Structural basis for the functions of dopamine receptors, neurotransmitter transporters, and sigma 1 receptor

多巴胺受体、神经递质转运蛋白和 sigma 1 受体功能的结构基础

• 批准号: 10267556
• 负责人: Lei Shi
• 金额: $ 157.85万
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10267556
• 关键词: Adrenergic Agonists; Adrenergic Receptor; Adverse effects; Affect; Affinity; Agonist; Allosteric Site; Amphetamines; Amyotrophic Lateral Sclerosis; Antidepressive Agents; Arrestins; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Models; Biophysics; Brain; C-terminal; CNR1 gene; Cannabis; Carrier Proteins; Characteristics; Citalopram; Cocaine; Complex; Coupled; Couples; Coupling; Crystallization; Crystallography; DRD2 gene; Detergents; Development; Dissociation; Dopamine; Dopamine Receptor; Drug Targeting; Environment; Exhibits; Functional disorder; G protein-coupled inwardly-rectifying potassium channel; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Goals; Heroin; Hippocampus (Brain); Human; Hydrophobicity; Imipramine; Impairment; In Vitro; Incidence; Indoles; Investigation; Lead; Ligand Binding; Ligand Binding Domain; Ligands; Mediating; Modeling; Modification; Molecular; Molecular Conformation; Mus; Mutagenesis; Mutation; N-terminal; Nervous system structure; Neurons; Neurotransmitters; Overdose; Pattern; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Play; Positioning Attribute; Protein Conformation; Proteins; Protocols documentation; Rattus; Reporting; Research; Research Subjects; Risperidone; Role; Serotonin; Serotonin Receptor 5-HT1A; Signal Transduction; Signaling Protein; Site; Structure; Structure-Activity Relationship; Substance Use Disorder; Surface; Temperature; Testing; Tetrahydrocannabinol; Therapeutic; Therapeutic Intervention; Toxic effect; Transducers; Treatment Efficacy; Vestibule; Western Blotting; addiction; base; conformational conversion; design; dopamine transporter; drug discovery; drug of abuse; eticlopride; extracellular; in silico; in vivo; inhibitor/antagonist; insight; migration; nervous system disorder; non-cannabinoid; novel; positive allosteric modulator; preservation; radioligand; receptor; recruit; response; serotonin transporter; sigma-1 receptor; simulation; small molecule; synergism; synthetic cannabinoid; synthetic opioid; therapeutic development; therapy development; trend; virtual

Aim 1. Structure-function investigations of G-protein coupled receptors By analyzing and simulating inactive conformations of the highly homologous dopamine D2 and D3 receptors (D2R and D3R) revealed by crystallography, we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na+ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the divergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D2R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands. We have previously identified a G protein-biased agonist of D2R that exhibits impaired -arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. In the new study, we show that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a micro-switch for regulating receptor interactions with -arrestin. As this residue is relatively conserved among class A GPCRs, we constructed analogous mutations within other GPCRs and found that these alterations similarly impaired -arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active state structure of the 2-adrenergic receptor (2R), to build 2R-WT and 2R-Y1995.38A models in complex with the full 2R agonist BI-167107 for MD simulations. These analyses identified conformational rearrangements in 2R-Y1995.38A that propagate from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in 2R-Y1995.38A, which is predicted to affect its interactions with -arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions resulting in biased signaling. When compared to cannabis, the misuse of SCs is associated with a higher incidence of serious adverse effects, suggesting the possible involvement of non-cannabinoid sites of action. Here, we find that indole-based SCs, AM2201 and JWH-018, display minimal orthosteric interaction with several serotonin (5-HT), dopamine, and adrenergic receptors, but act as positive allosteric modulators (PAMs) at the 5-HT1A receptor (5-HT1AR), which is not observed with 9-tetrahydrocannabinol. This suggests that certain biological effects of SCs might involve allosteric interactions with 5-HT1ARs. To test this hypothesis, we examined effects of AM2201 on 5-HT1AR agonist-activated G protein-coupled inwardly-rectifying potassium channel currents and the hypothermic response to 5-HT1AR stimulation in mice lacking the cannabinoid receptor 1. We found that both 5-HT1AR-mediated responses were potentiated by AM2201, suggesting that PAM activity at 5-HT1AR may represent a novel non-cannabinoid receptor mechanism underlying their adverse effects. Aim 2. Structural basis of atypical and allosteric modulators of DAT and SERT For SERT, it has been long known that there exists a low-affinity allosteric binding site for an inhibitor S-citalopram. Binding to the allosteric site impedes dissociation of antidepressants from the high-affinity site, which may enhance antidepressant efficacy. SERT crystal structures reveal two S-citalopram binding pockets in the central binding (S1) site and the extracellular vestibule (S2 site). Our combined in vitro and in silico analysis indicates that the bound S-citalopram or imipramine in S1 is allosterically coupled to the ligand binding to S2 through altering protein conformations. Remarkably, a novel SERT inhibitor Lu AF60097, the first high-affinity S2-ligand reported and characterized here, allosterically couples the ligand binding to S1 through a similar mechanism. Mutagenesis of selected Lu AF60097 interacting residues in this pose reduces its allosteric potency. Importantly, the in vivo allosteric inhibition of SERT by Lu AF60097 is demonstrated by the potentiated imipramine binding and increased hippocampal serotonin level in rats. Together, we show the molecular mechanism of the allosteric coupling between S1 and S2 binding and the first lead compound towards developing high-affinity SERT allosteric inhibitors. Our findings suggest that the role of the extracellular vestibule is evolutionarily conserved among NSS as a druggable binding site for small-molecule ligands, and can be exploited for drug discovery. Aim 3. Molecular mechanisms of 1R homomerization Western blot assays have been widely used to investigate protein oligomerizations. However, the unique topology of 1R renders several intertwined challenges in western blot. We have developed a western blot protocol without temperature denaturization to study the ligand binding effects on the oligomerization state of 1R. Using this approach, we observed an unexpected ladder-like incremental migration pattern of 1R, demonstrating preserved homomeric interactions in the detergent environment. We compared the migration patterns of the intact 1R construct and the C-terminally tagged 1R constructs, and found similar trends in response to drug treatments. In contrast, N-terminally tagged 1R constructs show opposite trends to that of the intact construct, suggesting distorted elicitation of the ligand binding effects on oligomerization. Together, our findings indicate that the N-terminus plays an important role in eliciting the impact of bound ligands, whereas the C-terminus is amenable for modifications for biochemical studies. The E102Q mutation of 1R has been found to elicit familial cases of amyotrophic lateral sclerosis. Despite reports of its downstream signaling consequences, the mechanistic details of the functional impact of E102Q at the molecular level are not clear. We investigated the molecular mechanism of the E102Q mutation with a spectrum of biochemical, biophysical, and pharmacological approaches. Our analysis of the interaction network of 1R indicates that a set of residues near E102 is critical for the integrity of the C-terminal ligand-binding domain. However, this integrity is not affected by the E102Q and E102A mutations, which is confirmed by the radioligand binding results. Instead, the E102 mutations disrupt the connection between the C-terminal domain and the N-terminal transmembrane helix (NT-helix). Results from BRET and western blot assays demonstrate that these mutations destabilize higher-order 1R oligomers, while our MD simulations based on a 1R crystal structure reveal a potential mechanism by which the mutations perturb the NT-helix dynamics. Thus, we propose that E102 is at a critical position in propagating the effects of ligand binding from the C-terminal domain to the NT-helix, while the latter may be involved in forming alternative oligomer interfaces, separate from the trimer interface in the crystal structures of 1R. Together, these results provide the first account of the molecular mechanism of 1R dysfunction caused by E102Q.

目的1。g蛋白偶联受体的结构-功能研究