Functional and Pharmacological Significance of Receptor Heteromers in the CNS

National Institute On Drug Abuse

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Abstract

Activation of cannabinoid CB1 receptors (CB1Rs) by agonists induces analgesia but also induces cognitive impairment mediated by heteromers of CB1Rs and serotonin 5-HT2A receptors (5-HT2AR) localized in the cerebral cortex. This side effect poses a serious drawback in the therapeutic use of cannabis for pain alleviation. Synthetic peptides with the amino acid sequence of transmembrane domains (TMs) of the CB1R which were predicted to bind to the 5HT2AR and alter the stability of the CB1R-5HT2AR heteromer, were previously shown to counteract CB1R agonist-induced cognitive impairment while preserving analgesia. It would therefore be desirable to obtain non-peptidic small molecules that could be orally administered and could disrupt CB1R-5-HT2A receptor heteromers. Using those heteromer-disruptive TM peptides as templates, we have designed nonpeptidic small molecules that prevent CB1R-5HT2AR heteromerization in bimolecular fluorescence complementation assays and the heteromerization-dependent allosteric modulations in cell signaling experiments [1]. These results provide a significant validation of TM peptides as tools for the study of GPCR heteromeric interfaces and proof-of-principle for the design of optimized ligand-based disruptors of the CB1R-5HT2AR heteromer, opening new perspectives for in vivo studies. We previously suggested that heteromerization of Gs with Gi protein-coupled receptors is necessary to sustain the canonical Gi-Gs antagonistic interaction at adenylyl cyclase (AC). These heteromers have a tetrameric quaternary structure, composed by two homodimers each one coupled to its corresponding G protein. We no described a new GPCR heterotetramer formed by the Gi-coupled μ-opioid receptor (MOR) and the Gs-coupled corticotropin releasing factor CRF1 receptor (CRF1R), which also sustains a canonical interaction at AC and reciprocal allosteric interactions between MOR and CRF1R ligands [2]. In addition, we found that CRF1R can also couple to Gq proteins in the MOR-CRF1R heteromer, providing the frame for also canonical Gi-Gq antagonistic interactions that include other effectors, such as phospholipase C and its calcium-dependent signaling, and which control glutamate release in the central amygdala (CeA) [2]. We previously found that MORs forming heteromers with galanin 1 receptors (Gal1Rs) mediate the dopaminergic effects of opioids and that methadone poorly activates the MOR-Gal1R heteromer, which can be responsible for its low abuse liability as compared to other opioids. Methadone has two enantiomers, R- and S-methadone and we recently demonstrated that S-methadone is responsible for the MOR-Gal1R heteromer-dependent weak dopaminergic effects of methadone. Thus, S-methadone binds but specifically loses its efficacy for the MOR that forms heteromers with Gal1R, acting as a competitive MOR antagonist. We now found that S-methadone is an effective agonist at the MOR-CRF1R heteromer [2]. The specific pharmacodynamic properties of the MOR-CRF1R heteromer, including its sensitivity to S-methadone, as well as its localization in the CeA suggest it might represent a significant pharmacological target for the analgesic, antistressor and antidepressant effects of opioids and the hyperalgesia of opioid withdrawal. Several dopaminergic compounds, including the clinically used pramipexole, are labelled as “preferential dopamine D3 receptor (D3R) agonists” based on their moderately higher affinity for the D3R versus other D2-like receptor subtypes. In rodents, these compounds typically produce locomotor depression with low doses and locomotor activation with higher doses, which has been assumed to be mediated by presynaptic D3Rs and postsynaptic striatal D2Rs, respectively. However, studies with selective pharmacological and genetic blockade of each dopamine receptor subtype suggest opposite roles. We have addressed this apparent conundrum by performing a comprehensive in vitro, in vivo and ex vivo pharmacological comparison of several preferential D3R agonists. Their differential properties reveal that their locomotor activating effects in mice are dependent on the striatal postsynaptic D3Rs forming heteromers with D1Rs, via their ability to potentiate β-arrestin recruitment by the D1R in the D1R-D3R heteromer [3]. The results also indicate that the locomotor depressant effects are largely dependent on their ability to activate presynaptic D2Rs [3]. More broadly, it is demonstrated that locomotor activity in mice depends on β-arrestin recruitment by the D1R in the striatal D1R-D3R heteromer [3]. These results can have implications for the treatment of L-dopa-induced dyskinesia and Restless Legs Syndrome (RLS). Our detailed analysis demonstrated that just considering the affinity of different ligands to determine the predominant receptor subtype involved in their behavioral and clinical outcomes can be misleading, since such an approach ignores other pharmacokinetic and pharmacodynamic factors that can significantly influence the final in vivo functional response of the agonist. Our results favor that striatal D2SRs localized in dopaminergic terminals and D2SRs and D4Rs localized in corticostriatal terminals, and not striatal presynaptic or postsynaptic D3Rs, are the main target responsible for the initial therapeutic effect of “preferential D3R agonists” in RLS [3]. They also favor that higher doses are thus more likely to activate striatal postsynaptic D3Rs forming heteromers with D1Rs [3]. It is possible that these pharmacological effects can explain why pramipexole and other “preferential D3R agonists” have significant clinical benefits at low doses, but that particularly with increasing doses, will progressively worsen or augment the symptoms (aka “augmentation”). This represents a clinical challenge which is promoting the search for alternatives to dopamine receptor agonists in RLS. In addition, a previous and a recent clinical study demonstrated that the effect of some alternative drugs, including α2δ ligands (such as gabapentin), the inhibitor of adenosine transport dipyridamole and the orexin receptor antagonist suvorexant, are significantly reduced in RLS patients with a previous exposure to dopamine receptor agonists [4]. Restlessness is a core symptom of not only RLS, but also neuroleptic-induced akathisia, and opioid withdrawal. These three conditions also share other clinical components suggesting some overlap in their pathophysiology. Recent prospective studies demonstrate the frequent incidence of RLS-like symptoms during opioid withdrawal and supervised prescription opioid tapering. Based on the therapeutic role of MOR agonists in the three clinical conditions and recent preclinical experimental data in rodents, we have provided a coherent and unifying neurobiological basis for the restlessness observed in these three clinical syndromes and have proposed a heuristic hypothesis of a key role of the specific striatal neurons that express MORs in akathisia/restlessness, which preferentially express D1Rs. Our hypothesis is that akathisia/restlessness is secondary to a preferential activation of these MOR-D1R neurons which are localized in the striatal striosomal compartment. More specifically, in RLS, we hypothesize that brain iron deficiency, which is a well-established initial pathogenetic mechanism, determines an increased sensitivity of the striosomal MOR-D1R neurons [4]. These neurons also co-express D3R and adenosine A1 receptors (A1Rs), which together with presynaptic A1Rs localized in corticostriatal nerve terminals seem to be the main targets for our previously demonstrated therapeutic effect of dipyridamole in RLS. We have also proposed that different degrees of the same process, an increased sensitivity of the MOR-D1R neuron, can lead to RLS symptoms, diminished response to dopamine receptor agonists and the phenomenon of augmentation [4]. These hypotheses are now being tested in an animal model of RLS, the rodent with dietary-induced brain iron deficiency.