Jun Takasaki

Molecular identification of nicotinic acid receptor.

Nicotinic acid and its derivative, Acipimox, have been widely used in the treatment of hyperlipidemia. Pharmacological studies have demonstrated that they exert the beneficial effect through the activation of a Gi-protein-coupled receptor on adipocyte, which has remained elusive to date. Here we show that a novel GPCR, designated HM74b because of its high similarity to HM74, is a receptor for nicotinic acid. HM74b mRNA is found in human, murine, and rat adipose tissues. Nicotinic acid and Acipimox inhibit forskolin-stimulated intracellular cAMP accumulation in human HM74b-expressing cells and activate GTP gamma S binding in a dose-dependent manner. [3H]Nicotinic acid specifically binds to HM74b-expressing membrane and its binding is replaced by Acipimox. This finding will open a new phase of research on the physiological role of nicotinic acid and will be a clue to develop novel antihyperlipidemic drugs.

Molecular cloning and characterization of prokineticin receptors.

Recent studies have identified two novel biofunctional proteins, termed prokineticin 1/EG-VEGF and prokineticin 2, which were mammalian homologues of mamba MIT1 and frog Bv8. Prokineticins have been demonstrated to exert their physiological functions through G-protein coupled receptors (GPCRs). In this study, we report the molecular identification of two endogenous prokineticin receptors, designated PK-R1 and PK-R2, through a search of the human genomic DNA database. PK-R1, locating in chromosome 2, and PK-R2, locating in chromosome 20p13, shared 87% homology, which was an extremely high value among known GPCRs. In functional assays, mammalian cells expressing PK-Rs responded to prokineticins in a concentration-dependent manner. Tissue distribution analysis revealed that expression of PK-R1 was observed in the testis, medulla oblongata, skeletal muscle and skin, while that of PK-R2 showed preferential expression in the central nervous system. The tissue distribution of PK-Rs reported in this paper suggests that the prokineticins play multifunctional roles in vivo.

Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule

Heterotrimeric GTP-binding proteins (G proteins) transmit extracellular stimuli perceived by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. Hundreds of GPCRs exist in humans and are the targets of a large percentage of the pharmaceutical drugs used today. Because G proteins are regulated by GPCRs, small molecules that directly modulate G proteins have the potential to become therapeutic agents. However, strategies to develop modulators have been hampered by a lack of structural knowledge of targeting sites for specific modulator binding. Here we present the mechanism of action of the cyclic depsipeptide YM-254890, which is a recently discovered Gq-selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of α subunit of Gq protein (Gαq) by inhibiting the GDP release from Gαq. X-ray crystal structure analysis of the Gαqβγ–YM-254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the Gαq. The binding stabilizes an inactive GDP-bound form through direct interactions with switch I and impairs the linker flexibility. Our studies provide a novel targeting site for the development of small molecules that selectively inhibit each Gα subunit and an insight into the molecular mechanism of G protein activation.

The novel G-protein coupled receptor SALPR shares sequence similarity with somatostatin and angiotensin receptors.

A cDNA encoding a novel G-protein coupled receptor (GPCR) was isolated from a human cerebral cortex cDNA library by low stringency hybridization screening. This putative seven-transmembrane domain receptor of 469 amino acids was designated SALPR (Somatostatin- and Angiotensin- Like Peptide Receptor). SALPR shares the highest amount of amino acid similarity with the somatostatin (35% with SSTR5) and angiotensin receptors (31% with AT1). Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the SALPR mRNA is predominantly expressed in human brain regions, particularly the substantia nigra and pituitary, although the mRNA can also be detected in the peripheral tissues, albeit at low levels. Chromosomal mapping by radiation hybrid analysis localized the human SALPR gene to the chromosome 5p15.1-5p14. Transient expression of SALPR in COS-1 cells did not produce any binding sites for somatostatin or angiotensin II, indicating the necessity for further study to discover its ligand and physiological significance.

The evolutionarily conserved G protein-coupled receptor SREB2/GPR85 influences brain size, behavior, and vulnerability to schizophrenia

The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3′ UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.

Gα(q/11)-coupled P2Y2 nucleotide receptor inhibits human keratinocyte spreading and migration

Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucle‐otides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y2 nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymogra‐phy analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y2 receptor activation also induces a dramatic dismantling of the actin network, the loss of a3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of av integrins and focal contact protein distribution. In addition, activation of P2Y2R prevents growth factor‐induced phosphorylation of Erk1,2 and Akt/PkB. The use of a specific pharmacological inhibitor (YM‐254890), the depletion of Ga(q/11) by siRNA, or the expression of a constitutively active Ga(q/11) mutant (Q209L) show that activation of Ga(q/11) is responsible for these ATP/ UTP‐induced effects. Finally, we report that ATP delays growth factor‐induced wound healing of keratino‐cyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing.— Taboubi, S., Milanini, J., Delamarre, E., Parat, F., Garrouste, F., Pommier, G., Takasaki, J., Hubaud, J‐C., Kovacic, H., Lehmann, M. Ga(q/11)‐coupled P2Y2 Nucleotide Receptor Inhibits Human Keratinocyte Spreading and Migration. FASEB J. 21, 4047–4058 (2007)

Biological properties of a specific Gαq/11 inhibitor, YM-254890, on platelet functions and thrombus formation under high-shear stress

1 The effects of YM‐254890, a specific Gαq/11 inhibitor, on platelet functions, thrombus formation under high‐shear rate condition and femoral artery thrombosis in cynomolgus monkeys were investigated. 2 YM‐254890 concentration dependently inhibited ADP‐induced intracellular Ca2+ elevation, with an IC50 value of 0.92±0.28 μM. 3 P‐selectin expression induced by ADP or thrombin receptor agonist peptide (TRAP) was strongly inhibited by YM‐254890, with IC50 values of 0.51±0.02 and 0.16±0.08 μM, respectively. 4 YM‐254890 had no effect on the binding of fibrinogen to purified GPIIb/IIIa, but strongly inhibited binding to TRAP‐stimulated washed platelets. 5 YM‐254890 completely inhibited platelet shape change induced by ADP, but not that induced by collagen, TRAP, arachidonic acid, U46619 or A23187. 6 YM‐254890 attenuated ADP‐, collagen‐, TRAP‐, arachidonic acid‐ and U46619‐induced platelet aggregation with IC50 values of <1 μM, whereas it had no effect on phorbol 12‐myristate 13‐acetate‐, ristocetin‐, thapsigargin‐ or A23187‐induced platelet aggregation. 7 High‐shear stress‐induced platelet aggregation and platelet‐rich thrombus formation on a collagen surface under high‐shear flow conditions were concentration dependently inhibited by YM‐254890. 8 The antithrombotic effect of YM‐254890 was evaluated in a model of cyclic flow reductions in the femoral artery of cynomolgus monkeys. The intravenous bolus injection of YM‐254890 dose dependently inhibited recurrent thrombosis without affecting systemic blood pressure or prolonging template bleeding time. 9 YM‐254890 is a useful tool for investigating Gαq/11‐coupled receptor signaling and the physiological roles of Gαq/11.

cDNA cloning and characterization of porcine histamine H4 receptor.

The cDNA encoding histamine H4 receptor was cloned from the porcine spleen cDNA library. Porcine H4 receptor, which shares 72% homology with its human counterpart, bound to histamine in receptor-expressing mammalian cells. Isolation of the porcine H4 receptor, which is important for understanding of the pharmacology, will aid in better interpretation of physiological role of this subtype of histamine receptor.

A functional G300S variant of the cysteinyl leukotriene 1 receptor is associated with atopy in a Tristan da Cunha isolate.

Atopy is a well-defined immune phenotype that is reported to be a risk factor for asthma. Among the many loci that contribute to a genetic predisposition to asthma, the cysteinyl leukotriene receptor genes and their variants have been important subjects of study because they are functionally and pharmacologically implicated in the atopy phenotype affecting many asthma subjects. Moreover, the product of cysteinyl-leukotriene 1 receptor gene (CysLT1), located at Xq13.2, is targeted by LT receptor antagonists. In our earlier association study, the M201V variant of the cysteinyl-leukotriene 2 receptor gene (CysLT2), located at 13q14, was implicated in atopic asthma. Here we report the screening of the coding region of the CysLT1, gene in the highly asthmatic Tristan da Cunha population. In this population, we discovered a CysLT1 G300S variant that is carried with a significantly higher frequency in atopics and asthmatics from the Tristan da Cunha population. Furthermore, we report the asthma independent association of the CysLT1 G300S variant with atopy. Subsequently, we compared the changes conferred by each SNP on CysLT function. The CysLT1 300S receptor interacts with LTD4 with significantly greater potency. For the 300S variant, a statistically significant decrease in the effector concentration for half-maximum response (EC50) for intracellular Ca2+ flux and total InsP generation is observed. Other aspects of the receptor function and activity, such as desensitization, pharmacologic profile in response to montelukast, and cellular localization, are unchanged. These in vitro analyses provide evidence that the 300S CysLT1 variant, found more commonly in atopics in the Tristan da Cunha population, encodes a functionally more sensitive variant.

Pharmacological properties of YM-254890, a specific G α q/11 inhibitor, on thrombosis and neointima formation in mice

The pharmacological properties of YM-254890, a specific G(alpha)q/11 inhibitor, on acute thrombosis and chronic neointima formation after vascular injury have been investigated. FeCl3 was used to induce vascular injury in the carotid artery of mice. For the thrombosis studies, the test drug was either intravenously or orally administered before vascular injury. For the neointima studies, the test drug was orally administered 1 h before and twice daily for 1 week after vascular injury. Histological analysis was then performed 3 weeks later. YM-254890 significantly inhibited ex vivo platelet aggregation 5 min after intravenous bolus injection at 0.03 mg/kg or more, and 1 h after oral administration at 1 mg/kg. YM-254890 significantly inhibited thrombus formation after intravenous bolus injection at 0.03 mg/kg as well as after oral administration at 1 mg/kg, but tail transection bleeding time was significantly prolonged at 0.1 mg/kg for intravenous injection and 3 mg/kg for oral administration. Furthermore, oral administration of YM-254890 dose-dependently inhibited neointima formation 3 weeks after vascular injury with significant effects at 1 mg/kg twice daily for 1 week. Clopidogrel also significantly inhibited neointima formation at its antithrombotic dose, but its inhibitory potency was less than that of YM-254890. However, YM-254890 significantly reduced systemic blood pressure at doses 3 times higher than those that produced significant inhibitory effects on thrombosis and neointima formation. Though the systemic use of YM-254890 may be limited, owing to its narrow therapeutic window, this unique compound is a useful research tool for investigating the physiological roles of G(alpha)q/11 .