stably transfected with human D 2 R (long form) and transiently cotransfected with the A 2A R double-tagged with hemagglutinin. Long term exposure to A 2A R and D 2 R agonists in D 2 R-cotransfected SH-SY5Y cells resulted in coaggregation, cointernalization and codesensitization of A 2A R and D 2 R. These results give a molecular basis for adenosine-dopamine antagonism at the membrane level and have implications for treatment of Parkinson's disease and schizophrenia, in which D 2 R are involved.
Evidence for the heterotetrameric structure of the adenosine A2A-dopamine D2 receptor complex
Biochemical Society Transactions, 2016
Heteromers of G-protein-coupled receptors (GPCRs) have emerged as potential novel targets for drug development. Accumulating evidence indicates that GPCRs can form homodimers and heteromers, with homodimers being the predominant species and oligomeric receptors being formed as multiples of dimers. Recently, heterotetrameric structures have been proposed for dopamine D1 receptor (D1R)–dopamine D3 receptor (D3R) and adenosine A2A receptor (A2AR)–dopamine D2 receptor (D2R) heteromers. The structural model proposed for these complexes is a heteromer constituted by two receptor homodimers. The existence of GPCR homodimers and heteromers provides a structural basis for inter-protomer allosteric mechanisms that might account for a multiplicity of unique pharmacological properties. In this review, we focus on the A2AR–D2R heterotetramer as an example of an oligomeric structure that is key in the modulation of striatal neuronal function. We also review the interfaces involved in this and oth...