Palmitoylation on the carboxyl terminus tail is required for the selective regulation of dopamine D2 versus D3 receptors.

Dopamine D2 receptor (D2R) and D3 receptor (D3R) possess highly conserved amino acid sequences but this study showed that D3R was more extensively palmitoylated than D2R. Based on this finding, the molecular basis of this selective palmitoylation of D3R was determined and the roles of palmitoylation in the regulation of D3R functions were investigated. D3R was palmitoylated on the cysteine residue on its carboxyl terminus tail, the last amino acid residue of D3R, and an exchange of the carboxyl terminus tail between D2R and D3R (D2R-D3C and D3R-D2C) resulted in the switching of the palmitoylation phenotype. When the consensus site for palmitoylation was mutated or the palmitoylation of D3R was inhibited by treatment with 2-bromopalmitate (2BP), a palmitoylation blocker, cell-surface expression, PKC-mediated endocytosis, agonist affinity, and agonist-induced tolerance of D3R were all inhibited. However, these changes were not observed when D3R palmitoylation was inhibited by replacing its carboxyl tail with that of D2R (D3R-D2C) or when the palmitoylation of D2R-D3C was inhibited by treatment with 2BP. Overall, this study shows that D3R is palmitoylated more extensively than D2R even though the carboxyl terminus tails of D2R and D3R are highly homologous, and thus provides a new clue regarding the consensus sequence for palmitoylation. This study also shows that palmitoylation controls various functionalities of D3R only when the receptor is in the intact D3R configuration.

Functional Regulation of Dopamine D3 Receptor through Interaction with PICK1.

PICK1, a PDZ domain-containing protein, is known to increase the reuptake activities of dopamine transporters by increasing their expressions on the cell surface. Here, we report a direct and functional interaction between PICK1 and dopamine D3 receptors (D3R), which act as autoreceptors to negatively regulate dopaminergic neurons. PICK1 colocalized with both dopamine D2 receptor (D2R) and D3R in clusters but exerted different functional influences on them. The cell surface expression, agonist affinity, endocytosis, and signaling of D2R were unaffected by the coexpression of PICK1. On the other hand, the surface expression and tolerance of D3R were inhibited by the coexpression of PICK1. These findings show that PICK1 exerts multiple effects on D3R functions.

Roles of Dopamine D2 Receptor Subregions in Interactions with ß-Arrestin2.

ß-Arrestins are one of the protein families that interact with G protein-coupled receptors (GPCRs). The roles of ß-arrestins are multifaceted, as they mediate different processes including receptor desensitization, endocytosis, and G protein-independent signaling. Thus, determining the GPCR regions involved in the interactions with ß-arrestins would be a preliminary step in understanding the molecular mechanisms involved in the selective direction of each function. In the current study, we determined the roles of the N-terminus, intracellular loops, and C-terminal tail of a representative GPCR in the interaction with ß-arrestin2. For this, we employed dopamine D2 and D3 receptors (D2R and D3R, respectively), since they display distinct agonist-induced interactions with ß-arrestins. Our results showed that the second and third intracellular loops of D2R are involved in the agonist-induced translocation of ß-arrestins toward plasma membranes. In contrast, the N- and C-termini of D2R exerted negative effects on the basal interaction with ß-arrestins.

Induction of human leukemia cell differentiation via PKC/MAPK pathways by arsantin, a sesquiterpene lactone from Artemisia santolina.

Sesquiterpene lactone compounds have received considerable attention in pharmacological research due to their therapeutic effects including anti-cancer and anti-inflammatory activities. In this report, we investigated the effect of arsantin, a sesquiterpene lactone compound present in Artemisia santolina, on cellular differentiation in the human promyelocytic leukemia HL-60 cell culture system. Arsantin significantly induced HL-60 cell differentiation in a concentration-dependent manner. Cytofluorometric analysis indicated that arsantin induced HL-60 cell differentiation predominantly into granulocytes. Both PKC and MAPK inhibitors suppressed the HL-60 cell differentiation induced by arsantin. Moreover, treatment with arsantin increased protein levels of PKCα and PKCßII isoforms, and also induced increased protein levels and phosphorylation form of MAPKs in HL-60 cells. Importantly, arsantin synergistically enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either low doses of 1,25-(OH)2D3 or ATRA. The ability to enhance the differentiation potential of 1,25-(OH)2D3 or ATRA by arsantin may improve outcomes in the therapy of acute promyelocytic leukemia.

Synthesis of C (6)-epimer derivatives of diacetoxy acetal derivative of santonin and their inducing effects on HL-60 leukemia cell differentiation.

Induction of differentiation is a new and promising approach to leukemia therapy, well illustrated by the treatment of acute promyelocytic leukemia with 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] or all-trans retinoic acid (ATRA). Using combination of either 1,25-(OH)(2)D(3) or ATRA and chemotherapy, adverse effects 1,25-(OH)(2)D(3) or ATRA such as hypercalcemic effects have decreased, and long-term survival has improved. In a previous study, we demonstrated that santonin could be chemically modified into a diacetoxy acetal derivative of santonin with strong differentiation-inducing activity. In this study, we further synthesized C(6)-epimer derivatives of diacetoxy acetal derivative of santonin and tested their effects on HL-60 cell differentiation. Some of the C(6)-epimer derivatives themselves induced increases in cell differentiation. Especially, (11S)-3,3-(ethylenedioxy) eudesmano-13-ol-6ß-acetate (7) was demonstrated to induce differentiation with larger than 80% of the cells attaining a differentiated phenotype. Importantly, 7 strongly enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either low doses of 1,25-(OH)(2)D(3) or ATRA. The ability to enhance the differentiation potential of 1,25-(OH)(2)D(3) or ATRA by 7 may improve outcomes in the therapy of acute promyelocytic leukemia.

Synthesis of DAAS derivatives and their enhancement of HL-60 leukemia cell differentiation.

DAAS is the diacetoxy acetal derivative of a-santonin and induces HL-60 cell differentiation into granulocytes. In this report, we investigated the structure-activity relationship (SAR) of DAAS derivatives in the differentiation of human HL-60 leukemia cells. Although its derivatives themselves had less effect on HL-60 cell differentiation than DAAS, the monoacetyl derivative, 2, mainly induced HL-60 cell differentiation. Moreover, compound 2 synergistically enhanced all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation when combined with 50 nM ATRA, a well-known differentiation inducer. This enhancing effect is similar to that of DAAS in ATRA-induced differentiation.

Chemical modification of santonin into a diacetoxy acetal form confers the ability to induce differentiation of human promyelocytic leukemia cells via the down-regulation of NF-kappaB DNA binding activity.

Many sesquiterpene lactone compounds either induce or enhance the cell differentiation of human leukemia cells. However, we reported in a previous study that santonin, a eudesmanolide sesquiterpene lactone, exerts no effects on the differentiation of leukemia cells. In this report, to evaluate the possibility of chemically modifying santonin into its derivatives with differentiation inducing activity, we synthesized a series of santonin derivatives, and determined their effects on cellular differentiation in the human promyelocytic leukemia HL-60 cell system. A diacetoxy acetal derivative of santonin (DAAS) was found to induce significant HL-60 cell differentiation in a dose-dependent manner, whereas santonin in its original form did not. The HL-60 cells were differentiated into a granulocytic lineage when exposed to DAAS. In addition, the observed induction in cell differentiation closely correlated with the levels of NF-kappaB DNA binding activity inhibited by DAAS. Both Western blot analyses and kinase inhibitor studies determined that protein kinase C, ERK, and phosphatidylinositol 3-kinase were upstream components of the DAAS-mediated inhibition of NF-kappaB binding activity in HL-60 leukemia cells. The results of this study indicate that santonin can, indeed, be chemically modified into a derivative with differentiation inducing abilities, and suggest that DAAS might prove useful in the treatment of neoplastic diseases.

Synthesis of diacetoxy acetal derivatives of santonin and their enhancing effects on HL-60 leukemia cell differentiation.

Several diacetoxy acetal analogues have been synthesized from santonin and assessed for their ability of inducing or enhancing the differentiation of human HL-60 leukemia cells. The compounds themselves had little effect on HL-60 cell differentiation. However, three analogues, 2a, 3a, and 5b, synergistically enhanced 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced HL-60 cell differentiation when combined with 5 nM of dihydroxyvitamin D3 [1,25-(OH)2D3], a well-known differentiation inducer. Especially, the compound 5b profoundly enhanced the 1,25-(OH)2D3]-induced HL-60 cell differentiation.

Synthesis and antiviral activity of novel trans-2,2-dimethyl cyclopropyl nucleosides.

Novel trans-2,2-dimethylcyclopropyl nucleosides were synthesized as potential antiviral agents. The key intermediate, 3, was synthesized via five steps from ethyl chrysanthemate and condensed with purine bases using the Mitsunobu reaction to give six cyclopropyl nucleosides. These synthesized nucleosides did not show any significant antiviral activity against HSV-1, HSV-2, EMCV, Cox B3, or VSV, at concentrations up to 100 microM.

Synthesis and antiviral activity of novel C-methyl branched cyclopropyl nucleosides.

A series of novel cyclopropyl nucleosides was synthesized using the highly stereoselective Simmons-Smith reaction starting from 1,2:5,6-di-O-isopropylidene-D-mannitol. The structural assignments of these nucleosides were determined by NMR studies and X-ray crystallography. All the synthesized nucleosides were assayed against several viruses.

Synthesis and antiviral activity of novel exomethylene cyclopropyl pyrimidine nucleosides.

A series of novel exomethylene cyclopropyl nucleosides have been synthesized starting from Feist's acid. Classical nucleophilic substitution conditions (K2CO3, 18-crown-6) of the tosylate 2 as well as Mitsunobu reaction (DEAD, PPh3) of alcohol 1 with pyrimidine bases afforded a series of novel cyclopropyl nucleosides. Compound 4b displayed moderate anti-HBV activity without any cytotoxicity up to 100 microM.