Masaki Kataoka

Subcellular localization of a novel G protein XLGαolf

XLGalpha(olf) was identified as a transcriptional variant of the heterotrimeric G protein, Galpha(olf). Previous work showed that XLGalpha(olf) couples with adenosine A2a receptor and dopamine D1 receptor in vitro. However, physiological functions of XLGalpha(olf) remain to be elucidated. In this study, we performed indirect immunofluorescence confocal analyses to examine the subcellular localization of XLGalpha(olf). With overexpression, surprisingly, many large endosomes resulted. We also observed that XLGalpha(olf) localizes at the Golgi apparatus. The N-terminal region of XLGalpha(olf) appears necessary for both endosome formation and the Golgi localization. The results indicate that XLGalpha(olf) and Galpha(olf) play distinctly separate roles. Moreover, XLGalpha(olf) colocalized with Rab3A and Rab8A, as well as partially with Rab11A, but not with other endocytotic endosomes. We could confirm the interaction between XLGalpha(olf) and Rab3A/Rab8A by co-immunoprecipitation experiments. Our study provides important clues toward understanding physiological functions of XLGalpha(olf).

Pharmacokinetics of a novelN-methyl-D-aspartate receptor antagonist (SM-18400): Identification of anN-acetylated metabolite and pre-clinical assessment ofN-acetylation polymorphism

Summary(S)-9-chloro-5-[p-aminomethyl-o-(carboxymethoxy)phenylcarbamoylmethyl]-6,7-dihydro-1H,5H-pyrido[1,2,3-de]quinoxaline-2,3-dion hydrochloride trihydrate (SM-18400) was given intravenously to rats and dogs and its pharmacokinetics was investigated. By LC/MS/MS analysis, the major metabolite in the rat serum was identified asN-acetylated SM-18400 (SM-NAc). In rats,AUC ratio of SM-NAc to SM-18400 was approximately 50%. However, 71% of the dose was excreted as unchanged SM-18400 and only 9.8% as SM-NAc in the urine and bile, indicating that the contribution ofN-acetylation clearance (CLNAc) to the total clearance (CLtot) is limited to 10–30% in rats. No SM-NAc or other metabolites were detected in the dog serum, urine or bile. Thein vitro intrinsic clearance (CLint, ml/min/mg cytosolic protein) ofN-acetyltransferase (NAT) activities of dog liver cytosol towards SM-18400 and hepaticN-acetylation clearance (CLNAc, ml/min/kg body weight) estimated by well-stirred model were both only 5% of the respective rat value, well reflecting the relativein vivo CLNAc/CLtot ratios.CLint values for human live cytosol samples (n=4) and estimatedCLNAc were all less than 18% and 7% of the rat, respectively. Based on these results, we concluded that theCLNAc/CLtot of human would be small enough to avoid major inter-individual variance in SM-18400 pharmacokinetics due toN-acetylation polymorphism. In addition, even a human liver cytosol sample lacking polymorphic NAT2 activity as determined by sulfamethazine (SMZ)N-acetylation analysis, proved capable of acetylating SM-18400, suggesting that NAT2 is not the major enzyme responsible forN-acetylation of SM-18400 in human. This fact would also reduce the risk ofN-acetylation polymorphism playing a role in clinical use of this drug.

XLGαolf regulates expression of p27Kip1 in a CSN5 and CDK2 dependent manner.

XLGα(olf) is an extra large transcriptional variant of the heterotrimeric G protein, Gα(olf), which we previously reported to be localized in the Golgi apparatus and interacted with Rab3A and Rab8A through its N-terminal region. However, many physiological functions of XLGα(olf) remain to be elucidated. In this study, performance of yeast two-hybrid screening with XLGα(olf) allowed isolation of COP9 signalosome subunit 5 (CSN5), known to regulate the p27(Kip1) protein level through a proteasome dependent pathway. Co-immunoprecipitation experiments followed by Western blotting also showed association of CSN5 with XLGα(olf) linked to down-regulation of p27(Kip1). Gene silencing of endogenous CSN5 by siRNA attenuated the XLGα(olf)-mediated down-regulation, which was also demonstrated to require CDK2. Both knock down of CDK2 and the treatment with a CDK2 inhibitor reversed the reduction of p27(Kip1) due to XLGα(olf). Our findings provide important clues to understanding physiological functions of XLGα(olf).