YM-254890, which was isolated from the culture broth of Chromobacterium sp., inhibits ADP-induced platelet aggregation and has antithrombotic and thrombolytic effects. YM-254890 blocks G␣ q/11 -coupled ADP receptor P2Y1-mediated Ca 2؉ mobilization. Here we report that YM-254890 is a selective G␣ q/11 inhibitor. YM-254890 blocked Ca 2؉ mobilization mediated by several G␣ q/11 -coupled receptors but not by G␣ i -or G␣ 15 -coupled receptor, indicating that phospholipase C␤ activation and subsequent signaling molecules are not the target of YM-254890. YM-254890 completely prevented the serum response factor (SRF)-mediated gene transcription induced by G␣ q R183C, which is constitutively active in a receptor-dependent manner because of its reduced k cat of GTP hydrolysis. Conversely, YM-254890 had only a modest effect on the SRF-mediated gene transcription by G␣ q Q209L, which is GTPase-deficient (activated) G␣ q . These suggested that the acting point of YM-254890 is receptor-G␣ q interaction or the subsequent guanine nucleotide exchange step. The fact that YM-254890 (i) inhibited the SRF-mediated gene transcription by G␣ qi5 , which interacts with G␣ i -coupled receptor and possesses the effector function of G␣ q , and (ii) had no effect on the K d value of high affinity [ These data indicate that YM-254890 blocks the exchange of GDP for GTP in G␣ q/11 activation. This novel G␣ q/11 -selective inhibitor is a promising and powerful tool for studying G␣ q/11 protein activation, G␣ q/11 -coupled receptor signaling, and G␣ q/11 -mediated biological events. G protein-coupled receptors (GPCR)1 and heterotrimeric G proteins, consisting of G␣, ␤, and ␥, transduce extracellular stimuli, such as hormones, neurotransmitters, chemokines, and other local mediators, into appropriate intracellular responses (1, 2). The activation of G␣ proteins is related to conformational change by guanine nucleotide interaction. The GPCRs, activated by the agonist, induced exchanges of GDP for GTP on the coupled G␣ subunit. The resultant G␣-GTP complex dissociates from the G␤␥ subunit and activates its downstream effectors, which in turn regulate various functions such as gene transcription, mitogenesis, metabolism, muscle contractile state, and ion channel regulation. The GTPase activity of ␣-subunit turns off effector signals by hydrolyzing G␣-GTP to G␣-GDP, which re-associates with G␤␥.There are over 20 G␣ subunits classified into subfamilies by its sequence homology and the downstream signal. These include the major four families, G␣ q/11 , G␣ s , G␣ i/o , and G␣ 12/13 . Main effector molecules of G␣ q/11 , G␣ s , G␣ i/o , and G␣ 12/13 are thought to be phospholipase C (PLC), adenylyl cyclase (activation), adenylyl cyclase (inhibition), and small GTPase families, respectively (3). Transient intracellular Ca 2ϩ mobilization is led by PLC␤ activation via the G␣ q/11 , G␣ 15/16 , or G␤␥ subunit with G␣ i . PLC␤ hydrolyzes phosphatidylinositol bisphosphate in the plasma membrane, and the generated inositol 1,4,5-trisphosphate (IP 3 ) activates ...

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 G q -selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of α subunit of G q protein (Gα q ) by inhibiting the GDP release from Gα q . X-ray crystal structure analysis of the Gα q βγ–YM-254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the Gα q . 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.

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Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1-and PKR2-gene-disrupted mice (Pkr1 ؊/؊ and Pkr2 ؊/؊ , respectively). Phenotypic analysis indicated that not Pkr1 ؊/؊ but Pkr2 ؊/؊ mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2 ؊/؊ mice. In addition, the Pkr2 ؊/؊ mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2 ؊/؊ mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2 ؊/؊ mice. The phenotype of the Pkr2 ؊/؊ mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.

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