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1.  Distinct Human and Mouse Membrane Trafficking Systems for Sweet Taste Receptors T1r2 and T1r3 
PLoS ONE  2014;9(7):e100425.
The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.
PMCID: PMC4100762  PMID: 25029362
2.  A competitive inhibitor traps LeuT in an open-to-out conformation 
Science (New York, N.Y.)  2008;322(5908):1655-1661.
Secondary transporters are workhorses of cellular membranes, catalyzing the movement of small molecules and ions across the bilayer, coupling substrate passage to ion gradients. However, the conformational changes that accompany substrate transport, the mechanism by which substrate moves through the transporter, and principles of competitive inhibition remain unclear. Here we use crystallographic and functional studies on LeuT, a model for neurotransmitter sodium symporters, to show that various amino acid substrates induce the same occluded conformational state, and that a competitive inhibitor, tryptophan, traps LeuT in an open-to-out conformation. In the Trp complex the extracellular gate residues, Arg30 and Asp404, define a second weak binding site for substrates as they permeate from extracellular solution to the primary substrate site, demonstrating how residues that participate in gating also mediate substrate permeation.
PMCID: PMC2832577  PMID: 19074341
3.  Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin 
Invertebrate Neuroscience   2008;8(2):71-81.
Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR–neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH–π interactions in the Ls-AChBP–CTD complex than in the Ls-AChBP–IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.
PMCID: PMC2413115  PMID: 18338186
Acetylcholine binding protein (Lymnaea stagnalis); Crystal structures; Neonicotinoids; Nicotinic acetylcholine receptors; Ion channels
4.  Capping protein binding to actin in yeast 
The Journal of Cell Biology  2004;164(4):567-580.
The mechanism by which capping protein (CP) binds barbed ends of actin filaments is not understood, and the physiological significance of CP binding to actin is not defined. The CP crystal structure suggests that the COOH-terminal regions of the CP α and β subunits bind to the barbed end. Using purified recombinant mutant yeast CP, we tested this model. CP lacking both COOH-terminal regions did not bind actin. The α COOH-terminal region was more important than that of β. The significance of CP's actin-binding activity in vivo was tested by determining how well CP actin-binding mutants rescued null mutant phenotypes. Rescue correlated well with capping activity, as did localization of CP to actin patches, indicating that capping is a physiological function for CP. Actin filaments of patches appear to be nucleated first, then capped with CP. The binding constants of yeast CP for actin suggest that actin capping in yeast is more dynamic than in vertebrates.
PMCID: PMC2171992  PMID: 14769858
cytoskeleton; cell motility; polymerization; assembly; Saccharomyces cerevisiae
5.  Cis3/Socs3/Ssi3 Plays a Negative Regulatory Role in Stat3 Activation and Intestinal Inflammation 
Immune and inflammatory systems are controlled by multiple cytokines, including interleukins (ILs) and interferons. These cytokines exert their biological functions through Janus tyrosine kinases and signal transducer and activator of transcription (STAT) transcription factors. We recently identified two intrinsic Janus kinase (JAK) inhibitors, JAK binding protein (JAB; also referred to as suppressor of cytokine signaling [SOCS1]/STAT-induced STAT inhibitor [SSI1]) and cytokine-inducible SH2 protein (CIS)3 (or SOCS3/SSI3), which play an essential role in the negative regulation of cytokine signaling. We have investigated the role of STATs and these JAK inhibitors in intestinal inflammation. Among STAT family members, STAT3 was most strongly tyrosine phosphorylated in human ulcerative colitis and Crohn's disease patients as well as in dextran sulfate sodium (DSS)-induced colitis in mice. Development of colitis as well as STAT3 activation was significantly reduced in IL-6–deficient mice treated with DSS, suggesting that STAT3 plays an important role in the perpetuation of colitis. CIS3, but not JAB, was highly expressed in the colon of DSS-treated mice as well as several T cell–dependent colitis models. To define the physiological role of CIS3 induction in colitis, we developed a JAB mutant (F59D-JAB) that overcame the inhibitory effect of both JAB and CIS3 and created transgenic mice. DSS induced stronger STAT3 activation and more severe colitis in F59D-JAB transgenic mice than in their wild-type littermates. These data suggest that hyperactivation of STAT3 results in severe colitis and that CIS3 plays a negative regulatory role in intestinal inflammation by downregulating STAT3 activity.
PMCID: PMC2195913  PMID: 11181699
Janus kinase; CIS/SOCS; interleukin 6; ulcerative colitis; negative regulation
6.  Isolation of Transformation Suppressor Genes by cDNA Subtraction: Lumican Suppresses Transformation Induced by v-src and v-K-ras 
Journal of Virology  2000;74(2):1008-1013.
We have reported that suppressive factors for transformation by viral oncogenes are expressed in primary rat embryo fibroblasts (REFs). To identify such transformation suppressor genes, we prepared a subtracted cDNA library by using REFs and a rat normal fibroblast cell line, F2408, and isolated 30 different cDNA clones whose mRNA expression was markedly reduced in F2408 cells relative to that in REFs. We referred to these as TRIF (transcript reduced in F2408) clones. Among these genes, we initially tested the suppressor activity for transformation on three TRIF genes, TRIF1 (neuronatin), TRIF2 (heparin-binding growth-associated molecule), and TRIF3 (lumican) by focus formation assay and found that lumican inhibited focus formation induced by activated H-ras in F2408 cells. Colony formation in soft agar induced by v-K-ras or v-src was also suppressed in F2408 clones stably expressing exogenous lumican without disturbing cell proliferation. Tumorigenicity in nude mice induced by these oncogenes was also suppressed in these lumican-expressing clones. These results indicate that lumican has the ability to suppress transformation by v-src and v-K-ras.
PMCID: PMC111623  PMID: 10623765

Results 1-6 (6)