Functional and Pharmacological Significance of Receptor Heteromers in the CNS

National Institute On Drug Abuse

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Abstract

We have recently reviewed the evidence that supports the existence of GPCR-effector macromolecular membrane assemblies (GEMMAs) comprised of specific GPCRs, G proteins, plasma membrane effector molecules and other associated transmembrane proteins that are pre-assembled prior to receptor activation by agonists, which then leads to subsequent rearrangement of the GEMMA components (1). The GEMMA concept offers an alternative and complementary model to the canonical collision-coupling model, allowing more efficient interactions between specific signaling components, as well as the integration of the concept of GPCR oligomerization (GPCR homomers and heteromers), and GPCR interactions with orphan receptors, truncated GPCRs and other membrane-localized GPCR-associated proteins (1). GPCR heteromers are often constituted by heterotetramers, with two different homodimers coupled to their preferred G protein. A common heterotetramer is constituted by a homodimer coupled to a Gs and the other coupled to a Gi protein, providing the framework for the canonical Gs-Gi antagonistic interaction at the adenyl cyclase (AC) level, by which the activation of a Gi-coupled receptor inhibits the activation of AC by a Gs-coupled receptor (1). This includes the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heterotetramer, where A2AR is the Gs-coupled and D2R is the Gi-coupled receptor. The A2AR-D2R heteromer is localized in the striatum, in the striato-pallidal neurons, and is one of the few GPCR heteromers that fulfills the established criteria for its existence in native tissue, in the brain. We have recently used the A2AR-D2R heteromer as an example to illustrate the implementation of the techniques that have mostly contributed to reveal GPCR oligomers in native tissue, which include immunogold electron microscopy, proximity ligation assay, resonance energy transfer between fluorescent ligands and the amplified luminescent proximity homogeneous assay (2). The A2AR-D2R heteromer constitutes a target for the treatment of Parkinsons disease, where it mediates the efficacy of A2AR antagonists, by potentiating the effect of L-DOPA and selective D2R agonists. This depends on allosteric interactions between the A2AR and D2R ligands simultaneously binding in their corresponding orthosteric sites in the A2AR-D2R heteromer. Heterobivalent drugs that simultaneously bind to A2AR and D2R orthosteric sites in the A2AR-D2R heteromer could then provide tools to probe the A2AR-D2R heteromer in situ and, possibly, to develop new effective therapeutic antiparkinsonian agents. We have designed and synthesized heterobivalent ligands for the A2AR-D2R heteromer with various spacer lengths (3). The indispensable simultaneous binding of these ligands to the two different orthosteric sites of the heteromer was evaluated by radioligand competition-binding assays in the absence and presence of specific peptides that disrupt the heteromer, label-free dynamic mass redistribution assays in living cells, and molecular dynamic simulations. We identified compound 26, with a spacer length of 43-atoms, as a true bivalent ligand that simultaneously binds to the two different orthosteric sites (3). With bioluminescence resonance energy transfer experiments, it could also be demonstrated that compound 26 favors the stabilization of the A2ARD2R heteromer (3) We previously demonstrated that the A2AR-D2R heterotetramer forms part of a GEMMA that includes AC (AC5 isoform), which depends on molecular interactions between transmembrane domains of AC5 and A2AR and D2R. We aldo demonstrated that the canonical Gs-Gi antagonistic interaction of A2AR and D2R ligands at AC5 level was dependent on the integrity of the A2AR-D2R heteotetramer-AC5 GEMMA. We then proposed a classification of allosteric interactions in receptor heteromers in types I, II and III (1). Type I corresponds to the interactions between orthosteric ligands of the two different GPCRs, by which the ligand of one GPCR changes the properties (affinity or efficacy) of the ligand for the other GPCR, such as the interactions between A2AR and D2R ligands in the A2AR-D2R heterotetramer. Type II corresponds to a ligand-independent interaction, where one of the GPCRs, without ligands, changes the properties of a ligand of the other GPCR. And type III, corresponds to an allosteric interaction through the GEMMA effector, such as the canonical Gs-Gi antagonistic interaction at the AC level. We also found that GPCR oligomerization can significantly modify G protein subtype coupling, when studying two GPCR heteromers localized in the ventral tegmental area, where they modulate dopaminergic neuronal function: the mu-opioid receptor (MOR)-galanin Gal1 receptor (Gal1R) heterotetramer (4) and the oligomeric complex formed by dopamine D1 receptor (D1R), ghrelin GHS-R1a and its non-functional truncated isoform GHS-R1b (5). Evaluating the role of specific heteromer-disrupting peptides with several in vitro techniques, including total internal reflection fluorescence microscopy, we found that MOR and Gal1R predominate as dimers, either in isolation or when forming MOR-Gal1R heteromers (4). However, heteromerization was associated with a change in the homomeric interfaces, which changed G protein coupling of Ga1R from Gi to Gs. Galanin and exogenous Gal1R agonists promoted activation of AC (cAMP formation) and the MOR-Gal1R heterotetramer dispayed a type III allosteric interaction at the AC level (4). In the D1R-GHS-R1a-GHS-R1b oligomeric complex, GHS-R1a, which canonically couples to Gq, couples to Gs and promotes AC activation (5). In this study we could also demonstrate that D1R does not directly heteromerize with GHS-R1a, but through GHS-R1b, establishing a new function for truncated GPCR isoforms, the linking of molecularly different GPCRs (5). Akathisia is used to define an urgent need to move. The primary component of akathisia is a sensory experience which acts as a drive or motivational state that compels the subject to move. Akathisia is the main symptom of the very prevalent Restless Legs Syndrome (RLS), where, more often, the sensory experience is an urgent need of specifically moving the legs. Brain iron deficiency (BID), not necessarily associated with peripheral iron deficiency, is a main initial pathogenetic mechanism in RLS. The rodent with BID constitutes a well-established animal model of RLS with both face and construct validity, as we recently assessed by consensus by a task force of the International RLS Study Group (IRLSSG) (6). As recently reviewed (7), we previously found that in the rodent with BID there is a specific increase in the sensitivity of cortico-striatal glutamatergic terminals to release glutamate. We also found that BID is associated with downregulation of adenosine A1 receptors (A1Rs) and upregulation of A2ARs in the rodent brain. In previous studies we demonstrated the existence of functionally significant adenosine A1R-A2AR heteromers in cortico-striatal terminals which modulate glutamate release and the ability of A1R to inhibit the strong constitutive activity of A2AR upon heteromerization. We therefore hypothesized that the BID-induced increased sensitivity of cortico-striatal terminals could be related to an increased constitutive activity of A2Rs, secondary to a change in the proportion of adenosine A1 and A2A receptors (A1R and A2AR) localized in those terminals in favor of A2AR (7). This led to the proposal of using adenosinergic drugs in RLS, which we could recently support with a randomized, placebo-controlled crossover study with the adenosine transport inhibitor dipyridamole (see previous annual report). By using a FACS-based synaptometric analysis we could now confirm our hypothesis and BID in the rat caused a significant decrease in the A1R/A2AR ratio in cortico-striatal terminals (8).