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

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Abstract

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-HT2A receptor (5-HT2AR). Using classical pharmacology and targeted signaling pathway assays, previous findings suggest that clozapine and other atypical antipsychotics behave principally as 5-HT2AR 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-HT2AR or the H452Y variant of the gene encoding the 5-HT2AR receptor. This naturally occurring variation within the 5-HT2AR 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 (10−5 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-HT2AR-H452Y construct. Together, these findings suggest that clozapine behaves as an agonist inducing phosphorylation of numerous pathways downstream of the 5-HT2AR, and that the single nucleotide polymorphism encoding 5-HT2AR-H452Y affects these clozapine-induced phosphorylation-dependent signaling networks.

Introduction

G protein-coupled receptors (GPCRs) are plasma membrane proteins composed of seven transmembrane-spanning domains that transmit signaling inputs including light, hormones, peptides and neurotransmitters from the extracellular milieu to a profusion of intracellular signaling cascades [1]. Most of the previous studies related to GPCR-dependent signaling focused their interest on their principal immediate downstream target: the heterotrimeric guanine nucleotide proteins (G proteins). Heterotrimeric G proteins are divided into four families according to their primary sequence and functional properties (Gs, Gi/o, Gq/11 and G12/13). The Gs proteins behave as stimulators of adenylyl cyclases (AC), resulting in increased levels of cyclic AMP (cAMP) and activation of protein kinase A (PKA), whereas the Gi/o proteins are usually defined as inhibitory with AC and potassium channels as their main targets. The Gq/11 proteins stimulate phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and Ca2+ release from intracellular stores via the phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3) signaling pathway, whereas G12/13 proteins act as activators of the RhoA small GTP binding protein and phospholipase D (PLD), which ultimately regulate cell shape and motility. Although these G protein-dependent mechanisms play fundamental roles in multiple cell processes, other pathways such as Akt, JAK-STAT, MAPK, mTOR, NF-κB, and TGF-β, are also crucial components of signaling cascades in multicellular organisms. It is then clear that the use of classical biochemical tools to study GPCR-dependent signaling, such as [35S]GTPγS binding, AC activity, or Ca2+ release from the endoplasmic reticulum, may not provide sufficient information about how GPCR activation upon agonist binding affects the activity of multiple signaling cascades within individual cells. This is particularly relevant in light of relatively recent findings suggesting that GPCRs may also signal through G protein-independent pathways such as those regulated by β-arrestin [2]. The primary role of β-arrestin is to bind phosphorylated GPCRs to induce receptor uncoupling from heterotrimeric G proteins. More recent findings also suggest that β-arrestin can also function as an adaptor protein that associates the tyrosine kinase Src via SH3 domain interactions, which leads to activation of the MAPK (mitogen-activated protein kinase) signaling cascade. However, most of the studies focused on GPCRs and β-arrestin use as readouts experimental assays that test direct binding between GPCR and β-arrestin using biophysical assays such bioluminescence resonance energy transfer (BRET), yet do not directly test whether this recruitment of β-arrestin affects signaling downstream [3]. Together, these findings point to the need of further investigations about signaling networks affected by GPCRs in living mammalian cells [1].

The serotonin (or 5-hydroxytryptamine, 5-HT) 5-HT2A receptor (5-HT2AR) is a class A GPCR whose canonical pathway upon agonist administration involves activation of Gq/11 proteins. This receptor is the target responsible for most of the signaling and behavioral effects induced by psychedelic drugs, such as lysergic acid diethylamide (LSD) and psilocybin and its active compound psilocin [4]. Additionally, drugs clinically used to reduce psychosis in patients with neuropsychiatric conditions, such as schizophrenia, Parkinson’s disease and bipolar disorder, show a high affinity for the 5-HT2AR [5], [6]. When testing canonical pathways downstream of 5-HT2AR, most of these second generation of atypical antipsychotic medications, such as clozapine, olanzapine and risperidone, behave as either neutral antagonists or inverse agonists [7], [8], [9]. However, most recent findings have suggested that, either indirectly through a physical interaction with other GPCRs, or directly via the 5-HT2AR, clozapine may show agonistic properties in both heterologous expression systems and in vivo in rodent models. Thus, clozapine was able to potentiate Gi/o protein-dependent signaling in cells expressing 5-HT2AR and the Gi/o protein-coupled metabotropic glutamate receptor 2 (mGluR2) as a GPCR heterocomplex [10]. Similarly, clozapine acts as an agonist at 5-HT2AR to activate Akt via a β-arrestin-independent mechanism [11].

Clozapine was described in 1958 as a “tricyclic antidepressant but with neuroleptic properties”. Today, clozapine is still an atypical antipsychotic that has been demonstrated to be superior for the treatment of refractory schizophrenia patients [7]. Interestingly, a naturally occurring single nucleotide polymorphism (SNP) within the 5-HT2AR (Htr2a) gene has repeatedly been associated with poor clozapine treatment response. Thus, schizophrenia patients carrying a H452Y polymorphism (rs6314) at the 5-HT2AR gene responded less to clozapine treatment, demonstrating that the Tyr452 allele occurred more frequently in the non-responders that in the responders [12], [13]. Using a proteomic approach based on peptide affinity chromatography followed by mass spectrometry and immunoblotting, previous findings by other groups showed a battery of proteins that interact with the C-terminal tail of the 5-HT2CR in mouse choroid plexus samples [14]. Although interesting, these studies were carried out under steady-state conditions, and therefore the effect of drug administration to the signaling machinery downstream of 5-HT2AR remains largely unexplored. Using transfected NIH3T3 mammalian cells as an experimental system, previous findings convincingly demonstrated that the H452Y polymorphism affects canonical pathways downstream 5-HT2AR, such as heterotrimeric G protein activation and phosphoinositide hydrolysis [15]. Herein we extend previous findings regarding the molecular pharmacology characterization of 5-HT2AR-H452Y expressed in mammalian cells. In addition, using a mass-spectometry (MS)-based phosphoproteomics method that enables the identification of thousands of phosphorylated peptides in a single experiment, we tested the effect of clozapine on kinase network statuses in HEK293 cells stably expressing 5-HT2AR or the variant 5-HT2AR-H452Y.

Section snippets

Materials

All the reagents for tissue culture procedures were obtained from Life Technologies (Waltham, MA, USA). [3H]ketanserin, [3H]myo-inositol (20.3 Ci/mmol) and RNA Binding YSi SPA Beads were purchased from PerkinElmer Life Science (Waltham, MA, USA). Doxycycline and lysergic acid diethylamide (LSD) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). 5-hydroxytryptamine (5-HT), (±)2,5-dimethoxy-4-iodoamphetamine (DOI), mianserin and clozapine were purchased from Tocris (Minneapolis, MN, USA).

Receptor fusions with fluorescent proteins and site-directed mutagenesis

Subcellular distribution in living cells and density of 5-HT2AR and 5-HT2AR-H452Y

Previous observations of the Flp-In T-Rex HEK293 cell line inducibly expressing 5-HT2AR by epifluorescence microscopy revealed a peculiar distribution of the fluorescent signal in the form of punctate aggregates all through the cytoplasmic area of the cells [16], [23]. This particular cellular distribution was confirmed in the present set of experiments as shown in Fig. 1A. By contrast, cells expressing 5-HT2AR-H452Y presented a fluorescent signal more homogeneously distributed and concentrated

Discussion

Single nucleotide polymorphisms resulting in the substitution of an amino acid in the primary structure of a given protein may confer alterations in its functionality at the physiological level. Particularly in the case of GPCRs, these polymorphisms could involve pharmacological implications such as alterations in the risk of developing a disease and/or alterations of the response to therapy [31]. One of the genetic variants of the human 5-HT2AR is a non-synonymous single nucleotide

CRediT authorship contribution statement

Sandra M. Martín-Guerrero: Software, Validation, Formal analysis, Investigation, Visualization. Paula Alonso: Validation, Formal analysis, Investigation. Alba Iglesias: Validation, Formal analysis, Investigation. Marta Cimadevila: Validation, Formal analysis, Investigation. José Brea: Supervision, Visualization. M. Isabel Loza: Resources, Supervision, Visualization, Funding acquisition. Pedro Casado: Supervision, Software, Visualization. David Martín-Oliva: Supervision, Visualization. Pedro R.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was funded by the following grants and agencies: Proyecto RTI2018-079344-BI00 (FEDER/Minsterio de Ciencia e Innovación – Agencia Estatal de Investigación) to J.F.L-G. NIH R01 MH084894, NIH R01 MH111940, and NIH P30 DA033934 to J.G.-M. S.M.M-G was funded by a grant from Ministerio de Educación, Cultura y Deporte, Spain (FPU14/02219). Spanish Ministry of Economy and Competitiveness (SAF2017-85225-C3-1-R) and the European Regional Development Fund (ERDF) to M.I.L.

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    Present address: Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London SE5 9RX, UK.

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