Functional and Pharmacological Significance of Receptor Heteromers in the CNS

National Institute On Drug Abuse

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Abstract

Experimental data accumulated during the last 10 years strongly support the existence and functional significance of GPCR oligomerization. We study the functional and pharmacological properties of G protein-coupled receptor (GPCR) heteromers localized in brain circuits that are dysfunctional in substance use disorders (SUD) and other neuropsychiatric disorders. During the last year we have reported new results on GPCR heteromers with translational significance for attention deficit hyperactivity disorder (ADHD), restless legs syndrome (RLS) and schizophrenia. The human dopamine D4 receptor gene (DRD4) displays a high number of polymorphisms in its coding sequence. The most extensive polymorphism is found in exon 3, which is a region that encodes the third intracellular loop of the receptor. This polymorphism consists of a variable number of tandem repeats of a 48-base pair sequence, with 211 repeats. The two most common polymorphisms contain 4 and 7 repeats, and they encode a dopamine D4 receptor (D4R) with 4 and 7 repeats of a proline-rich sequence of 16 amino acids (D4.4R and D4.7R). DRD4 polymorphisms have been associated with individual differences in personality traits and neuropsychiatric disorders, particularly between the gene encoding D4.7R and attention deficit hyperactivity disorder (ADHD) or substance use disorder (SUD). In fact, there is a clear association between D4.7R and the impulsivity traits action impulsivity and choice impulsivity, which are considered endophenotypes of ADHD and SUD. The alpha2A adrenoceptor (alpha2AR) gene has also been associated with impulsivity and ADHD. In fact, drugs targeting the alpha2AR, such as guanfacine, are commonly used in ADHD treatment. In view of the involvement of both D4R and alpha2AR in impulsivity and ADHD, their concurrent localization in cortical pyramidal neurons and the demonstrated ability of D4R to form functional heteromers with other G protein-coupled receptors, we investigated whether D4R could form functional heteromers with alpha2AR and if those heteromers would show different properties depending on the D4R variant (1). Using cortical brain slices from knock-in mice expressing a humanized D4.7R (hD4.7R mice) and from wild-type mice, we demonstrated that alpha2AR and D4R heteromerize and constitute a significant functional population of cortical alpha2AR and D4R (1). Moreover, in cortical slices from wild-type mice and in cells transfected with alpha2AR and D4.4R, we detected a negative crosstalk in the heteromer. This negative crosstalk was lost in the cortex from hD4.7R knock-in mice and in cells expressing the D4.7R polymorphic variant. In addition, only in the alpha2AR-D4.7R heteromer D4R ligands lost their efficacy at promoting G protein activation and signaling (1). Taken together, our results suggest that alpha2AR-D4R heteromers play a pivotal role in catecholaminergic signaling in the brain cortex and are likely targets for ADHD pharmacotherapy. The term akathisia is used to define a feeling of restlessness, an urgent need to move objectively perceived as psychomotor agitation, an inability to stay still. Akathisia is produced by a strong activation of the dopaminergic system, with psychostimulants, but it can also be associated with the treatment with dopamine receptor blocking agents (DRBAs) and represents a key symptom RLS. We have recently proposed a heuristic hypothesis about the mechanisms involved in akathisia, the apparent paradoxical association of akathisia with DRBAs, and the apparent paradoxical improvement of akathisia in RLS with dopamine receptor agonists (2). We proposed that akathisia depends on the alteration of specific presynaptic and postsynaptic mechanisms in the ventral striatum. Presynaptically, there is an increase in dopamine neurotransmission secondary to autoreceptor inactivation (with DRBA) or to a brain iron deficiency (BID)induced increase in glutamate release (in RLS). Postsynaptically, there is an increased activation of dopamine D1 receptors (D1R), which we previously demonstrated they form heteromers with dopamine D3 receptors (D3R) and with adenosine A1 receptors (A1R) (2). The rodent models of RLS are under revision. We have recently contributed to a report by the International Restless Legs Syndrome Study Group (IRLSSG) that provides consensus guidelines on rodent models of RLS (3). BID is a well-established initial pathogenetic mechanism in RLS, and the rodent with BID constitutes a valuable animal model of RLS. With this model we previously obtained evidence for a role of adenosine in RLS, a hypoadenosinergic state, with a decrease in the activation of presynaptic A1R that form heteromers with adenosine A2A receptors (A2AR), promoting an increase in glutamate release. We therefore suggested that an inhibitor of the adenosine transporter ENT1, such as dipyridamole, could ameliorate the clinical symptoms in RLS. In fact, this could be demonstrated in a randomized, placebo-controlled crossover clinical study (4), providing a significant new therapeutic approach for RLS. According to the adenosine hypothesis of schizophrenia, the classically associated hyperdopaminergic state may also be secondary to a loss of function of the adenosinergic system. Such a hypoadenosinergic state might either be due to a reduction of the extracellular levels of adenosine or to alterations in the density of postsynaptic A2AR or their degree of functional heteromerization with dopamine D2 receptors (D2R). In a recent study we provided preclinical and clinical evidence for this latter mechanism. Two animal models of schizophrenia endophenotypes, namely the phencyclidine (PCP) mouse model and the A2AR knockout mice, were used to establish correlations between behavioral and molecular studies (5). In addition, a new AlphaLISA-based method was implemented to detect native A2AR-D2R heteromers in mouse and human brains. First, we observed a reduction of pre-pulse inhibition in A2AR knockout mice, as previously observed in the PCP animal model of sensory gating impairment of schizophrenia, as well as a significant upregulation of striatal D2R without changes in A2AR expression in PCP-treated animals (5). In addition, PCP-treated animals showed a significant reduction of striatal A2AR-D2R heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of A2AR-D2R heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both D2R and A2AR were found to be upregulated (5). Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal A2AR-D2R heteromers (5). An alteration of the degree of A2AR-D2R heteromer formation might constitute a hallmark of schizophrenia, which indeed should be further studied to establish possible correlations with chronic antipsychotic treatments.