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 ...

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Hemocytes of the horseshoe crab (limulus) contain a family of arthropodous peptide antibiotics, termed the tachyplesin family, and antibacterial protein, called anti-LPS factor, of which the former is located in the small (S) granules and the latter in the large (L) granules of the hemocytes. In our ongoing studies on granular components, we have identified here a novel defensin-like substance present in both L- and S-granules. This substance strongly inhibits the growth of Gram-negative and -positive bacteria, and fungi, such as Candida albicans. The isolated substance, tentatively termed "big defensin," consists of 79 amino acid residues, of which the COOH-terminal 37 residues have a sequence similar to those of mammalian neutrophil-derived defensins, especially rat defensin. Characterization of the disulfide motif in big defensin indicated that the disulfide array is identical to that of beta-defensins from bovine neutrophils. One clear structural difference is that the limulus hemocyte-derived big defensin has an extension of the NH2-terminal hydrophobic sequence with 35 amino acid residues followed by the COOH-terminal cationic defensin portion. This amphipathic nature of big defensin seems likely to be associated with its potent antibacterial activity. Furthermore, antibacterial activities of the NH2-terminal hydrophobic region and the COOH-terminal defensin portion separated by tryptic digestion are significantly different: the former displays a more potent activity against Gram-positive bacteria, whereas the latter is more potent against Gram-negative bacteria. Big defensin, therefore, may prove to represent a new class of defensin family possessing two functional domains with different antimicrobial activities.