JP7492733B2 - Dopamine D2 receptor-dopa receptor interaction inhibitor peptide - Google Patents

Description

The present invention relates to a peptide that inhibits the interaction between the dopamine D2 receptor and the 3,4-dihydroxyphenylalanine (DOPA) receptor GPR143.

Dopamine is an important neurotransmitter in the brain, and one of its receptors, the D2 receptor, is a therapeutic target for Parkinson's disease and other neuropsychiatric disorders.

Dopa, a precursor of dopamine, is used as the gold standard for treating Parkinson's disease. However, there are many unsolved issues with dopa, such as side effects with unknown mechanisms associated with long-term administration (dyskinesia, low blood pressure, emotional disorders, etc.), loss of efficacy, and rapid fluctuations in efficacy. In addition, there is no consensus as to whether dopa itself suppresses the neurodegenerative process or, conversely, accelerates it. Traditionally, dopamine D2 agonists have been used to compensate for the shortcomings of dopa in the treatment of Parkinson's disease. However, they have been found to have lower efficacy and more side effects than dopa. In addition, D2 receptor inhibitors, which are used for drug addiction and schizophrenia, have been reported to cause many side effects, such as movement disorders and endocrine abnormalities.

GPR143 is a G protein-coupled receptor (GPCR) and has been identified as the causative gene for ocular albinism, a condition in which melanin pigmentation is impaired. The present inventors have demonstrated that GPR143 functions as a dopa receptor (Non-Patent Document 1: Hiroshima et al, Br J Pharmacol, 2014 (171, 2, 403-414). Furthermore, they have demonstrated that GPR143 in vascular smooth muscle regulates blood pressure by forming hetero-oligomers with the adrenergic α1 receptor (α1AR) (Non-Patent Document 2: Masukawa et al, JCI insight, 2017 (2, 18, e90903. doi: 10.1172/jci.insight.90903).

Hiroshima et al, Br J Pharmacol, 2014（171, 2, 403-414） Masukawa et al, JCI insight, 2017 (2, 18, e90903. doi: 10.1172/jci.insight.90903)

The present invention aims to provide new drugs that can treat psychiatric and neurological disorders such as Parkinson's disease by elucidating the functions of GPR143 and D2 receptors in dopamine signaling.

The inventors discovered that the dopa receptor GPR143 interacts with the D2 receptor, which is closely related to the pharmacological effects of dopa, and further invented a peptide sequence that inhibits this interaction and modifies the function of the D2 receptor. This is a completely new technology that has never been seen before.

The gist of the present invention is as follows.
(1) A peptide consisting of the amino acid sequence IPHYVTTYLPLLLVLVANPIL (SEQ ID NO: 1), or a pharma- ceutically acceptable salt or solvate thereof.
(2) The peptide according to (1), to which a cell membrane-permeable peptide has been added, or a pharma-ceutically acceptable salt or solvate thereof.
(3) A medicine comprising the peptide according to (1) or (2), or a pharma- ceutically acceptable salt or solvate thereof.
(4) A preventive and/or therapeutic agent for a disease involving a signal transduction pathway mediated by dopamine D2 receptor, comprising the peptide according to (1) or (2) or a pharma- ceutically acceptable salt or solvate thereof.
(5) The prophylactic and/or therapeutic agent according to (4), wherein the disease involving a signal transduction pathway mediated by dopamine D2 receptor is at least one disease selected from the group consisting of Parkinson's disease, schizophrenia, bipolar disorder, depression, mania, addiction, and attention deficit hyperactivity disorder.
(6) An inhibitor of a signal transduction pathway mediated by dopamine D2 receptor and dopa receptor GPR143, comprising the peptide according to (1) or (2) or a pharma- ceutically acceptable salt or solvate thereof.
(7) A method for screening a substance effective for preventing and/or treating a disease involving a signal transduction pathway mediated by dopamine D2 receptor, using inhibition of the interaction between dopamine D2 receptor and dopa receptor GPR143 as an index.
(8) The method according to (7), further comprising using as an indicator at least one effect selected from the group consisting of the following (a), (b) and (c) in cells co-expressing dopamine D2 receptor and dopa receptor GPR143:
(a) Inhibition of the decrease in cAMP production induced by dopamine D2 receptor agonists
(b) Inhibition of dopamine D2 receptor agonist-induced increase in GSK3β phosphorylation level
(c) Inhibition of an increase in the phosphorylation level of DARPP32 induced by a dopamine D2 receptor agonist. (9) The method according to (7) or (8), further comprising the use of inhibition of the action of a dopamine D2 receptor agonist and/or a dopamine D2 receptor antagonist in a non-human animal as an indicator.
(10) The method according to (9), wherein the effect of the dopamine D2 receptor agonist in a non-human animal is a decrease in behavioral response.
(11) The method according to (9), wherein the effect of the dopamine D2 receptor antagonist in a non-human animal is catalepsy.
(12) The method according to any one of (7) to (11), wherein the disease involving a signal transduction pathway mediated by a dopamine D2 receptor is at least one disease selected from the group consisting of Parkinson's disease, schizophrenia, bipolar disorder, depression, mania, addiction, and attention deficit hyperactivity disorder.

This invention is expected to lead to the creation of drugs that are more selective and have fewer side effects than agonists or antagonists that directly target G protein receptors (GPCRs), including dopamine receptors.

The interaction between GPR143 and the D2 receptor may be a new target for developing treatments for neuropsychiatric disorders.

Signaling through D2DR required striatal GPR143. (A) Summary effects of quinpirole (QNP, 0.01-0.1 mg/kg, i.p.) or saline (SAL) to induce locomotor activity (n=5-7, left). Time course (center) and total (right) locomotor activity in QNP (0.03 mg/kg, i.p.) or SAL to induce hypolocomotion in wild-type (WT) and Gpr143-/y (10) mice. (B) Summary effects of SKF81297 (SKF, 1.0-5.0 mg/kg, i.p.) or SAL to induce locomotor activity (n=6-8, left). Time course (middle) and total (right) locomotor activity in SKF (2.5 mg/kg, i.p.) or SAL induced hyperlocomotor activity in WT and Gpr143-/y (10) mice. (C) EGFP (a,b) and GPR143 immunoreactive (b,f) signals in the striatum of KO mice infected with AAV-DJ-GPR143-P2A-EGFP (a-d) or -EGFP (e-h). GPR143 signals (arrowheads) were observed in GPR143-P2A-EGFP (b) but not in the striatum expressing EGFP (f). Magnified images of the merge are shown in (d,h). Blue signal indicates DAPI. Scale bars: 50 (a-c,e-g), 20 μm (d,h). (D) Time course (left) and total (right) locomotor activity in quinpirole (0.03 mg/kg, i.p.) induced hypolocomotion in WT and GPR143-KO mice. (E) Changes in phosphorylated protein levels in the striatum after quinpirole (0.3 and 3 mg/kg, i.p.) treatment. Protein levels of pDARPP32 (T75), DARPP32, pGSK3β(S9), GSK3β, pERK (T42, 44), ERK, pAkt (T473), and Akt in the striatum of WT and GPR143-KO (KO) mice. Summary effects of quinpirole were shown by calculating the ratio of phosphoprotein/total protein, with WT 0 mg/kg control taken as 100%. All values are means ± SEM. *P<0.01, **P<0.01, ***P<0.001, one-way ANOVA with Tukey's multiple comparison test. Expression patterns of GPR143 and DRD1 or D2DR in the striatum. (A) In situ hybridization signals in mouse striatum. The blue signal of GPR143 mRNA was detected with the antisense (left) but not with the sense (right) probe. (B) Double staining of GPR143 mRNA (green) and DARPP32. Scale bar, 100 μm. The fifth transmembrane domain (TM5) of GPR143 is required for enhancement of D2DR signaling. (A) Effect of GPR143 and GPR37 on pGSK3β(S9) and GSK3β protein levels after treatment with quinpirole (QNP, 0.1 and 0.1 μM) in CHO cells expressing D2DR. (B) Summary of data from (A) (quantification and graphs). (C) Schematic of chimeric receptor. (D) Effect of GPR143 and chimeric receptor on pGSK3β(S9) and GSK3b protein levels after treatment with quinpirole (QNP, 0.01 and 0.1 μM) in D2DR expressing CHO cells. (E) Summary of data from (D) (quantification and graphs). (F) Effect of TAT-TM5 peptide on GPR143 enhancement of quinpirole-induced pGSK3β(S9). (G) Summary of data from (F) (quantification and graphs). All values are means ± SEM. *P <0.1, ** P<0.01, *** P<0.001, #P <0.01, two-way ANOVA with Bonfferoni's multiple comparison test. TAT-TM5 peptide inhibited D2DR signaling in CHO cells. (A) Effect of cell-penetrating peptide TAT fusion-TM5 (TAT-TM5) peptide on the interaction signal between D2DR and GPR143 in CHO cells co-expressing D2DR and GPR143 using in situ proximity ligation assay. Scale bar, 10 μm. (B) Quantitative analysis of the interaction signal in (A). (C) Effect of GPR143 on quinpirole (0.05-50 μM) binding to [3H]-raclopride binding (4 nM) in CHO cells expressing D2DRR with or without GPR143. (D) Effect of quinpirole (0.001-10 μM) on cAMP accumulation in CHO cells expressing D2DRR with or without co-expression of GPR143. (E) Effect of TAT-TM5 on quinpirole (10 nM)-induced decrease in cAMP accumulation in CHO cells expressing D2DRR with or without co-expression of GPR143. (F) Values are means ± SEM. *P<0.01, ** P<0.01, ***P<0.001, #P<0.01, two-way ANOVA with Bonfferoni's multiple comparison test. TM5-TAT peptide inhibited D2DR signaling in mouse striatum. (A) Effects of TAT-TM5 or TAT-control peptide (100 pmol, i.c.v.) on quinpirole (0.02 mg/kg, i.p.) induced hypolocomotion. (B) Summary of the effects in (A) (graphed). (C) Effect of TAT-TM5 on quinpirole (0.3 mg/kg, i.p.)-induced increase in pGSK3β and pDARPP32 protein levels in mouse striatum. (D) Summary of the effects in (C) (quantified and graphed). *P<0.1, **P<0.01, ***P<0.001, #P<0.01, one-way ANOVA with Tukey's multiple comparison test. Changes in pGSK3β and GSK3β protein levels in the frontal cortex (A) and hypothalamus (B) of WT and GPR143-KO mice after treatment with quinpirole (0.3 and 3 mg/kg, i.p.). The summarized effect of quinpirole was calculated by the ratio of pGSK3β/GSK3β. All values are means ± SEM. Effect of quinpirole (10 nM) on dopamine release in the striatum. Values are expressed as percentage of control. All values are means ± SEM. Changes in pGSK3β and GSK3β protein levels in CHO cells expressing D1 (A) and D3 (B) after treatment with quinpirole (1 and 10 μM). The summarized effect of quinpirole was calculated by the ratio of pGSK3β/GSK3β. Effect of TAT transmembrane (1-4, 6 and 7) peptides on the interaction of D2DR and GPR143 using in situ PLA (green). Blue indicates DAPI signal. Scale bar, 10 μm. Graph shows dot counts/cell of in situ PLA signal (n=6, two independent experiments). All values are mean ± SEM. Changes in cAMP accumulation in CHO cells expressing D1 (A) and D3 (B) after treatment with quinpirole (QNP, 0.001-10 μM). The summarized effects of quinpirole were calculated using mock-vehicle as a control. All values are means ± SEM. Effects of TAT-TM5 or TAT-control peptide (100 pmol, i.c.v) on the time course of catalepsy test after haloperidol (0.5 mg/kg, i.p.) treatment in WT and GPR143-KO mice. All values are means ± SEM. **P<0.01, #P<0.01, two-way ANOVA with Bonfferoni's multiple comparison test. Chromatogram of TAT-TM5. Mass spectrum of TAT-TM5.

The present invention is described in detail below.

The present invention provides a peptide consisting of the amino acid sequence IPHYVTTYLPLLLVLVANPIL (SEQ ID NO: 1), or a pharma- ceutically acceptable salt or solvate thereof.

The peptide of the present invention is a peptide encoding the fifth transmembrane domain (TM5) of mouse-derived GPR37. The amino acid sequence of GPR37 is registered in UniProt (https://www.uniprot.org/) as Prosaposin receptor GPR37 under the number Q9QY42, and the peptide corresponding to the region of amino acid numbers 192 to 212 is the peptide of the present invention.

The peptide of the present invention may be added with a cell membrane-permeable peptide. Examples of cell membrane-permeable peptides include TAT peptide and small molecule compounds having an equivalent effect.

The peptide of the present invention can be produced by known genetic engineering techniques. For example, the peptide of the present invention can be produced by obtaining DNA encoding the peptide of the present invention, incorporating the obtained DNA into an appropriate expression vector, and then introducing it into an appropriate host to produce it as a recombinant protein (see, for example, Kaoru Saigo and Yumiko Sano, CURRENT PROTOCOLS Compact Edition, Molecular Biology Experimental Protocols, I, II, III, Maruzen Co., Ltd.: Original; Ausubel, F.M. et al., Short Protocols in Molecular Biology, Third Edition, John Wiley & Sons, Inc., New York).

Alternatively, the peptides of the present invention can be produced according to known peptide synthesis methods.

The peptide of the present invention can inhibit the interaction between the dopamine D2 receptor and the dopa receptor GPR143. The peptide of the present invention can inhibit the signal transduction pathway mediated by the dopamine D2 receptor and the dopa receptor GPR143 by inhibiting the interaction between the dopamine D2 receptor and the dopa receptor GPR143, and can therefore be used as an inhibitor of the signal transduction pathway mediated by the dopamine D2 receptor and the dopa receptor GPR143. The peptide of the present invention can be used as a medicine, for example, as a preventive and/or therapeutic drug for diseases involving the signal transduction pathway mediated by the dopamine D2 receptor. Examples of diseases involving the signal transduction pathway mediated by the dopamine D2 receptor include Parkinson's disease, schizophrenia, bipolar disorder, depression, mania, addiction, attention deficit hyperactivity disorder, and the like, and examples of addiction include drug, alcohol, smoking, and gambling addiction. The peptide of the present invention may be administered in the form of a medicamentously acceptable salt or solvate, or in the form of a prodrug.

Examples of pharma- ceutically acceptable salts include acetates, trifluoroacetates, and salts of strong acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, etc.). These salts can be prepared by known methods.

The peptide of the present invention or a pharma- ceutically acceptable salt thereof may form a solvate with a solvent such as water or an organic solvent. Examples of organic solvents include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol, n-amyl alcohol, isoamyl alcohol, t-pentanol, ethyl acetate, acetone, tetrahydrofuran, chloroform, methylene chloride, propylene glycol, methyl ethyl ketone, and dimethyl sulfoxide. The solvate may be pharma-ceutically acceptable.

After being administered to a living body, a prodrug becomes a medicamentously active compound through the action of enzymes or metabolic hydrolysis. An example of a prodrug is an acid derivative known to those skilled in the art, such as an ester produced by reaction with a suitable alcohol, or an amide produced by reaction with a suitable amine.

The peptide of the present invention, its pharma- ceutically acceptable salt, solvate or prodrug can be administered locally or systemically, orally or parenterally, to animals (e.g., mammals such as humans, mice, rats, guinea pigs, rabbits, cats, dogs, pigs, monkeys, sheep, goats, cattle, horses, and birds such as chickens) either alone or together with a pharma- ceutical carrier, diluent or excipient in an appropriate dosage form. The dosage and frequency of administration vary depending on the subject, target disease, symptoms, administration route, and the like. The effective dosage obtained in mice (body weight about 30 g) is 100 pmol (intracerebroventricular administration, 0.4 μg: the molecular weight of TAT-TM5 is 4081), and the human equivalent dose is calculated based on the following formula:
Formula [1]
HED = Animal Dose (mg/kg) x (Animal Body Weight (kg) / Human Body Weight (kg)) ^0.33
The HED for a mouse weighing 60 kg was calculated from the dose (0.4 μg x 1000/30 = 13.32 μg)/kg for a mouse weighing 30 g.
HED = 13.32μg x (0.03 / 60) ^0.33
HED = 13.32μg x 0.0814 μg/kg = 1.08μg.
For example, when used for the prevention and treatment of adult psychiatric and neurological disorders (e.g., schizophrenia, side effects of dopa), the peptide of the present invention, or a pharma- ceutical acceptable salt or solvate thereof may be administered by intravenous injection (preferably continuously or every other day) at a single dose of usually about 1 to 100 μg/kg body weight, preferably about 1 to 10 μg/kg body weight, about 3 to 4 times a day, preferably about 1 to 2 times a day. Similar amounts can also be administered in other parenteral and oral administrations. When symptoms are particularly severe, the dose may be increased according to the symptoms.

Preparations for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such preparations can be manufactured by conventional methods and may contain carriers, diluents, or excipients commonly used in the pharmaceutical field. For example, carriers and excipients for tablets include lactose, starch, sucrose, magnesium stearate, etc.

Examples of preparations for parenteral administration include injections and suppositories, and the injections may be in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Such injections can be prepared in the usual manner, that is, by dissolving, suspending, or emulsifying the peptide of the present invention, its pharma- ceutically acceptable salt, or solvate in a sterile aqueous or oily liquid that is usually used for injections. Aqueous liquids for injection include physiological saline, isotonic liquids containing glucose and other adjuvants, and may be used in combination with appropriate solubilizing agents, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. Oily liquids include sesame oil, soybean oil, etc., and may be used in combination with benzyl benzoate, benzyl alcohol, etc. as solubilizing agents. The prepared injections are usually filled into appropriate ampoules. Suppositories for rectal administration can be prepared by mixing the peptide of the present invention, a pharma- ceutically acceptable salt or solvate thereof with a conventional suppository base.

The oral or parenteral formulations may be prepared in dosage unit forms that correspond to the dosage of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories, and each dosage unit form preferably contains about 200 to 300 mg of the peptide of the present invention, its pharma- ceutically acceptable salt, solvate, or prodrug.

The formulation may contain other active ingredients as long as they do not cause undesirable interactions when combined with the peptide of the present invention, its pharma- ceutically acceptable salt, solvate or prodrug.

The present invention also provides a method for screening for a substance effective in preventing and/or treating a disease involving a signal transduction pathway mediated by the dopamine D2 receptor, using inhibition of the interaction between the dopamine D2 receptor and the dopa receptor GPR143 as an index. The diseases involving a signal transduction pathway mediated by the dopamine D2 receptor have been described above.

To examine whether a candidate substance inhibits the interaction between the dopamine D2 receptor and the dopa receptor GPR143, it is useful to use the in situ PLA method. If the interaction signal between the dopamine D2 receptor and the dopa receptor GPR143 is reduced by adding the candidate substance to cells that co-express the dopamine D2 receptor and the dopa receptor GPR143, it can be determined that the candidate substance inhibits the interaction between the dopamine D2 receptor and the dopa receptor GPR143.

In the screening method of the present invention, at least one action selected from the group consisting of (a), (b) and (c) below in cells co-expressing dopamine D2 receptor and dopa receptor GPR143 can also be used as an indicator.
(a) Inhibition of the decrease in cAMP production induced by dopamine D2 receptor agonists
(b) Inhibition of dopamine D2 receptor agonist-induced increase in GSK3β phosphorylation level
(c) Inhibition of increase in phosphorylation level of DARPP32 induced by dopamine D2 receptor agonist Cells co-expressing dopamine D2 receptor and dopa receptor GPR143 can be prepared as follows. A CHO cell line (or an equivalent cell line) stably expressing D2DR-mCherry is prepared using a plasmid incorporating the G418 gene according to a standard method (Goshima et al, J Neurosci 13(2): 559-567, 1993), and the same stabilized cell line is further forced to express GPR143-flag using a plasmid containing the GPR143-flag in the insert, to prepare a cell line expressing both of these receptors. At this time, co-expression is confirmed using mCherry and flag as markers.

Examples of dopamine D2 receptor agonists include quinpirole, bromocriptine, cabergoline, sumanilole, ropinirole, talipexole, and apomorphine.

As shown in the Examples below, the present inventors have found that in cells co-expressing the dopamine D2 receptor and the dopa receptor GPR143, a dopamine D2 receptor agonist (e.g., quinpirole) inhibits forskolin-stimulated cAMP production. Therefore, if the inhibition of cAMP production is suppressed by adding a candidate substance to this test system, the candidate substance can be determined to be effective in preventing and/or treating a disease involving a signal transduction pathway mediated by the dopamine D2 receptor. The amount of cAMP can be calculated using a Direct cAMP ELISA kit (Enzo Life Science) according to a standard protocol.

In addition, as shown in the Examples below, the present inventors found that in cells co-expressing the dopamine D2 receptor and the dopa receptor GPR143, treatment with TAT-TM5 (a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to which the TAT peptide has been added) suppresses the increase in phosphorylation of GSK3β caused by the dopamine D2 receptor agonist (quinpirole). Therefore, if the increase in phosphorylation of GSK3β caused by the dopamine D2 receptor agonist (quinpirole) is suppressed by adding a candidate substance to cells co-expressing the dopamine D2 receptor and the dopa receptor GPR143, the candidate substance can be determined to be effective in preventing and/or treating diseases involving the signal transduction pathway mediated by the dopamine D2 receptor. The phosphorylation level of GSK3β can be measured by immunoblot analysis.

Furthermore, as shown in the Examples below, the present inventors found that the increase in the protein levels of pGSK3β (Ser9) and pDARPP32 (Thr75) induced by a dopamine D2 receptor agonist (quinpirole) was reduced in the striatum of mice administered TAT-TM5 (a peptide consisting of the amino acid sequence of SEQ ID NO: 1 to which the TAT peptide has been added). This indicates that the inhibition of the increase in the phosphorylation level of DARPP32 induced by a dopamine D2 receptor agonist in the striatum of mice administered a candidate substance can also be used as an indicator for screening. If the addition of a candidate substance inhibits the increase in the phosphorylation level of DARPP32 induced by a dopamine D2 receptor agonist in cells co-expressing the dopamine D2 receptor and the dopa receptor GPR143, the candidate substance can be determined to be effective in preventing and/or treating diseases involving signal transduction pathways mediated by the dopamine D2 receptor. The phosphorylation level of DARPP32 can be measured by immunoblot analysis.

In the screening method of the present invention, the inhibition of the action of dopamine D2 receptor agonists and/or dopamine D2 receptor antagonists in non-human animals can also be used as an indicator.

Examples of non-human animals include mice, rats, guinea pigs, rabbits, cats, dogs, birds such as chickens, pigs, monkeys, sheep, goats, cows, and horses.

Examples of dopamine D2 receptor antagonists include haloperidol, benperidol, chlorpromazine, droperidol, perazine, pimozide, sulpiride, spiperone, and thioridazine.

Examples of dopamine D2 receptor agonists include those mentioned above.

Examples of the effects of dopamine D2 receptor agonists in non-human animals include reduced behavioral responses, suppression of prolactin secretion, suppression of growth hormone, and lowering of blood pressure.

Examples of the effects of dopamine D2 receptor antagonists in non-human animals include catalepsy, inhibition of gastric acid secretion, increased gastrointestinal peristalsis, antiemesis, and decreased or increased sleep time (depending on the dose).

If inhibition of the action of dopamine D2 receptor agonists and/or dopamine D2 receptor antagonists is observed in a non-human animal administered a candidate substance, the candidate substance can be determined to be effective in preventing and/or treating a disease involving a signal transduction pathway mediated by the dopamine D2 receptor.

The present invention will now be described in more detail with reference to examples.
Example 1
Dopamine receptors can regulate a myriad of cellular responses to fine-tune the expression of dopamine-related behaviors and functions. However, the mechanisms of regulation of dopamine signaling are largely unknown. In this study, we show that dopa, an amino acid previously thought to be inactive, fine-tunes dopamine D2DR receptor (D2DR) signaling through an interaction between D2DR and GPR143. In GPR143 gene-deficient (Gpr143-/y) mice, the dopamine D2/3 receptor agonist quinpirole-induced hypolocomotion was attenuated compared to wild-type (WT) mice. Quinpirole increased phosphorylation of DARPP32 at T75 and GSK3β at S9 in the striatum, but this was attenuated in Gpr143-/y mice when compared to WT mice. These changes in D2DR signaling in Gpr143-/y mice were mimicked by disruption of the D2DR-GPR143 interaction with a peptide encoding the fifth transmembrane domain (TM5) and rescued by introduction of GPR143 into the dorsal striatum. These results provide evidence that DOPA regulates D2DR signaling through GPR143 in the striatal dopaminergic system.

The striatum is a key component of the basal ganglia, a group of brain nuclei that control a variety of psychomotor behaviors. The importance of the striatum to basal ganglia function is highlighted by neurological disorders in which striatal function is impaired. Dopamine receptors are involved in many neurological processes, including motivation, pleasure, cognition, memory, learning, and fine motor skills. There are five subtypes of dopamine receptors: D1DR, D2DR, D3DR, D4DR, and D5DR. Dopamine receptors exert a wide variety of functions and play a major role in both normal and pathological conditions (1-3). Drugs that act on D2DR are often used to alleviate symptoms caused by diseases such as Parkinson's disease (PD), schizophrenia, and depression. A limitation of the use of these drugs is that patients suffer from sometimes severe side effects. Dopamine receptors, especially D2DR, are the most important drug targets for schizophrenia and other similar psychiatric disorders, but there is a lack of drugs that can improve negative symptoms and social conformity, and their development is urgently needed. Selective targeting of specific D2DR-mediated signaling pathways may therefore lead to the development of improved treatments for these devastating diseases.

Dopamine receptors are G protein-coupled receptors that belong to two main families (1,4-7): the D1-like receptor subfamily (D1R), which includes the D1 and D5 receptors; and the D2-like subfamily, which includes the D2, D3, and D4 receptors. D1R activates adenylate cyclase (AC) through coupling with Gs/Golf, whereas D2DR couples to G i/o and releases Gα i/o and Gβγ subunits. The function of D2DR has been considered in terms of antagonizing cAMP-dependent signaling. In cAMP-dependent signaling, Gα i subunits bind and inhibit adenylate cyclase, preventing the production of cAMP and activation of protein kinase A (PKA). In striatal neurons, DARPP-32 is the primary target of PKA, and DARPP-32 is involved in many dopaminergic actions, including D2DR inhibition of the cAMP-dependent system. D2DR receptors also alter intracellular signaling through Gβγ subunits, which can act on many intracellular targets.

In PD, dopaminergic afferents to the striatum are lost, altering striatal output through direct and indirect pathways, resulting in impaired motor performance. L-3,4-dihydroxyphenylalanine (L-DOPA) is the most effective therapeutic agent for the treatment of PD and has been considered an inactive amino acid capable of acting through its conversion to dopamine (DA) by aromatic L-amino acid decarboxylase (AADC). We have proposed that DOPA is a neurotransmitter in the central nervous system (CNS) (8). Recently, it has been reported that the G-protein-coupled receptor (GPCR) GPR143 functions as a candidate L-DOPA receptor in vitro (9) and in vivo (10).

Dopa enhances dopamine D2DR receptor (D2DR)-mediated transmission in the midbrain (11). To investigate the in vivo role of GPR143 in dopaminergic neurotransmission, we investigated the effect of the D2DR agonist quinpirole on locomotor activity.

Quinpirole (0.01-0.1 mg/kg, i.p.) dose-dependently decreased spontaneous locomotion in WT mice. The decrease in spontaneous locomotion induced by quinpirole (0.03 mg/kg) was attenuated in Gpr143-/y mice compared to WT mice (Fig. 1A). On the other hand, the DRD1 agonist SKF81297 (1.0-5.0 mg/kg, i.p.) dose-dependently increased spontaneous locomotion in WT mice. The increase in spontaneous locomotion induced by SKF81297 (2.5 mg/kg) in Gpr143-/y mice was similar to that observed in WT mice (Fig. 1B). To ensure the specificity of GPR143 loss of function, we performed a re-expression assay of GPR143 in the striatum of Gpr143-/y (KO) mice using adeno-associated viruses encoding AAV-DJ-CAGGS-GPR143-P2A-EGFP or AAV-DJ-CAGGS-EGFP (Fig. 1C). Re-expression of GPR143-P2A-EGFP (Fig. 1D) in the striatum restored the quinpirole-induced decrease in locomotor activity when compared with the EGFP-expressing group (Fig. 1D), indicating that Gpr143 is involved in D2DR signaling in the striatum.

Next, we examined the localization of GPR143 in the striatum by in situ hybridization assay. GPR143 mRNA signals were detected with an antisense probe, but not with a sense probe (Fig. 2A). Again, GPR143 mRNA signals were expressed in the striatum, and these signals were expressed in DARPP32- and/or D2DR-immunoreactive positive cells (Fig. 2B, C). These results suggested that GPR143 regulates D2DR-mediated responses in the striatum.

To elucidate how GPR143 is involved in behavioral responses to D2DR agonists (Figure 1A), we first measured phosphorylated DARPP32, GSK3β, Akt, and ERK, downstream molecules of D2DR-mediated signaling, by immunoblotting in the striatum. The phosphorylation levels of DARPP32 (Thr75) and GSK3β (Ser9) were increased by quinpirole in the striatum of WT mice. The increase in phosphorylated protein levels by quinpirole was attenuated in Gpr143-/y mice (Figure 1E). Furthermore, phosphorylation of GSK3β was unchanged in the frontal cortex and hypothalamus (Figure 6). In contrast, the levels of p-Akt and p-ERK in Gpr143-/y mice were similar to those in WT mice (Figure 1E). Quinpirole acts on the D2DR autoreceptor and suppresses locomotor activity by negatively regulating dopamine synthesis and release (12). We next measured dopamine release from the striatum using in vivo microdialysis. Quinpirole (100 nM) inhibited dopamine release in Gpr143-/y and WT mice. However, unexpectedly, this effect was observed in Gpr143-/y and WT mice as well (Fig. 7). These findings suggested that the phenotypic defects in Gpr143-/y mice were due to loss of function of GPR143, which is mainly expressed in postsynaptic neurons in the striatum.

GPR143 couples to a1B adrenergic receptors in smooth muscle cells and sensitizes vasoconstrictor responses to sympathetic nerve activity (10). To investigate the possible interaction between D2DR and GPR143, we estimated D2DR-mediated signaling in a reconstituted system. Quinpirole increased p-GSK3β concentrations in CHO cells expressing D2DR-mCherry (Fig. 3). Quinpirole (0.1 μM)-induced increase in p-GSK3β levels in cells coexpressing D2DR and GPR143 was higher than in CHO cells expressing D2DR alone (Fig. 3B). This increase in GSK3β phosphorylation was not observed in cells coexpressing GPR143 with DRD1 or DRD3 (Fig. 8A, B). We next used chimeric receptors to determine the domains of GPR143 required for functional interaction with D2DR. Parkin-related endothelin receptor-like receptor (Pael-R) (GPR37) shares some properties in common with those of GPR143. GPR37 showed an overlapping expression pattern with GPR143, with poor tracking to the plasma membrane. GPR37 and GPR143 localize to Lewy bodies (13). However, unlike GPR143, GPR37 did not enhance quinpirole-induced GSK3β phosphorylation (Fig. 3A, B). Next, we constructed seven chimeric receptors in which each transmembrane (TM) domain of GPR143 was replaced by the corresponding TM domain of GPR37 (TM1-TM7) (Fig. 3C). Coexpression of the TM chimeric GPR143 mutants TM1, 2, 3, 4, 6, and 7 enhanced quinpirole-induced GSK3β phosphorylation in D2DR-expressing cells (Fig. 3D, E). In contrast, the GPR143 mutant TM5 did not enhance the phosphorylation of GSK3β by quinpirole (Fig. 3D, E). This finding indicated that the TM5 domain of GPR143 may play an important role in enhancing D2DR-mediated signaling in CHO cells co-expressing D2DR and GPR143 and may be required for the interaction between D2DR and GPR143. To analyze the function of GPCR heteromerization, we used an HIV transcriptional transactivator (TAT) fusion peptide to block the possibility of heteromerization between D2DR and GPR143. We examined the effect of TAT-TM5 on the enhancement of D2DR-mediated GSK3β phosphorylation by GPR143. Treatment with the TAT-TM5 peptide inhibited the increase in phosphorylation of GSK3β by quinpirole in CHO cells co-expressing D2DR and GPR143 (Fig. 3F, G). To determine whether the interaction between D2DR and GPR143 is required for the enhancement, we employed an in situ PLA method. In cells expressing both D2DR and GPR143, dot signals were observed (Fig. 4A, B). In contrast, no signal was observed in cells expressing D2DR alone (Fig. 4A). Treatment with TAT-TM5 peptide reduced the GPR143-D2DR interaction signal (number of dots/cell) in cells co-expressing D2DR and GPR143 (Fig. 4A, B). TAT-control peptide did not affect the interaction signal between D2DR and GPR143 (Fig. 4A, B). Other TAT-TM peptides, TAT-TM1, 2, 3, 4, 6 and 7 peptides, did not affect the enhancement of D2DR signaling by GPR143, respectively, and did not affect the interaction signal (Fig. 9). These results suggested that heteromerization of D2DR and GPR143 is required for GPR143 enhancement of D2DR signaling.

We also investigated whether the expression of GPR143 affected the binding affinity of quinpirole for the specific binding of the D2DR ligand, [3H]-raclopride, in D2DR-expressing cells, because heteromerization of GPCRs may modify ligand-binding properties. The binding affinity of quinpirole for [3H]-raclopride in cells coexpressing D2DR and GPR143 (Ki = 4.5, n = 4) was similar to that in cells expressing D2DR alone (Ki = 5.3, n = 4) (Figure 4B), thereby indicating that GPR143 does not affect the binding properties of D2DR ligands to D2DR. To further characterize D2DR-mediated signaling in cells coexpressing D2DR and GPR143, we measured cAMP production in cells expressing D2DR and cells coexpressing D2DR and GPR143. Quinpirole inhibited forskolin-stimulated cAMP production in CHO cells expressing D2DR alone and in CHO cells coexpressing D2DR and GPR143 (Fig. 4C, D). However, the inhibition by quinpirole was more markedly enhanced in cells coexpressing D2DR and GPR143 than in cells expressing D2DR alone (Fig. 4C). We also examined the cAMP response to SKF81297 in CHO cells expressing DRD1 with or without GPR143 (Fig. 10). There was no difference between the cAMP response in CHO cells expressing DRD1 or DRD3. Dopa had no effect on cAMP production in cells expressing D2DR alone. These findings suggest that GPR143 selectively functionally couples with D2DR, but not with DRD1 or DRD3, enhancing D2DR-mediated signaling.

To elucidate the in vivo relevance of the interaction between D2DR and GPR143, we examined the effect of TAT-TM5 on D2DR-mediated behavioral phenotypes. Quinpirole reduced locomotor activity in both vehicle- and TAT-TM5-treated mice. However, the suppression of locomotor activity by quinpirole was blunted in TAT-TM5-treated animals compared to vehicle-treated animals (Fig. 5A and B). The inhibitory effect of TAT-TM5 on D2DR-mediated effects was confirmed by the levels of pGSK3β (Ser9) and pDARPP32 (Thr75) in the striatum (Fig. 5C, D). Furthermore, TAT-TM5 administration (icv) suppressed haloperidol-induced catalepsy, a well-characterized D2DR-mediated behavioral response (Fig. S11).

In this study, we report the function of GPR143 as a regulatory component in dopamine signaling and demonstrate that D2DR/GPR143 complex formation is required to enhance dopamine-mediated GSK3β signaling generated by D2DR. Disruption of the D2DR/GPR143 interaction by TAT-TM5 may interfere with the functional coupling between D2DR/GPR143, thereby suppressing the GPR143-induced enhancement of D2DR signaling.

Thus, identification of the D2DR/GPR143 interaction potentially elucidates the mechanism of D2DR signaling diversity.

The physiological relevance of the interaction of D2DR with GPR143 and its regulation of GSK3β signaling is represented by the reduced behavioral response to the D2DR agonist quinpirole and the suppression of the D2DR antagonist haloperidol-induced catalepsy (Figures 1 and 11). This observation is particularly interesting due to ample evidence suggesting that dopa enhances D2DR-mediated actions (11, 14). Furthermore, although synthetic DA agonists have been included in the treatment of PD for decades, dopa remains the most effective therapeutic agent and is widely considered the gold standard in PD treatment (15). This study provides insight into the reasons and underlying mechanisms of the differences between dopa and dopamine agonists.

The data presented here do not exclude the possibility that the behavioral phenotype of GPR143 mice may be due to modulation of other neurotransmitter systems, such as serotonin or noradrenaline, by a similar mechanism. Further studies are needed to elucidate this possibility. However, it is noteworthy that GPR143 functionally interacts with D2DR but not with DRD1 or DRD3. D2DR is widely recognized to have important and diverse roles in dopaminergic transmission. However, compared with DRD1, the physiological consequences of activated D2DR on neurons are less understood (3). We observed that there were D2DR-positive or GPR143-positive, and D2DR and GPR143 double-positive neurons in the striatum. Thus, we provide evidence that dopa-GPR143 signaling may contribute to the diverse functions of D2DR in dopaminergic transmission in the brain.

Drugs that act on the dopamine D2DR receptor (D2DR) are often used to alleviate symptoms caused by diseases such as Parkinson's disease, schizophrenia, and depression. However, a limitation of the use of these drugs is that they sometimes plague patients with severe side effects. Selective targeting of specific D2DR-mediated signaling pathways may lead to the development of improved treatments for these devastating diseases.

Method

Ethics statement All animal care and experimental procedures were carried out in accordance with the recommendations in the Guide for Care and Use of Laboratory Animals at Yokohama City University Graduate School of Medicine (Permit Numbers: FA-14-046 and FA-17-012). Throughout the experiments, every effort was made to minimize the number of animals used and their suffering.

Animals: Gpr143 gene-deficient mice were previously generated (10) on a C57BL/6J genetic background. Litters of GPR143-KO and WT male mice were generated by mating female KO heterozygotes with male WT. All mice (6–12 weeks old) weighing 20–25 g were used and housed in a temperature (23 ± 1°C)- and humidity (55%)-controlled room. They had free access to laboratory mouse chow and water and were maintained on a light-dark cycle (07.00–19.00 h light). Mice were acclimatized to handling by picking them up once a day for at least 3 days before experiments were performed.

Cell culture
CHO cells were obtained from the American Type Culture Collection (Manassas, VA, USA). CHO cells were cultured in F-12 Ham medium. These media were supplemented with 10% FBS, penicillin (100 U mL-1) and streptomycin (100 μg mL-1) and incubated at 37 °C in a humidified atmosphere with 5% CO2. Cells were treated with TAT-TM5 peptide for 15 min before application of quinpirole and/or dopa.

Drugs and TAT-TM peptide
SKF81297 and quinpirole were purchased from Tcris (Bristol, UK). Dopa was purchased from Nacalai Tesque (Kyoto, Japan). The TAT-TM peptide was synthesized on a peptide synthesizer. The sequence of the GPR143 peptide was as follows;
Control TAT, YGRKKRRQRRR (SEQ ID NO:2):
TAT-TM1, VFHALCLGSGTLRLVLGLLQLYGRKKRRQRRR (SEQ ID NO: 3):
TAT-TM2, YGRKKRRQRRRLLGCLGIVIRSTVWIAYPEFI (SEQ ID NO: 4):
TAT-TM3, LLYSACFWWLFCYAVDVYLVIYGRKKRRQRRR (SEQ ID NO: 5):
TAT-TM4, YGRKKRRQRRRILLYHIMAWGLAVLLCVEGAV (SEQ ID NO: 6):
TAT-TM5, IPHYVTTYLPLLLVLVANPILYGRKKRRQRRR (SEQ ID NO: 7):
TAT-TM6, YGRKKRRQRRRIMLVLIACWLSNIINESLLFY (SEQ ID NO: 8):
TAT-TM7, FIMGILNPAQGLLLSLAFYGWYGRKKRRQRRR (SEQ ID NO: 9)
Figure 12 shows the chromatogram of TAT-TM5, in which a peak was detected at a retention time of 17.333 minutes. The compound corresponding to this peak was identified by mass spectrometry (calculated value: 3904.72, measured value: 3904.6) (Figure 13).

Surgery
To perform reversion analysis of GPR143 in GPR143-KO mice, we microinjected adeno-associated virus-DJ (AAV-DJ) containing mouse GPR143.
Mice were anesthetized with isoflurane and head-fixed.

Behavioral analysis For measurements of locomotor activity, mice were allowed to acclimate to the room for at least 1 h before testing. Mice were then individually placed in chambers (50 × 50 × 40 cm3) under 70 lux lighting conditions. The distance traveled (cm) was recorded 60 min before and after treatment with quinpirole or SKF81297. For measurements of haloperidol (0.5 mg/kg, ip)-induced catalepsy, the horizontal bar test was used to assess catalepsy time. The animals' forepaws were placed on a horizontal bar 5 cm above the floor, and the time spent in position was measured. Activity in the open field was quantified using imaging software (TimeOFCR4: O'Hara & Co., Ltd, Tokyo, Japan). Intracerebroventricular (icv) treatment with TAT-peptide was performed 15 min before treatment with quinpirole or haloperidol.

Plasmid construction
In situ hybridization and immunochemistry
For in situ hybridization analysis, mice were deeply anesthetized with isoflurane and intracardially perfusion-fixed with 4% paraformaldehyde/PBS pH 7.4 (4% PFA).

After perfusion, the brains were rapidly removed and stored in 4% PFA overnight at 4°C. After cryoprotection in 10% sucrose for 2 h, 20% for 2 h, and 30% overnight, the brains were embedded in a mixture of O.C.T compound (Sakura Finetechnical). The striatum, including the brain region, was sliced at a thickness of 20 μm. After fixation in 4% PFA for 10 min, the sections were treated with proteinase K for 30 min and then acetylated with 0.45% acetic anhydride in triethylamine hydrochloride. The tissues were then hybridized overnight at 65°C in a solution containing digoxigenin (DIG)-labeled riboprobes. The antisense probe was obtained by linearizing a plasmid containing mouse GPR143 (Genbank accession number 1-949 of 010951.3) in Bluescript SK (+) by PCR using M13 primers (M13-F, 5'-CCCAGTCACGACGTTGTAAAACG (SEQ ID NO: 10); M13-R, AGCGGAATAACATTCACAAGGG (SEQ ID NO: 11)) and transcribing with T3 polymerase. The sense control probe was obtained by transcribing with T7 polymerase. Nonspecific staining was prevented by prior incubation of the tissue with PBS containing 10% bovine serum albumin and normal sheep serum for 90 min at room temperature. The tissue was then incubated overnight at 4°C in anti-DIG conjugated with alkaline phosphatase. The alkaline phosphatase reaction was visualized using nitroblue tetrazolium and 5-bromo-4-chloro-3'-indolyl phosphate substrate. For fluorescent ISH, the interaction signal between D2DR and GPR143 was visualized by Duolink in situ PLA kit (Olink Bioscience) according to the manufacturer's protocol as described above. Specific signals were analyzed using an Olympus FV100 confocal laser scanning microscope. For in situ PLA, the interaction signal between D2DR and GPR143 was detected in cells co-expressing D2DR and GPR143 (CHO cells expressing D2DR-mCherry transfected with GPR143-flag plasmid) using anti-D2DR antibody (Frontier Institute, D2R-Rb-Af960) and antibody against flag (Sigma-Aldrich, Monoclonal ANTI-FLAG® M2 antibody).

Immunoblot analysis Brain samples were harvested after 45 min treatment with quinpirole (0.3 and 3 mg/kg, ip). Peptides were administered via icv for 15 min before quinpirole treatment. Samples were homogenized in 300 μl immunoprecipitation buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 0.1% Nonidet P-40, and 0.1% protease inhibitors). For cultured cells, CHO cells were harvested after 10 min treatment with quinpirole at 37°C and lysed in immunoprecipitation buffer. Protein amounts were quantified by Bradford protein assay (Bio-Rad). Briefly, equal amounts of protein (20 μg) dissolved in SDS 4× sample buffer containing DTT (50 μM) were separated by SDS-PAGE (9%) and then transferred to polyvinylidene fluoride membranes (Millipore).

Samples were then used for immunoblot analysis with anti-GSK (#12456, 1:6,000 dilution), pGSK (#9336, 1:6,000 dilution), Akt (#9272, 1:6,000 dilution) pAkt (#9271, 1:6,000 dilution), ERK (#9101, 1:3,000 dilution), pERK (#9102, 1:3,000 dilution), pDARPP32 (#2301, 1:3,000 dilution) (Cell Signaling Technology), or DARPP32 (#, 1:3,000 dilution) antibodies. After labeling with the primary antibodies, the membranes were washed and incubated with a secondary anti-rabbit IgG antibody conjugated to HRP (GE Healthcare). Antibody-antigen complexes were identified using Luminata Forte Western chemiluminescent HRP substrate (Millipore) or Western BLoT ultrasensitive HRP substrate (Takara).

cAMP assay
CHO cells expressing D2DR-mCherry were seeded at 1 × ¹⁰⁶ cells in 12-well culture plates. After plating overnight, cells were transfected with 2.5 mg each of GPR143-flag and free-flag plasmids using Fugene6 (Promega). After 2 days of culture, cells were treated with rolipram (10 μM) for 30 min at 37°C. Then, cells were treated with forskolin (10 μM) and quinpirole (0.001–10 μM) for 30 min at 37°C. After incubation, cells were lysed using 0.1 N HCl containing 0.1% Triton X-100. The amount of cAMP was calculated using the Direct cAMP ELISA kit (Enzo Life Science) according to standard protocols. The results are expressed as a percentage of the free-flag vehicle-treated control.

Receptor binding assay
CHO cells expressing D2DR-mCherry were seeded at 1 × ¹⁰⁶ cells in 10 cm culture dishes. After plating the cells overnight, they were transfected with 5 mg of GPR143-flag and free-flag plasmids using Fugene6 (Promega). After 2 days of incubation, the cells were harvested in ice-cold binding buffer [25 mM Tris, 150 mM NaCl, 5 mM EDTA (pH 7.4)] and aliquoted at 0.4 × ¹⁰⁶ cells/mL. The cells were incubated for 1 h in binding buffer containing [3H]-raclopride (5 nM, PekinElmer) and quinpirole (0.005–50 μM, final concentrations). The cells were washed three times with ice-cold binding buffer. The specific binding of [3H]-raclopride was calculated by subtracting the value of [3H]-raclopride binding in the presence of 1 μM quinpirole.

Data analysis results are presented as mean ± SEM of independent experiments. Data fitting and statistical analysis were performed by unpaired two-group Student's t-test or one-way or two-way ANOVA between multiple groups, followed by Tukey's or Bonferroni's multiple comparison test. All analyses were performed with GraphPad Prism 7.

References

1. Beaulieu JM, Gainetdinov RR, and Caron MG. Akt/GSK3 signaling in the
Action of psychotropic drugs. Annu Rev Pharmacol Toxicol. 2009;49:327-47.
2. Kreitzer AC. Physiology and pharmacology of striatal neurons. Annu Rev
Neurosci. 2009;32:127-47.
3. Nicola SM, Surmeier J, and Malenka RC. Dopaminergic modulation of neuronal
Excitability in the striatum and nucleus accumbens. Annu Rev Neurosci.
2000;23:185-215.
4. Beaulieu JM, Espinoza S, and Gainetdinov RR. Dopamine receptors - IUPHAR
Review 13. Br J Pharmacol. 2015;172(1):1-23.
5. Gainetdinov RR, Premont RT, Bohn LM, Lefkowitz RJ, and Caron MG.
Desensitization of G protein-coupled receptors and neuronal functions.
Rev Neurosci. 2004;27:107-44.
6. Missale C, Nash SR, Robinson SW, Jaber M, and Caron MG. Dopamine
receptors: from structure to function. Physiol Rev. 1998;78(1):189-225.
7. Urs NM, Caron MG. The physiological relevance of functional selectivity in
Dopamine signalling. Int J Obes Suppl. 2014;4(Suppl 1):S5-8.
8. Misu Y, Goshima Y. Is L-dopa an endogenous neurotransmitter? Trends
Pharmacol Sci. 1993;14(4):119-23.
9. Lopez VM, Decatur CL, Stamer WD, Lynch RM, and McKay BS. L-DOPA is
An endogenous ligand for OA1. PLoS Biol. 2008;6(9):e236.
10. Masukawa D, Koga M, Sezaki A, Nakao Y, Kamikubo Y, Hashimoto T, et al.
L-DOPA sensitizes vasomotor tone by modulating the vascular
alpha1-adrenergic receptor. JCI Insight. 2017;2(18).
11. Gantz SC, Levitt ES, Llamosas N, Neve KA, and Williams JT. Depression of
Serotonin Synaptic Transmission by the Dopamine Precursor L-DOPA. Cell Rep.
2015;12(6):944-54.
12. Ford CP. The role of D2-autoreceptors in regulating dopamine neuron activity
and transmission. Neuroscience. 2014;282:13-22.
13. Goshima Y, Masukawa D, Kasahara Y, Hashimoto T, and Aladeokin AC.
l-DOPA and Its Receptor GPR143: Implications for Pathogenesis and Therapy
in Parkinson's Disease. Front Pharmacol. 2019;10:1119.
14. Nakamura S, Yue JL, Goshima Y, Miyamae T, Ueda H, and Misu Y.
Non-effective dose of exogenously applied L-dopa itself stereoselectively
Potentiates postsynaptic D2 receptor-mediated locomotor activities of conscious
rats. Neurosci Lett. 1994;170(1):22-6.
15. Castrioto A, Thobois S, Carnicella S, Maillet A, and Krack P. Emotional
Manifestations of PD: Neurobiological basis. Mov Disord. 2016;31(8):1103-13.

The present invention can be used to develop medicines for preventing and/or treating a group of diseases in which D2 receptors are involved in the pathology, such as Parkinson's disease and neuropsychiatric disorders.

<SEQ ID NO: 1> Shows the amino acid sequence of a peptide encoding the fifth transmembrane domain (TM5) of GPR37.
IPHYVTTYLPLLLVLVANPIL
<SEQ ID NO: 2> Shows the amino acid sequence of TAT peptide.
YGRKKRRQRRR
<SEQ ID NO: 3> Shows the amino acid sequence of TAT-TM1.
VFHALCLGSGTLRLVLGLLQLYGRKKRRQRRR
<SEQ ID NO: 4> Shows the amino acid sequence of TAT-TM2.
YGRKKRRQRRRLLGCLGIVIRSTVWIAYPEFI
<SEQ ID NO: 5> Shows the amino acid sequence of TAT-TM3.
LLYSACFWWLFCYAVDVYLVIYGRKKRRQRRR
<SEQ ID NO: 6> Shows the amino acid sequence of TAT-TM4.
YGRKKRRQRRRILLYHIMAWGLAVLLCVEGAV
<SEQ ID NO: 7> Shows the amino acid sequence of TAT-TM5.
IPHYVTTYLPLLLVLVANPILYGRKKRRQRRR
<SEQ ID NO: 8> Shows the amino acid sequence of TAT-TM6.
YGRKKRRQRRRIMLVLIACWLSNIINESLLFY
<SEQ ID NO: 9> Shows the amino acid sequence of TAT-TM7.
FIMGILNPAQGLLLSLAFYGWYGRKKRRQRRR
<SEQ ID NO: 10> This shows the base sequence of the M13 primer (M13-F).
5'-CCCAGTCACGACGTTGTAAAACG
<SEQ ID NO: 11> This shows the base sequence of the M13 primer (M13-R).
AGCGGAATAACATTCACAAGGG

Claims (6)

A peptide consisting of the amino acid sequence IPHYVTTYLPLLLVLVANPIL (SEQ ID NO: 1), or a pharma- ceutically acceptable salt or solvate thereof. A peptide having an amino acid sequence of IPHYVTTYLPLLLVLVANPIL (SEQ ID NO: 1) to which a TAT peptide having an amino acid sequence of YGRKKRRQRRR (SEQ ID NO: 2) has been added at the C-terminus , or a pharma- ceutically acceptable salt or solvate thereof. A medicine comprising the peptide according to claim 1 or 2, or a pharma- ceutically acceptable salt or solvate thereof. A preventive and/or therapeutic agent for a disease involving a signal transduction pathway mediated by dopamine D2 receptors, comprising the peptide according to claim 1 or 2, or a pharma- ceutical acceptable salt or solvate thereof. The prophylactic and/or therapeutic drug according to claim 4, wherein the disease involving a signal transduction pathway mediated by dopamine D2 receptors is at least one disease selected from the group consisting of Parkinson's disease, schizophrenia, bipolar disorder, depression, mania, addiction, and attention deficit hyperactivity disorder. An inhibitor of a signal transduction pathway mediated by dopamine D2 receptor and dopa receptor GPR143, comprising the peptide according to claim 1 or 2, or a pharma- ceutical acceptable salt or solvate thereof.