Anatomical Transcriptome of G Protein-Coupled Receptors Leads to the Identification of a Novel Therapeutic Candidate GPR52 for Psychiatric Disorders

<div><p>Many drugs of abuse and most neuropharmacological agents regulate G protein-coupled receptors (GPCRs) in the central nervous system (CNS)_ENREF_1. The striatum, in which dopamine D1 and D2 receptors are enriched, is strongly innervated by the ventral tegmental area (VTA), which is the origin of dopaminergic cell bodies of the mesocorticolimbic dopamine system_ENREF_3 and plays a central role in the development of psychiatric disorders_ENREF_4. Here we report the comprehensive and anatomical transcript profiling of 322 non-odorant GPCRs in mouse tissue by quantitative real-time PCR (qPCR), leading to the identification of neurotherapeutic receptors exclusively expressed in the CNS, especially in the striatum. Among them, GPR6, GPR52, and GPR88, known as orphan GPCRs, were shown to co-localize either with a D2 receptor alone or with both D1 and D2 receptors in neurons of the basal ganglia. Intriguingly, we found that GPR52 was well conserved among vertebrates, is Gs-coupled and responsive to the antipsychotic drug, reserpine. We used three types of transgenic (Tg) mice employing a <i>Cre-lox</i> system under the control of the GPR52 promoter, namely, GPR52-LacZ Tg, human GPR52 (hGPR52) Tg, and hGPR52-GFP Tg mice. Detailed histological investigation suggests that GPR52 may modulate dopaminergic and glutamatergic transmission in neuronal circuits responsible for cognitive function and emotion. In support of our prediction, GPR52 knockout and transgenic mice exhibited psychosis-related and antipsychotic-like behaviors, respectively. Therefore, we propose that GPR52 has the potential of being a therapeutic psychiatric receptor. This approach may help identify potential therapeutic targets for CNS diseases.</p></div

Anatomically comprehensive profiling of mouse GPCR mRNA expression reveals the CNS specific clusters.

<p><b>A,</b> Six CNS specific clusters were highlighted by red square. <b>B,</b> Cluster 4, 11, and 12 indicated by red numbers in (<b>A</b>) were enlarged. Red and Green indicates orphan GPCRs of our interest and adhesion GPCRs, respectively. <b>C,</b> Pie charts of the numbers of orphan and ligand-known GPCRs in all and six CNS specific clusters.</p

Distributions of mGPR52 mRNA in wild type mice and LacZ signals in mGPR52-LacZ Tg mice.

<p>mGPR52 mRNA.</p><p>+++, highest; ++, moderate density; +, low density;</p><p>mGPR52-LacZ Tg mice.</p><p>○, LacZ-positive.</p

Gene expression distribution and protein localization of GPR52.

<p><b>A,</b> GPR52 mRNA is abundantly expressed in human brain. GPR52 mRNAs were quantified by qPCR throughout human tissues. Data represent the ratios of GPR52 to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA. <b>B–C,</b> ISH investigation of GPR52 mRNA was performed in rat striatum and medial prefrontal cortex. Double-ISH analysis of GPR52 with DRD1/DRD2 in adult male rats. Localizations of GPR52 and DRD1/DRD2 mRNAs were shown as brown and blue signals, respectively, in striatum (<b>B</b>) and medial prefrontal cortex (<b>C</b>). Allow indicates the double-stained neurons. Bar: 25 µm. <b>D,</b> Distribution of LacZ signals in GPR52-LacZ Tg mouse brain. Serial frontal brain sections (rostral → caudal) were stained with X-Gal in GPR52-LacZ Tg mouse. Black characters on the left side of the pictures show LacZ-positive cell bodies and fibers while purple characters on the right side show the fibers. Results and abbreviations were summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090134#pone-0090134-t002" target="_blank">Table 2</a>. Bar: 1 mm. <b>E,</b> Double detections of GFP signals with DRD2 in frontal brain sections of hGPR52-GFP Tg mouse. Green and red colors show GFP and DRD2 immunopositive signals, respectively. Bar: 200 µm.</p

Behavioral characterizations of hGPR52 Tg and GPR52 KO mice.

<p><b>A,</b> Spontaneous locomotor activities of hGPR52 Tg and non-Tg mice for 16 hours were monitored under the controlled condition of light. Animals were exposed to light for 12 hours during the experiment. <b>B,</b> Effects of MAP on locomotor activity of hGPR52 Tg and non-Tg mice were shown. Mean±SEM (n = 7–10). **p<0.01, Student's t-test. <b>C-D,</b> Exploratory activity (left) and time spent (right) in the open-field test over 5min (<b>C</b>), and startle reflex (left) and prepulse inhibition of the startle reflex (right) (<b>D</b>) in GPR52 KO and WT mice. Mean±SEM (n = 10). *p<0.05, **p<0.01, Student's t-test.</p

Gene expression of GPCRs enriched in striatum.

<p><b>A,</b> Descending order of ratio of mouse striatal expression per total of all the examined tissue expressions for each GPCR was presented using data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090134#pone.0090134.s010" target="_blank">Table S1</a>. <b>B,</b> Rat brain expressions of the 3 orphan GPCRs and D1 and D2 receptors were quantified by qPCR.</p

GPR52 is well conserved among vertebrates.

<p><b>A,</b> Alignments of GPR52 amino acid sequences. Human GPR52 shares 99.7%, 95.8%, 95.2%, 94.7% and 91.9% identities with its chimpanzee, bovine, mouse, rat and chicken orthologues. <b>B,</b> Phylogenic tree showing protein sequence relationship of the human GPR52 and other GPCRs. CHRM: Muscarinic acetylcholine receptor families. HRH: Histamine receptor families. MTNR: Melatonin receptor families. DRD: Dopamine receptor families. HTR: 5-hydroxytryptamine receptor families. TAAR: Trace amine-associated receptor families.</p

GPR52 is a Gs-coupled receptor activated by antipsychotics reserpine.

<p><b>A,</b> The chemical structure of reserpine. <b>B–C,</b> Reserpine evoked calcium influx via GPR52 in HEK293 stably expressing mutated CNG channels (<b>B</b>). Calcium influx was elicited by reserpine from 1 to 100 µM when GPR52 was expressed. Meanwhile, Reserpine failed to exert calcium influx when the orphan GPCR, GPRC5D, was expressed (<b>C</b>). Representative dose-response curves were shown in <b>B</b> and <b>C</b>. <b>D,</b> GPR52-expressing CHO cells induced cAMP rise in a dose-dependent manner by reserpine from 10 to 100 µM whereas Mock- and TGR5-CHO cells failed to respond to reserpine. TGR5 is a Gs-coupled receptor<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090134#pone.0090134-Kawamata1" target="_blank">[53]</a>. Mean ± SEM (n = 4). *p<0.025, Williams test. <b>E,</b> Internalization of GPR52-GFP was induced by reserpine. GFP-fused GPR52 in CHO cells was stimulated by 30 µM recerpine for 30 min whereas GFP-fused FPRL1 did not. Bar: 20 µm.</p

Distribution of GPR52 mRNA in the rat central nervous system.

<p>+++, highest; ++, moderate density; +, low density;</p