br GPCRs form heterotetramers In addition to

GPCRs form heterotetramers In addition to homodimers and heterodimers, for several family A receptor heteromers, experimental evidence supports the presence of higher order rearrangements as tetrameric structures, comprised of two different homodimers, each able to signal with its preferred G protein, that can act as the conduit of allosteric interactions between orthosteric ligands (Ferré, 2015; Ferré et al., 2016). As an example, Guitart et al. (2014), by combining BiFC and luminescence complementation with BRET and complemented donor-acceptor resonance Tarafenacin in transfer (CODA-RET) assays, demonstrated the existence of a tetrameric D1R-D3R heteromer, comprised of D1R and D3R homodimers able to couple to Gs and Gi proteins, respectively. Moreover, these authors identified that the TM5–TM6 interface is essential for the stability of this structure and also demonstrated ligand-induced allosteric interactions, such as positive cross-talk and cross-antagonism, which constitute specific biochemical characteristics of the D1R-D3R heteromer. These allosteric modulations may have implications for the treatment of several neuropsychiatric disorders, because this heteromer is being considered as a target for drug development in l-DOPA-induced dyskinesia and drug addiction (Cortés, Moreno, et al., 2016; Ferré, Lluis, Lanciego, & Franco, 2010; Fiorentini, Busi, Spano, & Missale, 2010). Another example of tetrameric structures emerged from a study on A1R–A2AR heteromers in primary cultures of rat astrocytes. This complex was found to couple to both Gi/o and Gs proteins, resulting in cross-antagonism. The A1R agonist increased Gi/o activity, which was suppressed by an A2AR agonist. Reciprocally, Gs activation was restricted to A2AR and was abolished by the binding of an A1R antagonist, consisting with the existence of A1R–A2AR heteromers of two homodimers, thus enabling the binding of two distinct G proteins (Cristóvão-Ferreira et al., 2013). BRET assays and computer modeling to monitor receptor oligomerization revealed 2:2 receptor heteromers, suggesting a rhombus-shaped tetramer arrangement connected by a TM4–TM5 interface for homodimerization and a TM5–TM6 for heterodimerization (Navarro et al., 2016). In this heterotetramer are bound two different interacting G proteins, Gi and Gs, highlighting the function of the tetramer as a possible Gs/Gi mediated switching mechanism by which low and high concentrations of adenosine fine-tune glutamate release (Farran, 2017). In the case of the A2AR–D2R tetrameric complex, A2AR and D2R preferentially couple to Gs and Gi proteins, respectively. This heterotetramer has been detected in transfected cells and mammalian striatum, and has been found to be implicated in PD, schizophrenia and drug abuse (Ferré, 2015; Trifilieff et al., 2011). In this heteromeric complex, D2R agonists exert a negative allosteric modulation, decreasing the affinity and intrinsic efficacy of A2AR ligands (Trincavelli et al., 2012). Reciprocally, any orthosteric A2AR ligand, agonist or antagonist, including the well-known caffeine, exerts a negative allosteric modulation, decreasing the affinity and intrinsic efficacy of any orthosteric D2R ligand, agonist or antagonist (Azdad et al., 2009; Bonaventura et al., 2015). This could explain the behavioral depressant effects of adenosine analogues and the psychostimulant effects of caffeine on [11C] raclopride binding in human striatum (Casadó-Anguera et al., 2016; Ferré, 2016; Gaitonde & González-Maeso, 2017; Volkow et al., 2015). Altogether, different experimental results on A2AR–D2R heteromer can be better explained by the heterotetrameric model, a necessary frame for the canonical antagonistic Gs-Gi interaction (Ferré, 2015; Ferré et al., 2016). Very recently, in mammalian transfected cells, using synthetic peptides with amino acid sequences of all TM domains of A2AR and D2R and the putative TMs of AC5, we have provided clear evidence for the existence of functional pre-coupled complexes of A2AR and D2R homodimers, their cognate Golf and Gi proteins and AC5 (Navarro et al., 2018). We first identified a symmetrical TM6 interface for the A2AR and D2R homodimers and a symmetrical TM4–TM5 A2AR–D2R heteromeric interface. Likewise, we also found asymmetrical interfaces formed by TMs of the receptors and putative TMs of AC5 which rearrange upon agonist exposure. Computational analysis indicated the existence of a minimal functional complex formed by two A2AR–D2R heterotetramers and two AC5 molecules (Ferré, Bonaventura, et al., 2018). In fact, this quaternary structure suggests the possible formation of zig-zagged arranged higher-order oligomeric structures, a higher-order linear arrangement of GPCR heteromers and effectors (Navarro et al., 2018). Finally, we could demonstrate that this macromolecular complex allows the canonical Gs-Gi antagonistic interaction at the level of AC5, and acts as an integrative device of adenosine and dopamine neurotransmission (Ferré, Bonaventura, et al., 2018).