Functional and Pharmacological Significance of Receptor Heteromers in the CNS

National Institute On Drug Abuse

Investigators

Linked publications, trials & patents

Abstract

We focused our studies on our previously reported striatal adenosine and dopamine receptor heteromers and established new proofs of concept about the properties of G protein-coupled receptor (GPCR) heteromers. Those included the adenosine A2A receptor (A2AR) heteromers and the dopamine D4 receptor (D4R) heteromers localized presynaptically in corticostriatal glutamatergic terminals, and the postsynaptic A2AR-dopamine D2 receptor (D2R) and D1R-D3R heteromers. Presynaptic A2AR heteromers include the A1R-A2AR and the A2AR-cannabinoid CB1 receptor (CB1R) heteromers. We previously demonstrated that one of their different properties is the ligand-independent ability of the A1R, but not the CB1R, to dampen the strong constitutive activity of the A2AR (1). Nevertheless, activation of the CB1R in the A2AR-CB1R heteromers does counteract the constitutive activity of the A2AR, which we previously found as the main mechanism by which cannabinoids inhibit glutamate release at the corticostriatal terminal (1,2). Our view of the adenosinergic control of presynaptic corticostriatal transmission is as follows: first, under conditions of low basal adenosine tone, adenosine mostly activates A1R (which has more affinity for adenosine than A2AR), which promotes inhibition of glutamate release; second, under conditions of high concentration of synaptic adenosine, which occurs upon strong activation of the corticostriatal neuron (which releases synaptic ATP that rapidly converts into synaptic adenosine), the A2AR is also activated, which allosterically dampens A1R and CB1R signaling in the corresponding heteromers (1,2). This view allows to resolve the conundrum of the ability of systemically administered THC to promote striatal glutamate and secondarily dopamine release (through the local microcircuit that involves the corticostriatal terminal, the cholinergic interneuron, and the dopaminergic terminal (1,2)). THC promotes a significant astrocytic-dependent activation of the corticostriatal neuron, which leads to a significant synaptic formation of adenosine and activation of A2AR, which counteracts the effect of THC at the A2AR-CB1R heteromer, altogether facilitating striatal glutamate release (2). We have postulated that THC-induced activation of the corticostriatal neuron is the main mechanism responsible for its dopamine-releasing and rewarding effects (2). In agreement, presynaptic A2AR antagonists counteract THC-induced glutamate and dopamine release in rats and THC-self-administration in squirrel monkeys (2). From our integrative analysis of the role of corticostriatal adenosinergic transmission, it becomes of importance to underscore the significant role of the constitutive activity of A2AR, which is present in the A2AR-CB1R heteromer and absent in the A1R-A2AR heteromer, in providing a basal sensitivity of the corticostriatal terminal. This allowed to explain the ability of A2AR inverse agonists (like SCH-442416) and inability of A2AR neutral antagonists (like KW-6002 and caffeine) to counteract stimulated glutamate release by corticostriatal terminals (1,2). It also allowed to explain changes in the sensitivity of the terminal upon stoichiometric changes in the A1R/A2AR expression ratio in favor of A2AR, with implications for the pathogenesis of Restless Legs Syndrome (see previous Annual Report). Finally, it also allowed to provide a neuropathological explanation for the possible role of the G2797.44S mutation of A1R in the early onset Parkinsons disease. Thus, we could demonstrate that this single mutation in TM7 of the A1R leads to a significant disruption of A1R-A2AR heteromers, with the concomitant released constitutive activity of the A2AR (3,4). When considering the stoichiometry of GPCR forming heteromers, we should also consider their stability, the dynamics of the association and dissociation of their protomers. We have provided a proof of concept for the ability of ligands to differentially alter this stability. We examined the effect of exposure to three prototypical antipsychotic drugs on A2AR-D2R heteromerization in mammalian cells using a NanoBiT assay (5). After 16 h of exposure, a significant increase in the density of A2AR-D2R heteromers was found with haloperidol and aripiprazole, but not with clozapine. On the other hand, clozapine, but not haloperidol or aripiprazole, was associated with a significant decrease in A2AR-D2R heteromerization after 2 h of treatment. Computational binding models of these compounds revealed distinctive molecular signatures that explain their different influence on heteromerization (5). We proposed that an increase in A2AR-D2R heteromerization is involved in the extrapyramidal side effects of antipsychotics, while the specific clozapine-mediated destabilization of A2AR-D2R heteromerization can explain its low liability to induce those side effects (5). The functional role of D4R and its main polymorphic variants has become evident with the demonstration of heteromers of D4R that control the function of frontal corticostriatal neurons (6). Those include heteromers with the alpha2A adrenoceptor (a2AR) and with the D2R, localized in their cortical somato-dendritic region and striatal nerve terminals, respectively (6). By using biophysical and cell-signaling methods and heteromer-disrupting peptides in mammalian transfected cells and rat brain slice preparations, we provided evidence for a new functionally relevant D4R heteromer, the a1AR-D4R heteromer, which is also preferentially localized in the corticostriatal glutamatergic terminals (7). Significant differences in allosteric modulations between heteromers of a1AR with the D4.4R and D4.7R polymorphic variants could be evidenced with the analysis of G protein-dependent and independent signaling (7). We proposed that the D4.4R variant provides a gain of function of the a1AR-mediated noradrenergic stimulatory control of corticostriatal glutamatergic neurotransmission, which could result in a decrease in the vulnerability for impulse control-related neuropsychiatric disorders and increase in the vulnerability for posttraumatic stress disorder (7). A main rationale for the role of GPCR heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. We provided a proof of concept for this rationale by demonstrating that heteromerization of D1R and D3R influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4116 (8). By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist, cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4116 specifically acted as a beta-arrestin-biased agonist in the D1R-D3R heteromer (8). Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats (8), highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinsons disease.