Genetic variants in dopamine receptors influence on heterodimerization in the context of antipsychotic drug action

CHAPTER NINE

Genetic variants in dopamine receptors influence on heterodimerization in the context of antipsychotic drug action Agata Faron-Góreckaa,∗, Maciej Ku
smidera, Joanna Solicha, Andrzej Góreckib, Marta Dziedzicka-Wasylewskaa,b a Department of Pharmacology, Maj Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland b Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krako´w, Poland ∗ Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Effect of SNPs on GPCRs dimerization References Further reading

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Abstract Human dopamine D2 receptor (D2R) gene has polymorphic variants, three of them alter its amino acid sequence: Val96Ala, Pro310Ser and Ser311Cys. Their functional role never became the object of extensive studies, even though there are some evidence that they correlate with schizophrenia. The present work reviews data indicating that these mutations play a role in dimer formation with dopamine D1 receptor (D1R), with the strongest effect observed for Ser311Cys variant. Similarly, the affinity for antipsychotic drugs of this genetic variant depends on whether it is expressed together with D1R or not. Better understanding of altered ability of genetic variants of D2R to form dimers with D1R, as well as of altered affinity for antipsychotic drugs, depending on the absence or presence of the second dopamine receptor is of great importance—since these two receptors are not always co-expressed in the same cell. It may well be that targeting new compounds toward the D1R-D2R dimers, which the most probably form under conditions of excessive dopamine release, will result in antipsychotic drugs devoid of serious side effects.

Progress in Molecular Biology and Translational Science, Volume 169 ISSN 1877-1173 https://doi.org/10.1016/bs.pmbts.2019.11.008

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2020 Elsevier Inc. All rights reserved.

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1. Introduction The classical dopamine hypothesis of schizophrenia proposed that hyperactivity of dopamine transmission is responsible for the positive symptoms (hallucination, delusions) observed in this disorder. This hypothesis was supported by the strong correlation between the clinical effectiveness and the ability to block dopamine D2 (D2R) receptors.1–3 It has also been demonstrated that deficit in dopamine transmission in the prefrontal cortex and lack of stimulation of D1 (D1R) receptors induces cognitive impairments reminiscent of those observed in schizophrenia patients.4 These two receptors appear to be important also because of their physical interaction. Dopamine D1 and D2 receptors exist as receptor homo or heterodimers complexes that can exhibit pharmacological and functional properties distinct from their constituent receptors. Dopamine D1R-D2R heterodimers were first identified in vivo by co-immunoprecipitation in the rat striatum5 and soon thereafter confirmed by studies on HEK 293 cells line,6 and subsequently in primary striatal neuronal culture by fluorescence resonance transfer (FRET) studies using confocal microscopy.7 The D1R-D2R heterodimers have been shown to exhibit pharmacological and cell signaling properties distinct from its constituent receptors5,7–9 and the expression of dopamine D1R-D2R heterodimers in the mesocorticolimbic system and basal ganglia nuclei10–12 suggest that this receptor complex may have etiological significance in disorders characterized by abnormal dopamine signaling. For example, the calcium signaling elicited by the dopamine receptors heterodimers, through activation of Gq/11 protein and phospholipase C (PLC), resulted in the activation of calcium calmodulin kinase II13,14 and consequently increased expression of brain-derived neurotrophic factor (BDNF) in the nucleus accumbens (Nac) and ventral tegmental area (VTA).7,14 Moreover, a potential role of D1R-D2R heterodimers in schizophrenia has been suggested since altering of Ca2+-signaling has been reported in post-mortem studies of brains obtained from schizophrenic patients.15 The affinity of ligands to the dopamine receptors has been shown to be highly dependent on co-existing homo- or heterodimers.16–20 In addition to the interaction described above, dopamine D2R can form dimers with other dopamine receptors: D3R,21 D4R22 and D5R23 as well as with other GPCRs (http://www.gpcr-hetnet.com). The pharmacological profile of atypical antipsychotic drugs action indicates that the interaction between dopamine D2 and serotonin 5-HT1A or 5-HT2A receptors seems to be

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important.24,25 It is well established that dopamine D2 and serotonin 5-HT1A and 5-HT2A receptors play an important role in neurotransmission and that alterations in their functioning have been implicated in many human neurological and psychiatric disorders, including schizophrenia. All of these receptors are co-localized on the same neuron and form heterodimers.18,25–29 For a long time, the main focus has been attributed to the antipsychotic effects of D2R blockade (in the extended striatum), what alleviated the positive symptoms of schizophrenia. Further studies have shown the significant effects of antipsychotic drugs that may be beneficial for negative (cognitive) symptoms via a combined effect on dopamine and serotonin and other classes of neuroreceptors.30 Significant evidence indicates that, in fact, the balance between the properties of dopamine D2R and serotonin receptors has a profound effect on the profile of these drugs in preclinical models.31 However, in studies related to the potential role of GPCR receptor dimers, it appears that the key role of these interactions plays dopamine D2R. This receptor is also a crucial player in schizophrenia, so naturally gene encoding this receptor is considered a candidate risk gene of this disease.31a Recently, the data obtained from genome-wide associated study (GWAS) further confirmed the involvement of this receptor in schizophrenia.31b However, the results of the research indicate that the role of D2R polymorphisms in schizophrenia still remains controversial. The first association between schizophrenia and the genetic variant of D2R was observed for missense nucleotide (SNP, single nucleotide polymorphism) change causing an amino acid substitution of serine into cysteine at codon 311: rs1801028 (Ser311Cys) in the Japanese population, which was described by Arinami et al.32 Soon after, other studies appeared, confirming33–38 or negating39–43 correlations of this polymorphism with schizophrenia. It seems that origin and heredity are responsible for the lack of homogeneity of the described results.44,45 There are a number of polymorphisms within the D2R gene, although only a few appear to be potentially important for schizophrenia. One of them is polymorphism that also occurs within exon 7 is rs1800496 (Pro310Ser) for which family-based association studies strongly implicated a risk for schizophrenia in Han Chinese from Taiwan.46 However, this genetic variant is not widely studied in the context of association with schizophrenia. In other in vitro studies it has been shown that C957T polymorphism rs6277 (Pro319Pro/C >T) has marked functional consequences for D2R mRNA stability and dopamine-regulated D2R expression.47 Moreover, the C957T affected striatal D2R binding in

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healthy humans48 and this SNP also correlated with schizophrenia.47,49,50 Another genetic variant potentially relevant in schizophrenia is polymorphism rs1799732
141C Ins/Del, which is located in the promoter sequence, downstream of D2R coding sequence. It has been demonstrated to alter gene expression in vitro and striatal receptor density in vivo, and it has a replicable effect on antipsychotic treatment response.51 On the other hand, in a meta-analysis performed by Glatt et al.,51a this genetic variant was not associated with schizophrenia. Recent study also indicated a lack of association of this genetic variant with schizophrenia.38 Besides the association between
141C Ins/Del mutation and clozapine response52 another SNP rs1800497, also known as the TaqIA (or Taq1A) polymorphism of the dopamine D2R, has been identified within exon 8 of the ankyrin repeat and kinase domain containing 1 (ANKK1) gene, 10 kb away from D2R gene in the 30 untranslated region. This polymorphism, which leads to a substitution of glutamic acid for a basic lysine (Glu713Lys) that may alter substrate-binding specificity,53 likely modulates the function and expression of D2R due to its close proximity. Despite the fact that this SNP is localized to the ANKK1 gene, it seems to be in linkage disequilibrium with several D2R genetic variants, which could potentially explain a dopaminergic role in the etiopathogenesis of schizophrenia.54 However, recent meta-analysis showed a lack of correlation of this polymorphism with schizophrenia.38 Among the genes encoding other dopamine receptors, equally important in the context of schizophrenia, is the gene encoding for D3R dopamine receptor. Especially one polymorphism, rs6280, also known as Ser9Gly, has its influence: it has been demonstrated that binding affinity of selective ligands for D3R is higher, as has been shown for mutant homozygote.55 However, meta-analysis studies are also inconsistent.55a–58 On the other hand in a study of patients treated with olanzapine, those who were rs6280(C;C) homozygotes had greater positive symptom remission,59 while having a minor role or lack of association in clozapine response.60 This result might eventually become more clearly defined with increased sample size.61 As stated by Scharfetter,62 although a single polymorphism may not play a significant role or be marginal, the accumulation of these polymorphisms may already play a crucial role in both the development of schizophrenia as well as in response to treatments. From this point of view, it is interesting to analyze the effect of SNP on the interaction between receptors for which they have been shown to form functional dimers relevant to this disease.

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2. Effect of SNPs on GPCRs dimerization For several polymorphisms of D2R described above the molecular mechanism of their modulation of dopamine action are known or have been hypothesized.63 However, do these polymorphisms affect the dimerization process? It seems that particular attention should be focused on genetic variants located in the regions responsible for potential dimer formation. A number of experiments were used to indicate the location of the interaction interface between D2R during dimer formation. Molecular modeling indicates transmembrane domains (TM): TM4–TM5–TM7–TM1 interface as a region of physical interaction.64 Also, recent studies using molecular dynamics (MD) simulations have shown the conformational changes specifically occurring in TM5 and TM6, resulting from bound D2R antagonist and involving inward Tyr1995.48 and Phe3906.52 sidechain conformations, which may alter the TM4–TM5 D2R homodimer interface.65 In experiments carried out in living cells based on the cysteine-crosslinking technique, the only TM4 was indicated as a region of physical impact on D2R dimerization. It has been shown that the crosslinking site in D2R is Cys-1684.58 located at the extracellular end of TM4 directly adjacent to these residues. However, the accessibility of adjacent residues in TM4 was affected by ligand binding, implying that the interface has functional significance.17 A similar result has been demonstrated by performing a series of mutants cleaving subsequent domains of the D2R.66 Like the full-length receptor, D2R truncation mutants containing the TM4 and 5 domains or consisting of the TM1–4 domains were able to form dimers. In contrast, truncation mutants lacking TM4, such as a mutant consisting of TM1–3 domains, were only visualized as monomers. In addition, the authors have shown that disruption of the TM4 helical structure by introducing a proline residue into the truncation mutant of the TM4 and 5 domains prevented dimer formation. Interestingly, the same mutation at the full-length receptor and the D2TM2-CT truncation mutant had no detectable effect on dimer formation. This observation indicated the presence of one or more additional intermolecular interactions at the full-length receptor that was absent or weaker in the D2TM4-ICL3 truncation mutant. Therefore, although the TM4 domain contains critical structural requirements for dimerization of the D2R, there are additional interfaces involved in the dimerization process.66 All of these highly different approaches indicate the participation of the only TMs regions on the process of D2R dimerization. In some contradiction

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with this conclusion remain results indicating the significant impact of single-site mutation for the dimerization process, despite localization of these mutations beside transmembrane regions.16,67 However, the significance of these mutations is easy to understand due to the extremely dynamic nature of receptor proteins in general.68 Therefore, the introduced mutation may disturb this dynamics, leading to a change in the accessibility of appropriate helices. It has been clearly demonstrated that the dynamics of the receptor is significantly changed and crucial upon ligand binding.68 Indication of protein dynamics as an important factor in the dimerization process can also be confirmed by the aforementioned cysteine-crosslinking experiments since these studies indicated also that the ligand binding led to a significant change in TM4 helix availability. Furthermore, it has been shown that besides the transmembrane domains of GPCRs, the N- or C-tail could play a role in dimer formation, e.g., the serine 374 exchanged for alanine in the C-terminal domain of the A2AR has an impact on the D2R-A2AR heteromer.27,28 Ciruela et al. postulated that heterodimerization of the D2R-A2AR strongly depends on electrostatic interaction between an Arg-rich epitope from ICL3 of the D2R (217RRRKR222) and two adjacent Asp residues (DD401-402) or a phosphorylated Ser (S374) residue in the C-tail of the A2AR.69 In the case of dopamine receptors heterodimerization it has been observed that two acidic residues in the C-terminal end of the D1R, as well as the Arg-rich region of ICL3 of the D2R, do not seem to take part in homodimerization, but they do influence D1R-D2R dimerization.70,71 Similar interactions were also described by Jackson et al.72 for the D2R and the cannabinoid CB1 receptors and by Lee et al.73 for the ionotropic glutamate NMDA (NR1 subunit) and the D1R. The importance of the ICL3 region in the interaction of D1RD2R receptors has also been demonstrated in spectacular studies by Pei et al. It has been demonstrated, using various GST fusion proteins containing an ICL3 or carboxyl tail, that the GST-D2LIL3 protein (Lys211-Gln344) precipitated D1R of the solubilized rat striatum. In addition, it has been shown that the peptide containing D2LIL3-29-2 disrupts D1R-D2R dimers, the excess of which was observed in the post-mortem brain of depression patients.74 As has been mentioned in the introduction the role of D1R-D2R dimers has been suggested in schizophrenia as well as in the action of antipsychotic drugs. It has been shown that this dimer can be dissociated by clozapine16 and this effect depended on the time and the concentration of the drug. The higher concentration of clozapine (10
6 M) increased the degree of

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D1R-D2R receptor dimerization, while the lower concentration of the drug (10
9 M) attenuated it. The effects of both concentrations of clozapine (10
6 and 10
9 M), although opposite, were significant only during shorter time, i.e., 30 min of the drug presence in the incubation medium, while both effects ceased to be apparent following 120 min. Our unpublished data with other drugs: chlorpromazine and risperidone indicated that for these antipsychotics the higher concentration (10
6 M) decreased D1R-D2R dimers and this effect was not dependent on the incubation time. Interestingly, the lower concentration of these drugs did not have any impact on D1R-D2R dimers (Table 1). Błasiak et al. have shown that polymorphic mutations of D2R play a role in formation of dimer with D1R.67 In this study three polymorphisms of D2R have been analyzed: Val96Ala, Pro310Ser and Ser311Cys. First of them is localized in the TM2 in the region responsible for ligand binding, while the last two are located in ICL3, so they can be responsible for changes in protein function. Studies using FLIM-FRET approach indicate that only Val96Ala and Ser311Cys polymorphisms affected the D1R-D2R dimers. Val96Ala is not widely studied as opposed to the Ser311Cys variant, which has a dominant effect of risk of schizophrenia.75,76 As shown by Yao et al.,38 rs1801028 (Ser311Cys) is a clear target in the diagnosis and treatment of schizophrenia. Moreover, for this SNP, family-based association study strongly implicates the haplotype containing this locus as a risk for schizophrenia.46 It has been also shown that patients with G allele display severe cognition disorder and positive symptoms characteristic for schizophrenia.77 It has been found that Ser311Cys genotype was associated with superior risperidone response in patients with acutely exacerbated schizophrenia.78 This observation was supported by studies indicating that schizophrenia patients carrying Ser311Cys polymorphism had a shorter duration of hospitalization or were less likely to be treatment-resistant than those without this SNP.32 In the context of these reports we can suppose that the uncoupling of D1RD2R dimers may be crucial in the treatment of schizophrenia, and the occurrence of the Ser311Cys mutation additionally supports this process. It has been shown that the interaction of D1R-D2RSer311Cys is lower than with native D2R, but more sensitive to stimulation with a ligand SKF83959 acting on these dimers, manifested by increased calcium secretion.67 Therefore, it seems that excessive stimulation of D1R-D2R dimers, causing excessive calcium secretion, is unfavorable, and the observed D1R-D2RSer311Cys decoupling normalizes this condition. Data obtained in our studies indicate that antipsychotic drugs (chlorpromazine, risperidone, clozapine) have an

Table 1 Summary of energy transfer measurements by fluorescence lifetime microscopy in HEK 293 cells incubation with antipsychotic drugs. Species Drug concentration Gpp(NH)p Incubation time Transfer efficiency hEi  S.E.M. (%)

D2-ECFP

–

D2-ECFP & D1-EYFP

– μM

– 10

30
6

M 1200

– nM

– 10 –

0

30
6

0

M 1200

Clozapine

Risperidone

Chlorpromazine

–

–

–

3.73  0.70

3.94  0.70

3.94  0.70

5.43  0.66∗

1.26  0.63∗∗

0.97  0.62∗∗

5.85  0.61∗∗

1.74  0.75∗

1.81  0.71∗

3.38  0.69

0.83  0.59∗∗

1.93  0.60∗

1.25  0.80∗

3.68  0.69

3.17  0.56

4.36  0.65

3.60  0.50

3.39  0.83

4.63  0.84

3.92  0.71

3.32  0.47

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The fluorescence decays from the single HEK 293 cell, transfected with the dopamine D2R-CFP construct were observed 48 h after transfection, using a 40 CFI60 objective (with 1.5 intermediate magnification) and Nikon fluor cube with dichroic beam splitter at 455 nm combined with exciter FF01-438/24, and emitter FF01483/32 interference filters (both from Semrock, Rochester, NY, USA). In case of measurements performed on cells co-transfected with the D1R-YFP construct, the presence of the acceptor was verified using Nikon fluor cube YEL GFP C57111 appropriate for YFP spectral properties after illumination of 100 W T-DH lamp. The average efficiency of energy transfer hEi was calculated from the average donor lifetime in the presence hτdai or absence hτdi of the acceptor: hEi ¼ 1
hhττdad ii. The statistical significance was evaluated using a one-way ANOVA, followed by a Dunnett’s test for post hoc comparison. *P < 0.05; **P < 0.01 versus D2R-CFP/D1R-YFP; # D2-ECFP & D1-EYFP with clozapine and Gpp(NH)p incubation (300 ) versus D2-ECFP & D1-EYFP with clozapine (300 ).

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impact on dopamine receptors dimers. Moreover, all of the examined drugs uncouple D1R-D2R heterodimer complex. Interestingly, for both chlorpromazine and risperidone, this effect is apparent only at high concentration of the drugs and does not depend on the incubation time. For clozapine this effect is subtle, the uncoupling of D1R-D2R heterodimers have been observed only in the lower concentration, which can be explained by the drug action on the receptors remaining in the active state and only in the short time (Table 1). These data can be analyzed in the context of allosteric modulation, an important paradigm that has emerged in recent years, which is the phenomenon of “functional selectivity” also referred to as “biased agonism.”79–81 The “functional selectivity” can produce cell-specific agonism. Moreover, such ligands can have many different efficacies for the many behaviors that the receptor can exhibit, leading to breakdown in the common classifications of agonist and antagonist.82 For example, ligands can show bias for either G protein- (G protein-biased) or β-arrestin-mediated (β-arrestin-biased) signaling.81 In the light of this phenomenon it can be postulated than clozapine can act on the D1R-D2R heterodimer complex like a “biased ligand.” The effect of uncoupling of D1R-D2R dimers reversely depends on drug concentration. It has been also described by the mathematical model, “the asymmetric/symmetric three-state dimer model,” in which three states, one inactive and two actives, are considered.83 The active states differentiate themselves by the asymmetric or symmetric array of the protomers within the dimer, with either one (R*R) or both (R*R*) of the protomers being active. The asymmetric active state was assigned to a G protein-mediated signaling pathway, in turn the symmetric active state is responsible for the β-arrestin, the Src- or any other accessory protein-dependent pathway present in the system. It is interesting that this model also described activating signaling pathway depending on ligand concentrations. In the context of the fractional functional response described by Rovira et al. we can suppose that clozapine which uncouples the D1R-D2R dimers only in the low concentration and short time can activate the G-protein-dependent pathway, while the high concentration of this drug and longer incubation would activate other signaling pathways. The pharmacological function of genetic variation in D2R receptor has never been the object of extensive studies. Cravchik et al. showed that the affinity of the Ser311Cys variant for dopamine was approximately twofold lower than for native D2R, while no significant differences were detected in the binding affinity for the antagonist between the D2R and the expressed genetic variant.84 Neuroleptics of the phenothiazine group (chlorpromazine

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and thioridazine) showed lower potency for Ser311Cys.85 Data obtained by Faron-Go´recka et al. from the in vitro competition binding study for clozapine strongly suggest that there are two class of binding sites for clozapine for both dopamine receptors (D1R and D2R). Additionally, these results have indicated that the affinity of D1R for clozapine strongly depended on the presence of dopamine D2R in the same cell. Moreover, for Ser311Cys variant only one class of binding site was observed, while upon co-expression with native D1R, two binding sites for clozapine were observed.16 The traditional view of a high-affinity receptor—resulting from the “ternary complex” theory—defines it as a G protein-bound receptor that is capable of binding an agonist (Fig. 1).86 Therefore, it is postulated that the low-affinity state determines the receptor not associated with the G protein. According to classical view of monomeric receptors both “the two-state model” and “the ternary complex model” have been sufficient. Nonetheless in the light of receptor dimerization to explain ligand binding and activation mechanism of GPCRs, “the two-state dimer receptor model” proposed by Franco et al.87 is more straightforward. For this model ligand binding by the first and the second protomers can be different and each of the equilibrium constants are considered as apparent values, reflecting the affinity of active receptor dimers and isomerization constants between their active and non-active states. Based on the obtained values of the equilibrium constants, dimer cooperative index (Dc) is calculated.88 Absence, positive, and negative cooperativity for ligand binding are defined as those experimental conditions making Dc ¼ 0, Dc > 0, and Dc < 0, respectively.

Fig. 1 The ternary complex model proposed by De Lean et al.86 The free receptor and bound to the G protein remains in equilibrium (right panel). These states have different affinity for the ligand and are in equilibrium with the unbound receptor (left panel).

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The curves obtained by fitting our unpublished data are runs similarly like isotherms got from the two-state dimer model fit. In our results, when native dopamine receptors are co-expressed in the same cell, the cooperativity is strongly negative and stable regardless of antipsychotic drugs (Dc ¼ c.a.
2.5) (Fig. 2). Results obtained in the competition binding study for risperidone on 3 [ H]spiperone in D2R and D1R/D2R were fitted to two binding sites model. When dopamine D1R and mutation DS311C R were co-expressed 2 in the same cells, only one binding site was fitted. Hill coefficients for this result is about 1. Of note, the lack of a biphasic binding curve is not sufficient evidence of the simplest binding model. In this case, the Hill coefficient may be decisive, since its value different from unity indicate a more complex system. A change in this parameter can be related to modification of the allosteric control between protomers, leading to a modification of cooperativity.89 According to the our data obtained by the two-state dimer receptor model, the parameters indicating the strong negative cooperativity when D2R receptors is co-expressed with D1R and in the case of expression of DS311C R (DCrisperidone cooperativity index
2.85 and 2.36, respectively). 2 Also the competition data for clozapine on [3H]spiperone were fitted well by a two binding site model and the Ki values did not differ when dopamine D2R was expressed alone or co-expressed together with dopamine D1R. However, when D2R mutant (DS311C ) was expressed alone, only 2 low-affinity binding site for clozapine was observed. The data obtained by using the dimer site model are similar to the parameters obtained by the two independent site model. The values for dimer cooperativity index Dcclozapine indicate the negative cooperativity; however, in the case of co-expressing D1R and DS311C R in the same cells, the Dcclozapine and the 2 KDAB parameters statistically increased. This parameter is changing in the situation of expressed DS311C or 2 co-expressed mutant receptor DS311C with native D1R, which indicate a 2 weakening of the negative cooperativity. Therefore, one might conclude that we observed a kind of allosteric modulation by one receptor in the dimer to the second. In the case of displacement [3H]SCH 23390 by risperidone the reduced affinity observed for co-expressed receptors in the same cell could be dependent on the impact of ligand binding to monomeric receptors D1 and D2, though in the case of binding using [3H]spiperone can be seen clearly that the affinity of risperidone depends on the allosteric modulation of D1R to D2R. The observed changes in the affinity of neuroleptics for the DS311C receptor and its effect on the potential interaction 2

Fig. 2 The competition binding results of chlorpromazine on [3H]spiperone reveal a distinct pharmacology of the D2 receptor. For D2R in all experimental models (D2R, D2R/D1R, DS311C R and D1R/DS311C R) two binding sites were observed. The dimer cooperativity index DCchlorpromazine 2 2 increased to
0.81, which indicates that a weakly negative cooperativity exists in the binding of chlorpromazine to DS311C R. 2

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with D1R (when they are simultaneously produced in the cell) may be of great importance in understanding the relationship between this genetic variant and correlation with the occurrence of schizophrenia or different sensitivity to the used therapies. Other genetic variants of dopamine D2R might be equally important; however, this subject needs additional studies. Nevertheless, better understanding of altered ability of genetic variants of D2R to form dimers with D1R, as well as of altered affinity for antipsychotic drugs, depending on the absence or presence of the second dopamine receptor is of great importance—since these two receptors are not always co-expressed in the same cell. It may well be that targeting new compounds toward the D1R-D2R dimers, which the most probably form under conditions of excessive dopamine release, will result in drugs devoid of serious side effects.

References 1. Creese I, Burt DR, Snyder SH. Dopamine receptors and average clinical doses. Science. 1976;194:546. 2. Seeman P, Lee T. Antipsychotic drugs: direct correlation between clinical potency and presynaptic action on dopamine neurons. Science. 1975;188:1217–1219. 3. Seeman P. Are dopamine D2 receptors out of control in psychosis? Prog Neuropsychopharmacol Biol Psychiatry. 2013;46:146–152. 4. Goldman-Rakic PS, Selemon LD. Functional and anatomical aspects of prefrontal pathology in schizophrenia. Schizophr Bull. 1997;23:437–458, Review. 5. Lee SP, So CH, Rashid AJ, et al. Dopamine D1 and D2 receptor Co-activation generates a novel phospholipase C-mediated calcium signal. J Biol Chem. 2004;279:35671–35678. 6. Dziedzicka-Wasylewska M, Faron-Go´recka A, Andrecka J, Polit A, Ku
smider M, Wasylewski Z. Fluorescence studies reveal heterodimerization of dopamine D1 and D2 receptors in the plasma membrane. Biochemistry. 2006;45:8751–8759. 7. Hasbi A, Fan T, Alijaniaram M, et al. Calcium signaling cascade links dopamine D1-D2 receptor heteromer to striatal BDNF production and neuronal growth. Proc Natl Acad Sci U S A. 2009;106:21377–21382. 8. Rashid AJ, So CH, Kong MM, et al. D1-D2 dopamine receptor heterooligomers with unique pharmacology are coupled to rapid activation of Gq/11 in the striatum. Proc Natl Acad Sci U S A. 2007;104:654–659. 9. Verma V, Hasbi A, O’Dowd BF, George SR. Dopamine D1-D2 receptor heteromermediated calcium release is desensitized by D1 receptor occupancy with or without signal activation: dual functional regulation by G protein-coupled receptor kinase 2. J Biol Chem. 2010;285:35092–35103. 10. Perreault ML, Hasbi A, Alijaniaram M, et al. The dopamine D1-D2 receptor heteromer localizes in dynorphin/enkephalin neurons: increased high affinity state following amphetamine and in schizophrenia. J Biol Chem. 2010;285:36625–36634. 11. Perreault ML, O’Dowd BF, George SR. Dopamine receptor homooligomers and heterooligomers in schizophrenia. CNS Neurosci Ther. 2011;17:52–57. 12. Rico AJ, Dopeso-Reyes IG, Martı´nez-Pinilla E, et al. Neurochemical evidence supporting dopamine D1-D2 receptor heteromers in the striatum of the long-tailed macaque: changes following dopaminergic manipulation. Brain Struct Funct. 2017;222: 1767–1784.

292

Agata Faron-Górecka et al.

13. Ng J, Rashid AJ, So CH, O’Dowd BF, George SR. Activation of calcium/calmodulindependent protein kinase II alpha in the striatum by the heteromeric D1-D2 dopamine receptor complex. Neuroscience. 2010;165:535–541. 14. Perreault ML, Fan T, Alijaniaram M, O’Dowd BF, George SR. Dopamine D1-D2 receptor heteromer in dual phenotype GABA/glutamate-coexpressing striatal medium spiny neurons: regulation of BDNF, GAD67 and VGLUT1/2. PLoS One. 2012;7: e33348. 15. Lidow MS. Calcium signaling dysfunction in schizophrenia: a unifying approach. Brain Res Rev. 2003;43:70–84. Review. 16. Faron-Go´recka A, Go´recki A, Ku
smider M, Wasylewski Z, Dziedzicka-Wasylewska M. The role of D1-D2 receptor hetero-dimerization in the mechanism of action of clozapine. Eur Neuropsychopharmacol. 2008;18:682–691. 17. Guo W, Shi L, Javitch JA. The fourth transmembrane segment forms the interface of the dopamine D2 receptor homodimer. J Biol Chem. 2003;278:4385–4388. 18. Łukasiewicz S, Faron-Go´recka A, Kędracka-Krok S, Dziedzicka-Wasylewska M. Effect of clozapine on the dimerization of serotonin 5-HT(2A) receptor and its genetic variant 5-HT(2A)H425Y with dopamine D(2) receptor. Eur J Pharmacol. 2011;659:114–123. 19. Maggio R, Aloisi G, Silvano E, Rossi M, Millan MJ. Heterodimerization of dopamine receptors: new insights into functional and therapeutic significance. Parkinsonism Relat Disord. 2009;15(suppl 4):S2–S7. 20. Vivo M, Lin H, Strange PG. Investigation of cooperativity in the binding of ligands to the D(2) dopamine receptor. Mol Pharmacol. 2006;69:226–235. 21. Scarselli M, Novi F, Schallmach E, et al. D2/D3 dopamine receptor heterodimers exhibit unique functional properties. J Biol Chem. 2001;276:30308–30314. 22. Borroto-Escuela DO, Van Craenenbroeck K, Romero-Fernandez W, et al. Dopamine D2 and D4 receptor heteromerization and its allosteric receptor-receptor interactions. Biochem Biophys Res Commun. 2011;404:928–934. 23. So CH, Verma V, Alijaniaram M, et al. Calcium signaling by dopamine D5 receptor and D5-D2 receptor hetero-oligomers occurs by a mechanism distinct from that for dopamine D1-D2 receptor hetero-oligomers. Mol Pharmacol. 2009;75:843–854. 24. Łukasiewicz S, Błasiak E, Szafran-Pilch K, Dziedzicka-Wasylewska M. Dopamine D2 and serotonin 5-HT1A receptor interaction in the context of the effects of antipsychotics—in vitro studies. J Neurochem. 2016;137:549–560. 25. Szlachta M, Ku
smider M, Pabian P, et al. Repeated clozapine increases the level of serotonin 5-HT(1A)R heterodimerization with 5-HT(2A) or dopamine D(2) receptors in the mouse cortex. Front Mol Neurosci. 2018;11:40. 26. Albizu L, Holloway T, Gonza´lez-Maeso J, Sealfon SC. Functional crosstalk and heteromerization of serotonin 5-HT2A and dopamine D2 receptors. Neuropharmacology. 2011;61(4):770–777. 27. Borroto-Escuela DO, Romero-Fernandez W, Tarakanov AO, et al. Characterization of the A2AR-D2R interface: focus on the role of the C-terminal tail and the transmembrane helices. Biochem Biophys Res Commun. 2010;402:801–807. 28. Borroto-Escuela DO, Romero-Fernandez W, Tarakanov AO, et al. Dopamine D2 and 5-hydroxytryptamine 5-HT(₂A) receptors assemble into functionally interacting heteromers. Biochem Biophys Res Commun. 2010;401:605–610. 29. Łukasiewicz S, Polit A, Kędracka-Krok S, Wędzony K, Ma
ckowiak M, DziedzickaWasylewska M. Hetero-dimerization of serotonin 5-HT(2A) and dopamine D(2) receptors. Biochim Biophys Acta. 2010;1803:1347–1358. 30. Newman-Tancredi A, Kleven MS. Comparative pharmacology of antipsychotics possessing combined dopamine D2 and serotonin 5-HT1A receptor properties. Psychopharmacology (Berl). 2011;216:451–473.

Influence of genetic variants on heterodimerization

293

31. Newman-Tancredi A. The importance of 5-HT1A receptor agonism in antipsychotic drug action: rationale and perspectives. Curr Opin Investig Drugs. 2010;11:802–812. Review. (a) Noble EP. D2 dopamine receptor gene in psychiatric and neurologic disordersand its phenotypes. Am J Med Genet B Neuropsychiatr Genet. 2003;116B(1):103–125. Review. (b) Schizophrenia Working Group of the Psychiatric Genomics Consortium. Biological insights from 108 schizophrenia-associated genetic loci. Nature. 2014;511: 421–427. 32. Arinami T, Itokawa M, Enguchi H, et al. Association of dopamine D2 receptor molecular variant with schizophrenia. Lancet. 1994;343:703–704. 33. Arinami T, Itokawa M, Aoki J, et al. Further association study on dopamine D2 receptor variant S311C in schizophrenia and affective disorders. Am J Med Genet. 1996;67:133–138. 34. Glatt SJ, J€ onsson EG. The Cys allele of the DRD2 Ser311Cys polymorphism has a dominant effect on risk for schizophrenia: evidence from fixed- and random-effects meta-analyses. Am J Med Genet B Neuropsychiatr Genet. 2006;141B:149–154. 35. Itokawa M, Arinami T, Toru M. Advanced research on dopamine signaling to develop drugs for the treatment of mental disorders: Ser311Cys polymorphisms of the dopamine D2-receptor gene and schizophrenia. J Pharmacol Sci. 2010;114:1–5. 36. J€ onsson EG, Sill
en A, Vares M, Ekholm B, Terenius L, Sedvall GC. Dopamine D2 receptor gene Ser311Cys variant and schizophrenia: association study and meta-analysis. Am J Med Genet B Neuropsychiatr Genet. 2003;119B:28–34. 37. Liu ZW, Liu JL, An Y, Zhang L, Wang YM. Association between Ser311Cys polymorphism in the dopamine D2 receptor gene and schizophrenia risk: a meta-analysis in Asian populations. Genet Mol Res. 2012;11:261–270. 38. Yao J, Pan YQ, Ding M, Pang H, Wang BJ. Association between DRD2 (rs1799732 and rs1801028) and ANKK1 (rs1800497) polymorphisms and schizophrenia: a meta-analysis. Am J Med Genet B Neuropsychiatr Genet. 2015;168B:1–13. Review. 39. Chen CH, Chien SH, Hwu HG. No association of dopamine D2 receptor molecular variant Cys311 and schizophrenia in Chinese patients. Am J Med Genet. 1996;67(4):418–420. 40. Crawford F, Hoyne J, Cai X, et al. Dopamine DRD2/Cys311 is not associated with chronic schizophrenia. Am J Med Genet. 1996;67(5):483–484. 41. Shaikh S, Collier D, Arranz M, Ball D, Gill M, Kerwin R. DRD2 Ser311/Cys311 polymorphism in schizophrenia. Lancet. 1994;343:1045–1046. 42. Spurlock G, Williams J, McGuffin P, et al. European Multicentre Association Study of Schizophrenia: a study of the DRD2 Ser311Cys and DRD3 Ser9Gly polymorphisms. Am J Med Genet. 1998;81:24–28. 43. Watanabe Y, Shibuya M, Someya T. DRD2 Ser311Cys polymorphism and risk of schizophrenia. Am J Med Genet B Neuropsychiatr Genet. 2015;168B(3):224–228. 44. He H, Wu H, Yang L, et al. Associations between dopamine D2 receptor gene polymorphisms and schizophrenia risk: a PRISMA compliant meta-analysis. Neuropsychiatr Dis Treat. 2016;12:3129–3144. 45. Kaur G, Singh Chavan B, Gupta D, et al. An association study of dopaminergic (DRD2) and serotoninergic (5-HT2) gene polymorphism and schizophrenia in a North Indian population. Asian J Psychiatr. 2019;39:178–184. 46. Glatt SJ, Faraone SV, Lasky-Su JA, Kanazawa T, Hwu HG, Tsuang MT. Family-based association testing strongly implicates DRD2 as a risk gene for schizophrenia in Han Chinese from Taiwan. Mol Psychiatry. 2009;14:885–893. 47. Monakhov M, Golimbet V, Abramova L, Kaleda V, Karpov V. Association study of three polymorphisms in the dopamine D2 receptor gene and schizophrenia in the Russian population. Schizophr Res. 2008;100:302–307.

294

Agata Faron-Górecka et al.

48. Hirvonen MM, Lumme V, Hirvonen J, et al. C957T polymorphism of the human dopamine D2 receptor gene predicts extrastriatal dopamine receptor availability in vivo. Prog Neuropsychopharmacol Biol Psychiatry. 2009;33:630–636. 49. Betcheva ET, Mushiroda T, Takahashi A, et al. Case-control association study of 59 candidate genes reveals the DRD2 SNP rs6277 (C957T) as the only susceptibility factor for schizophrenia in the Bulgarian population. J Hum Genet. 2009;54:98–107. 50. Gonza´lez-Castro TB, Herna´ndez-Dı´az Y, Jua´rez-Rojop IE, et al. The role of C957T, TaqI and Ser311Cys polymorphisms of the DRD2 gene in schizophrenia: systematic review and meta-analysis. Behav Brain Funct. 2016;12:29. 51. Lencz T, Robinson DG, Napolitano B, et al. DRD2 promoter region variation predicts antipsychotic-induced weight gain in first episode schizophrenia. Pharmacogenet Genomics. 2010;20:569–572. (a) Glatt SJ, Faraone SV, Tsuang MT. DRD2–141C insertion/deletion polymorphism is not associated with schizophrenia: results of a metaanalysis. Am J Med Genet B Neuropsychiatr Genet. 2004;128B(1):21–23. 52. Malhotra AK, Goldman D. Benefits and pitfalls encountered in psychiatric genetic association studies. Biol Psychiatry. 1999;45(5):544–550. Review. 53. Comings DE, Comings BG, Muhleman D, et al. The dopamine D2 receptor locus as a modifying gene in neuropsychiatric disorders. JAMA. 1991;266:1793–1800. 54. Arab AH, Elhawary NA. Association between ANKK1 (rs1800497) and LTA (rs909253) genetic variants and risk of schizophrenia. Biomed Res Int. 2015;2015:821827. 55. Lundstrom K, Turpin MP. Proposed schizophrenia-related gene polymorphism: expression of the Ser9Gly mutant human dopamine D3 receptor with the Semliki Forest virus system. Biochem Biophys Res Commun. 1996;225:1068–1072. (a) J€ onsson EG, Flyckt L, Burgert E, Crocq MA, Forslund K, Mattila-Evenden M, Rylander G, Asberg M, Nimgaonkar VL, Edman G, Bjerkenstedt L, Wiesel FA, Sedvall GC. Dopamine D3 receptor gene Ser9Gly variant and schizophrenia: association study and meta-analysis. Psychiatr Genet. 2003;13(1):1–12. 56. Nanko S, Sasaki T, Fukuda R, et al. A study of the association between schizophrenia and the dopamine D3 receptor gene. Hum Genet. 1993;92:336–338. 57. Qi XL, Xuan JF, Xing JX, Wang BJ, Yao J. No association between dopamine D3 receptor gene Ser9Gly polymorphism (rs6280) and risk of schizophrenia: an updated meta-analysis. Neuropsychiatr Dis Treat. 2017;13:2855–2865. eCollection 2017. 58. Sa´iz PA, Garcı´a-Portilla MP, Arango C, et al. Genetic polymorphisms in the dopamine-2 receptor (DRD2), dopamine-3 receptor (DRD3), and dopamine transporter (SLC6A3) genes in schizophrenia: data from an association study. Prog Neuropsychopharmacol Biol Psychiatry. 2010;34:26–31. 59. Adams DH, Close S, Farmen M, Downing AM, Breier A, Houston JP. Dopamine receptor D3 genotype association with greater acute positive symptom remission with olanzapine therapy in predominately caucasian patients with chronic schizophrenia or schizoaffective disorder. Hum Psychopharmacol. 2008;23:267–274. 60. Malhotra AK, Goldman D, Buchanan RW, et al. The dopamine D3 receptor (DRD3) Ser9Gly polymorphism and schizophrenia: a haplotype relative risk study and association with clozapine response. Mol Psychiatry. 1998;3:72–75. 61. Hwang R, Zai C, Tiwari A, et al. Effect of dopamine D3 receptor gene polymorphisms and clozapine treatment response: exploratory analysis of nine polymorphisms and metaanalysis of the Ser9Gly variant. Pharmacogenomics J. 2010;10:200–218. 62. Scharfetter J. Dopamine receptor polymorphisms and drug response in schizophrenia. Pharmacogenomics. 2001;2:251–261. Review. 63. Doehring A, Kirchhof A, L€ otsch J. Genetic diagnostics of functional variants of the human dopamine D2 receptor gene. Psychiatr Genet. 2009;19:259–268. 64. Kaczor AA, J€ org M, Capuano B. The dopamine D2 receptor dimer and its interaction with homobivalent antagonists: homology modeling, docking and molecular dynamics. J Mol Model. 2016;22:203.

Influence of genetic variants on heterodimerization

295

65. Wouters E, Marı´n AR, Dalton JAR, Giraldo J, Stove C. Distinct dopamine D₂ receptor antagonists differentially impact D₂ receptor oligomerization. Int J Mol Sci. 2019; 20(7):1686. 66. Lee SP, O’Dowd BF, Rajaram RD, Nguyen T, George SR. D2 dopamine receptor homodimerization is mediated by multiple sites of interaction, including an intermolecular interaction involving transmembrane domain 4. Biochemistry. 2003;42: 11023–11031. 67. Błasiak E, Łukasiewicz S, Szafran-Pilch K, Dziedzicka-Wasylewska M. Genetic variants of dopamine D2 receptor impact heterodimerization with dopamine D1 receptor. Pharmacol Rep. 2017;69:235–241. 68. Manglik A, Kim TH, Masureel M, et al. Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell. 2015;161:1101–1111. 69. Ciruela F, Burguen˜o J, Casado´ V, et al. Combining mass spectrometry and pull-down techniques for the study of receptor heteromerization. Direct epitope-epitope electrostatic interactions between adenosine A2A and dopamine D2 receptors. Anal Chem. 2004;76:5354–5363. 70. Łukasiewicz S, Faron-Go´recka A, Dobrucki J, Polit A, Dziedzicka-Wasylewska M. Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D1 and D2 receptor oligomerization. FEBS J. 2009;276:760–775. 71. O’Dowd BF, Ji X, Nguyen T, George SR. Two amino acids in each of D1 and D2 dopamine receptor cytoplasmic regions are involved in D1-D2 heteromer formation. Biochem Biophys Res Commun. 2012;417:23–28. 72. Jackson SN, Wang HY, Woods AS. Study of the fragmentation patterns of the phosphate-arginine noncovalent bond. J Proteome Res. 2005;4:2360–2363. 73. Lee FJ, Xue S, Pei L, et al. Dual regulation of NMDA receptor functions by direct protein-protein interactions with the dopamine D1 receptor. Cell. 2002;111:219–223. 74. Pei L, Li S, Wang M, et al. Uncoupling the dopamine D1-D2 receptor complex exerts antidepressant-like effects. Nat Med. 2010;16:1393–1395. 75. Allen NC, Bagade S, McQueen MB, et al. Systematic meta-analyses and field synopsis of genetic association studies in schizophrenia: the SzGene database. Nat Genet. 2008;40:827–834. 76. Shi J, Gershon ES, Liu C. Genetic associations with schizophrenia: meta-analyses of 12 candidate genes. Schizophr Res. 2008;104:96–107. 77. Hori H, Ohmori O, Shinkai T, Kojima H, Nakamura J. Association analysis between two functional dopamine D2 receptor gene polymorphisms and schizophrenia. Am J Med Genet. 2001;105:176–178. 78. Lane HY, Lee CC, Chang YC, Lu CT, Huang CH, Chang WH. Effects of dopamine D2 receptor Ser311Cys polymorphism and clinical factors on risperidone efficacy for positive and negative symptoms and social function. Int J Neuropsychopharmacol. 2004;7:461–470. 79. Kenakin T. Biased agonism. F1000 Biol Rep. 2009;1:87. 80. Keov P, Sexton PM, Christopoulos A. Allosteric modulation of G protein-coupled receptors: a pharmacological perspective. Neuropharmacology. 2011;60:24–35. 81. Violin JD, Lefkowitz RJ. Beta-arrestin-biased ligands at seven-transmembrane receptors. Trends Pharmacol Sci. 2007;28:416–422. 82. Kenakin T. Functional selectivity and biased receptor signaling. J Pharmacol Exp Ther. 2011;336:296–302. 83. Rovira X, Pin JP, Giraldo J. The asymmetric/symmetric activation of GPCR dimers as a possible mechanistic rationale for multiple signalling pathways. Trends Pharmacol Sci. 2010;31:15–21. 84. Cravchik A, Sibley DR, Gejman PV. Functional analysis of the human D2 dopamine receptor missense variants. J Biol Chem. 1996;271:26013–26017.

296

Agata Faron-Górecka et al.

85. Cravchik A, Sibley DR, Gejman PV. Analysis of neuroleptic binding affinities and potencies for the different human D2 dopamine receptor missense variants. Pharmacogenetics. 1999;9:17–23. 86. De Lean A, Stadel JM, Lefkowitz RJ. A ternary complex model explains the agonistspecific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor. J Biol Chem. 1980;255:7108–7117. 87. Franco R, Casado´ V, Mallol J, et al. The two-state dimer receptor model: a general model for receptor dimmers. Mol Pharmacol. 2006;69:1905–1912. 88. Giraldo J. On the fitting of binding data when receptor dimerization is suspected. Br J Pharmacol. 2008;155:17–23. 89. Moreno Delgado D, Møller TC, Ster J, et al. Pharmacological evidence for a metabotropic glutamate receptor heterodimer in neuronal cells. elife. 2017;29:6.

Further reading 90. Chidiac P, Green MA, Pawagi AB, Wells JW. Cardiac muscarinic receptors. Cooperativity as the basis for multiple states of affinity. Biochemistry. 1997;36:7361–7379. 91. Schizophrenia Working Group of the Psychiatric Genomics Consortium. Biological insights from 108 schizophrenia-associated genetic loci. Nature. 2014;511:421–427.