GPCR oligomers in pharmacology and signaling

G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of more than half of the current therapeutic drugs on the market. When activated by an agonist, the GPCR undergoes conformational changes that facilitate its interaction with heterotrimeric G proteins, which then relay signals to downstream intracellular effectors. Although GPCRs were thought to function as monomers, many studies support the hypothesis that G protein coupling involves the formation of GPCR homo- and/or hetero-complexes. These complex systems have been suggested to exhibit specific signaling cascades, pharmacological, internalization, and recycling properties. In this review, we summarize recent advances in our understanding of the structure, function and dynamics of GPCR complexes, as well as the findings obtained in animal models

Influence of 2,5-dimethoxy-4-iodoamphetamine (DOI) on ethanol preference and consumption in C57BL/6 male mice

Substance use disorders (SUD) account for a large number of mental health diagnosis in the United States and around the world. Approximately 13.6 million adults 26 or older and 5.1 million young adults (ages 18-24) battled with a SUD in 2017. Alcohol Use Disorder (AUD) alone effects nearly 6% of the adult population within the United States. This creates a substantial burden on the individual, with alcohol being the third-leading cause of preventable death in the United States. Few treatments for AUD exist, with no new FDA-approved therapeutic treatments within the last 15 years. Additionally, the limited treatments we do have are estimated to produce sustained abstinence in less than 20% of individuals. Psychedelics, such as lysergic acid diethylamide (LSD), psilocybin and 2,5-dimethoxy-4-iodoamphetamine (DOI) affect processes related to cognition, perception and sensory processing. Recently, it has been demonstrated that serotonin 5-HT2A receptor agonists, such as psilocybin, can be useful in attenuating substance abuse. As an example, clinical findings have demonstrated the ability of psilocybin to decrease heavy drinking days in alcoholic heavy drinkers. Studies utilizing both rats and mice have also suggested the ability of DOI to decrease ethanol preference and consumption in a two-bottle choice paradigm of drinking behavior. The present study aimed to assess the ability of two acute doses of DOI (2 mg/kg and 5 mg/kg) on ethanol preference and consumption using the two-bottle choice paradigm. To test this, 15 adult male C57BL/6 mice were kept on a reverse light cycle and trained on the two-bottle choice procedure, in which they were allowed to drink for four weeks to obtain a baseline reading of drinking behavior. Following these four weeks, mice were assigned to either the treatment group or vehicle group based on weight and baseline drinking behavior. On the first day of the fifth and sixth weeks, mice were injected intraperitoneally with a dose of DOI or saline vehicle 30 minutes prior to access to 20% ethanol. The amount consumed of both water and ethanol on drinking days was measured at 2- and 24-hours and analyzed to calculate consumption and preference. Overall, our findings suggest that DOI did not affect ethanol consumption or preference at the 2- or 24-hour measures. The DOI-treated group showed no difference from the vehicle-treated group after receiving an either moderate (2 mg/kg) or high (5 mg/kg) dose of DOI. There was an overall interaction of time and treatment in both 2- and 24-hour fluid consumption in the groups. Further studies are warranted using DOI or other psychedelics and other paradigms for assessing drinking behavior to understand the effects of psychedelics in modulating substance use behavior.https://scholarscompass.vcu.edu/gradposters/1156/thumbnail.jp

Elucidation of molecular kinetic schemes from macroscopic traces using system identification

Overall cellular responses to biologically-relevant stimuli are mediated by networks of simpler lower-level processes. Although information about some of these processes can now be obtained by visualizing and recording events at the molecular level, this is still possible only in especially favorable cases. Therefore the development of methods to extract the dynamics and relationships between the different lower-level (microscopic) processes from the overall (macroscopic) response remains a crucial challenge in the understanding of many aspects of physiology. Here we have devised a hybrid computational-analytical method to accomplish this task, the SYStems-based MOLecular kinetic scheme Extractor (SYSMOLE). SYSMOLE utilizes system-identification input-output analysis to obtain a transfer function between the stimulus and the overall cellular response in the Laplace-transformed domain. It then derives a Markov-chain state molecular kinetic scheme uniquely associated with the transfer function by means of a classification procedure and an analytical step that imposes general biological constraints. We first tested SYSMOLE with synthetic data and evaluated its performance in terms of its rate of convergence to the correct molecular kinetic scheme and its robustness to noise. We then examined its performance on real experimental traces by analyzing macroscopic calcium-current traces elicited by membrane depolarization. SYSMOLE derived the correct, previously known molecular kinetic scheme describing the activation and inactivation of the underlying calcium channels and correctly identified the accepted mechanism of action of nifedipine, a calcium-channel blocker clinically used in patients with cardiovascular disease. Finally, we applied SYSMOLE to study the pharmacology of a new class of glutamate antipsychotic drugs and their crosstalk mechanism through a heteromeric complex of G protein-coupled receptors. Our results indicate that our methodology can be successfully applied to accurately derive molecular kinetic schemes from experimental macroscopic traces, and we anticipate that it may be useful in the study of a wide variety of biological systems

Characterization of dopamine D2 receptor coupling to G proteins in postmortem brain of subjects with schizophrenia

Background: Alterations of dopamine D-1 (D1R) and D-2 receptor (D2R) are proposed in schizophrenia but brain neuroimaging and postmortem studies have shown controversial results in relation to D1R and D2R density. Besides, scarce information on the functionality of brain D1R and D2R is available. The present study characterized G-protein activation by D1R and D2R agonists in postmortem human brain. Furthermore, D2R functional status was compared between schizophrenia and control subjects. Methods: G-protein receptor coupling was assessed in control caudate nucleus and frontal cortex by [S-35]GTP gamma S-binding stimulation induced by increasing concentrations (10(-10)-10(-3) M) of dopamine, and the selective dopaminergic agonists SKF38393 (D1R) and NPA (D2R). Concentration-response curves to NPA stimulation of [S-35]GTP gamma S binding were analyzed in antipsychotic-free (n = 10) and antipsychotic-treated (n = 7) schizophrenia subjects and matched controls (n = 17). Results: In caudate, [S-35]GTP gamma S-binding responses to agonists were compatible with the existence of functional D2R. In contrast, stimulations in cortex showed responses that did not correspond to D1R or D2R. [S-35]GTP gamma S-binding activation by NPA in caudate displayed biphasic curves with similar profile in schizophrenia (EC50H = 7.94 nM; EC50L = 7.08 mu M) and control (EC50H = 7.24 nM; EC50L = 15.14 mu M) subjects. The presence or absence of antipsychotic medication did not influence the pharmacological parameters. Conclusions: Feasibility of functional evaluation of dopamine receptors in postmortem human brain by conventional [S-35]GTP gamma S-binding assays appears to be restricted to signalling through inhibitory G(i/o) proteins. These findings provide functional information about brain D2R status in subjects with schizophrenia and do not support the existence of D2R supersensitive in this mental disorder.This work was supported by the Spanish State Research Agency and EDR Funds (SAF-2017-88126-R to JJM;PID2019-106404RB-100 to LFC), the Basque Government (IT1211/19 to JJM ; ELKARTEK Programme KK-2019/00049 to RD-A), and the National Institutes of Health (R01MH084894 & NIH-R01MH111940 to JG-M)

Glucocorticoid receptor dysregulation underlies 5-HT2AR-dependent synaptic and behavioral deficits in a mouse neurodevelopmental disorder model.

Prenatal environmental insults increase the risk of neurodevelopmental psychiatric conditions in the offspring. Structural modifications of dendritic spines are central to brain development and plasticity. Using maternal immune activation (MIA) as a rodent model of prenatal environmental insult, previous results have reported dendritic structural deficits in the frontal cortex. However, very little is known about the molecular mechanism underlying MIA-induced synaptic structural alterations in the offspring. Using prenatal (E12.5) injection with polyinosinic-polycytidylic acid potassium salt as a mouse MIA model, we show here that upregulation of the serotonin 5-HT2A receptor (5-HT2AR) is at least in part responsible for some of the effects of prenatal insults on frontal cortex dendritic spine structure and sensorimotor gating processes. Mechanistically, we report that this upregulation of frontal cortex 5-HT2AR expression is associated with MIA-induced reduction of nuclear translocation of the glucocorticoid receptor (GR) and, consequently, a decrease in the enrichment of GR at the 5-HT2AR promoter. The translational significance of these preclinical findings is supported by data in postmortem human brain samples suggesting dysregulation of GR translocation in frontal cortex of schizophrenia subjects. We also found that repeated corticosterone administration augmented frontal cortex 5-HT2AR expression and reduced GR binding to the 5-HT2AR promoter. However, virally (adeno-associated virus) mediated augmentation of GR function reduced frontal cortex 5-HT2AR expression and improved sensorimotor gating processes via 5-HT2AR. Together, these data support a negative regulatory relationship between GR signaling and 5-HT2AR expression in the mouse frontal cortex that may carry implications for the pathophysiology underlying 5-HT2AR dysregulation in neurodevelopmental psychiatric disorders.National Institutes of Health R01MH084894 (to J. G.-M.), R01MH111940 (to J. G.-M.), NIH-N01DA-17-8932 (to P. M. B.), NIH-N01DA-19-8949 (to P. M. B.), and F30MH116550 (to J. M. S.), and Basque Government IT1211-19 (to J. J. M.) participated in the funding of this study. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

G protein-coupled receptor-effector macromolecular membrane assemblies (GEMMAs)

G protein-coupled receptors (GPCRs) are the largest group of receptors involved in cellular signaling across the plasma membrane and a major class of drug targets. The canonical model for GPCR signaling involves three components the GPCR, a heterotrimeric G protein and a proximal plasma membrane effector that have been generally thought to be freely mobile molecules able to interact by 'collision coupling'. Here, we synthesize evidence that supports the existence of GPCR-effector macromolecular membrane assemblies (GEMMAs) comprised of specific GPCRs, G proteins, plasma membrane effector molecules and other associated transmembrane proteins that are pre-assembled prior to receptor activation by agonists, which then leads to subsequent rearrangement of the GEMMA components. The GEMMA concept offers an alternative and complementary model to the canonical collision-coupling model, allowing more efficient interactions between specific signaling components, as well as the integration of the concept of GPCR oligomerization as well as GPCR interactions with orphan receptors, truncated GPCRs and other membrane-localized GPCR-associated proteins. Collision-coupling and pre-assembled mechanisms are not exclusive and likely both operate in the cell, providing a spectrum of signaling modalities which explains the differential properties of a multitude of GPCRs in their different cellular environments. Here, we explore the unique pharmacological characteristics of individual GEMMAs, which could provide new opportunities to therapeutically modulate GPCR signaling

His452Tyr polymorphism in the human 5-HT2A receptor affects clozapine-induced signaling networks revealed by quantitative phosphoproteomics

Antipsychotic drugs remain the current standard for schizophrenia treatment. Although they directly recognize the orthosteric binding site of numerous monoaminergic G protein-coupled receptors (GPCRs), these drugs, and particularly second-generation antipsychotics such as clozapine, all have in common a very high affinity for the serotonin 5-HT receptor (5-HTR). Using classical pharmacology and targeted signaling pathway assays, previous findings suggest that clozapine and other atypical antipsychotics behave principally as 5-HTR neutral antagonists and/or inverse agonists. However, more recent findings showed that antipsychotics may also behave as pathway-specific agonists. Reversible phosphorylation is a common element in multiple signaling networks. Combining a quantitative phosphoproteomic method with signaling network analysis, we tested the effect of clozapine treatment on the overall level of protein phosphorylation and signal transduction cascades in vitro in mammalian cell lines induced to express either the human 5-HTR or the H452Y variant of the gene encoding the 5-HTR receptor. This naturally occurring variation within the 5-HTR gene was selected because it has been repeatedly associated with schizophrenia patients who do not respond to clozapine treatment. Our data show that short time exposure (5 or 10 min) to clozapine (10 M) led to phosphorylation of numerous signaling components of pathways involved in processes such as endocytosis, ErbB signaling, insulin signaling or estrogen signaling. Cells induced to express the H452Y variant showed a different basal phosphoproteome, with increases in the phosphorylation of mTOR signaling components as a translationally relevant example. However, the effect of clozapine on the functional landscape of the phosphoproteome was significantly reduced in cells expressing the 5-HTR-H452Y construct. Together, these findings suggest that clozapine behaves as an agonist inducing phosphorylation of numerous pathways downstream of the 5-HTR, and that the single nucleotide polymorphism encoding 5-HTR-H452Y affects these clozapine-induced phosphorylation-dependent signaling networks

Dopaminergic control of ADAMTS2 expression through cAMP/CREB and ERK: molecular effects of antipsychotics

© The Author(s) 2019.A better understanding of the molecular mechanisms that participate in the development and clinical manifestations of schizophrenia can lead to improve our ability to diagnose and treat this disease. Previous data strongly associated the levels of deregulated ADAMTS2 expression in peripheral blood mononuclear cells (PBMCs) from patients at first episode of psychosis (up) as well as in clinical responders to treatment with antipsychotic drugs (down). In this current work, we performed an independent validation of such data and studied the mechanisms implicated in the control of ADAMTS2 gene expression. Using a new cohort of drug-naïve schizophrenia patients with clinical follow-up, we confirmed that the expression of ADAMTS2 was highly upregulated in PBMCs at the onset (drug-naïve patients) and downregulated, in clinical responders, after treatment with antipsychotics. Mechanistically, ADAMTS2 expression was activated by dopaminergic signalling (D1-class receptors) and downstream by cAMP/CREB and mitogen-activated protein kinase (MAPK)/ERK signalling. Incubation with antipsychotic drugs and selective PKA and MEK inhibitors abrogated D1-mediated activation of ADAMTS2 in neuronal-like cells. Thus, D1 receptors signalling towards CREB activation might participate in the onset and clinical responses to therapy in schizophrenia patients, by controlling ADAMTS2 expression and activity. The unbiased investigation of molecular mechanisms triggered by antipsychotic drugs may provide a new landscape of novel targets potentially associated with clinical efficacy.This work was supported by: SAF2016-76046-R and SAF2013-46292-R (MINECO and FEDER) to B.C.F., PI16/00156 (isciii and FEDER) to J.P.V., LUCHAMOS POR LA VIDA project to F.R.J. and J.P.V., SAF2017-83702-R (MINECO and FEDER), Red TERCEL RD12/0019/0024 (ISCIII) and GVA-PROMETEO 2018/041 (Generalitat Valenciana) to S.M. J.P.V. is supported by the RyC research programme (RYC-2013-14097) and F.R.J. by the predoctoral research programme (BES-2014-070615), from MINECO and FEDER