G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that sense extracellular signal and activate intracellular signaling pathways. The serotonin 5HT2A receptor (or 2AR) is one of the GPCRs coupled to Gq proteins, activating PLC and hydrolyzing PIP2. This hydrolysis causes a diffusion of bound PIP2 away from the channel binding site resulting in G protein-gated inwardly rectifying K+ channel (GIRK) inhibition and a downstream stimulation of Ca2+ release from endoplasmic reticulum stores. Previous experiments have demonstrated that the serotonin 5HTA receptor is a target of serotonergic psychedelic drugs, such as LSD, and partially mediates the action of many atypical antipsychotic drugs. However, the portion responsible for the functional activity and response of these drugs is unknown. The purpose of this study was to functionally characterize four deconstructed analogs of risperidone, an atypical antipsychotic agent, using two assays: by application to 5HT2A receptors in Xenopus oocytes and by calcium epifluorescence imaging in a HEK293 cell line stably expressing 2AR. Our experiments revealed that two analogs, RHV-006 and RHV-008, are partial agonists by themselves and greatly antagonize the effects of serotonin. RHV-006 and RHV-008 contain the piperidine and benzisoxizole ring systems of risperidone. RHV-023 and RHV-026, on the other hand, are more efficacious agonists than RHV-006 and RHV-008 but display a non-antagonistic effect with serotonin. RHV-023 and RHV-026 contain both the piperidine and benzisoxizole ring systems in addition to part of the diazabicyclo ring, thus containing more of risperidone’s structure than RHV-006 and RHV-008.

G-protein coupled receptors (GPCRs) implicated in disease are the predominant pharmaceutical targets. Growing evidence suggests that GPCRs form homo- and heteromeric complexes, resulting in allosteric functional changes. Ligands targeting one receptor can alter the function of the other receptor or receptors. Knowledge of these functional changes will provide unique opportunities to treat diseases. We examined two GPCR heteromers implicated in psychosis: mGlu2R-5HT2AR and D2R-5HT2AR. Using whole-cell patch clamp, we studied HEK-293 cells stably transfected with mGlu2R and 5HT2AR. Maximal heteromer formation allows inverse agonists to increase the G-protein activity of the opposite receptor, while sub-maximal heteromer formation does not. However, similar results are obtained in sub-maximal heteromer cells by applying a combination of a mGlu2R synthetic agonist with a 5HT2AR anti-psychotic drug. These results confirm our oocyte results, now in a mammalian cell line. Using two-electrode voltage clamp, we also investigated the allosteric changes upon heteromerization of D2R-5HT2AR in oocytes injected with appropriate cRNAs. Heteromer formation in the presence of dopamine or serotonin results in an increase in G-protein activity of each receptor while the simultaneous presence of both neurotransmitters further increases the G-protein activity. The addition of synthetic agonists or anti-psychotics decreases the G-protein activity of the opposite receptor while agonizing or antagonizing its target receptor, respectively. Maximal allosteric effects upon D2R-5HT2AR formation only occur at a specific cRNA injection ratio, but partial effects exist at other ratios. Our data suggest that allosteric functional changes upon heteromerization are physiologically relevant and are mostly different when comparing mGlu2R-5HT2AR to D2R-5HT2AR.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第17931号 / 生博第294号 / 新制||生||38(附属図書館) / 30751 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 渡邉 大, 教授 松崎 文雄 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The group II metabotropic glutamate receptors are known for their involvement in various psychiatric disorders. The mGluR2 in particular is linked with etiology of schizophrenia especially in the context of crosstalk with 5-HT2A. Thus, the mGluR2 has attracted attentions for its potential therapeutic applications. Despite numerous physiological evidences on the actions of mGluR2, its mechanism is still unclear to this day. It is partially due to the lack of understanding in characteristics of mGluR2 homodimer which is its functionally active form. Therefore, the characterization of dimeric interaction serves as a foundation to advanced understanding of the role of mGluR2. On that note, the role of the conserved cysteine residue (C121) in the ligand binding domain of mGluR2 has been evaluated in this study as they are known to play a critical part in homodimer formation. Collectively, C121 has been shown to affect the dimerization, subcellular localization, and pharmacokinetics of mGluR2. Lastly, the effect of mGluR2 on mouse behavior was examined in a partial effort to elucidate its role in crosstalk with 5-HT2A.

Schizophrenia is a severe mental disorder affecting ~1% of world population. Two G protein coupled receptors (GPCRs): Gi-coupled dopamine D2 receptor (D2R), and Gq-coupled serotonin 2A receptor (2AR), are targeted by the typical and atypical antipsychotic drugs to treat schizophrenia. These two receptors have been shown to co-localize in brain regions relevant to schizophrenia, including the ventral tegmental area (VTA), striatum, and prefrontal cortex (PFC). Studies in our lab characterized the integrated signaling of the D2R-2AR heteromer and found that both the Gi activity of D2R and the Gq activity of 2AR were potentiated in response to dopamine (DA) and serotonin (5-HT), whereas the potency of the typical antipsychotic drug (APD) haloperidol antagonizing Gi and Gq signaling was also enhanced. Using a peptide mimicking the transmembrane (TM) domain 5 of D2R, we showed disruption of the formation and function of the D2R-2AR heteromer in heterologous systems and ex vivo brain slices. Our functional and mutagenesis data suggested that D2R and 2AR heteromerize though a symmetric TM5,6-TM5,6 interface, and a network of Pi-Pi stacking interaction among eight conserved aromatic residues of D2R and 2AR may underlie the mechanism for the functional cross-talk between D2R and 2AR. Based on these results, we built a structural model for the D2R-2AR heteromer recapitulating its functional cross-talk characteristics. We are presently pursuing behavioral experiments to investigate the effectiveness of antipsychotic drugs on the function of the D2R-2AR heteromer in animal models of psychosis. Our overall study shows a dual role of the D2R-2AR heteromer in schizophrenia-associated psychosis and sheds light on the development of future therapeutic drugs for schizophrenia and other psychotic diseases.

Opioid Receptors (ORs) are involved in the pathophysiology of several neuropsychiatric conditions yet remain an untapped therapeutic resource. Although only mu-, delta-, and kappa-OR types have been cloned, additional subtypes result from complexes generated by direct receptor-receptor interactions. Mu- and delta-ORs form a heteromeric receptor complex with unique pharmacological and signalling properties distinct from those of mu- and delta-OR homomers. In these studies, we sought to characterize the ligand binding pocket and agonist-induced internalization profile of the mu-delta heteromer, to investigate mu-delta heteromer-specific signalling in brain, and to interrogate the contribution of this receptor complex to opioid-mediated behavioural effects. In competition radioligand binding studies, delta-agonists displaced high affinity mu-agonist binding from the mu-delta heteromer but not the muOR homomer, suggestive of delta-agonists occupying or allosterically modulating the muOR ligand binding pocket within the heteromer. Delta-agonists induced internalization of the mu-delta heteromer in a dose-dependent, pertussis toxin resistant, and muOR- and deltaOR-dependent manner from the cell surface via the clathrin and dynamin endocytic machinery. Agonist-induced internalization of the mu-delta heteromer persisted following chronic morphine treatment conditions which desensitized the muOR homomer. Using Galpha-specific GTPgammaS binding assays, we demonstrated that mu-delta heteromer signalling previously characterized in cell lines was present in the striatum and hippocampus, and did not desensitize following prolonged morphine treatment conditions which desensitized muOR homomer-mediated signalling. Since delta-agonists which also target the mu-delta heteromer possess antidepressant-like and anxiolytic-like properties, we investigated the role of this receptor complex in mood regulation. We devised a strategy to selectively analyze the effects of the mu-delta heteromer by dissociating it using a specific interfering peptide aimed at a sequence implicated in mu-delta heteromerization. The interfering peptide abolished the unique pharmacological and trafficking properties of delta-agonists at the mu-delta heteromer and dissociated this receptor complex in vitro. Intra-accumbens administration of the interfering peptide disrupted the mu-delta interaction in vivo and allowed for isolation of the mu-delta heteromer contribution to the mood-regulatory effects of a delta-agonist with activity at the heteromer. Activation of the mu-delta heteromer in the nucleus accumbens produced antidepressant-like and anxiolytic-like actions in animal models of depression and anxiety.

Opioid Receptors (ORs) are involved in the pathophysiology of several neuropsychiatric conditions yet remain an untapped therapeutic resource. Although only mu-, delta-, and kappa-OR types have been cloned, additional subtypes result from complexes generated by direct receptor-receptor interactions. Mu- and delta-ORs form a heteromeric receptor complex with unique pharmacological and signalling properties distinct from those of mu- and delta-OR homomers. In these studies, we sought to characterize the ligand binding pocket and agonist-induced internalization profile of the mu-delta heteromer, to investigate mu-delta heteromer-specific signalling in brain, and to interrogate the contribution of this receptor complex to opioid-mediated behavioural effects. In competition radioligand binding studies, delta-agonists displaced high affinity mu-agonist binding from the mu-delta heteromer but not the muOR homomer, suggestive of delta-agonists occupying or allosterically modulating the muOR ligand binding pocket within the heteromer. Delta-agonists induced internalization of the mu-delta heteromer in a dose-dependent, pertussis toxin resistant, and muOR- and deltaOR-dependent manner from the cell surface via the clathrin and dynamin endocytic machinery. Agonist-induced internalization of the mu-delta heteromer persisted following chronic morphine treatment conditions which desensitized the muOR homomer. Using Galpha-specific GTPgammaS binding assays, we demonstrated that mu-delta heteromer signalling previously characterized in cell lines was present in the striatum and hippocampus, and did not desensitize following prolonged morphine treatment conditions which desensitized muOR homomer-mediated signalling. Since delta-agonists which also target the mu-delta heteromer possess antidepressant-like and anxiolytic-like properties, we investigated the role of this receptor complex in mood regulation. We devised a strategy to selectively analyze the effects of the mu-delta heteromer by dissociating it using a specific interfering peptide aimed at a sequence implicated in mu-delta heteromerization. The interfering peptide abolished the unique pharmacological and trafficking properties of delta-agonists at the mu-delta heteromer and dissociated this receptor complex in vitro. Intra-accumbens administration of the interfering peptide disrupted the mu-delta interaction in vivo and allowed for isolation of the mu-delta heteromer contribution to the mood-regulatory effects of a delta-agonist with activity at the heteromer. Activation of the mu-delta heteromer in the nucleus accumbens produced antidepressant-like and anxiolytic-like actions in animal models of depression and anxiety.