The present study aims to define the role of postsynaptic density (PSD)-95 in the regulation of dopamine (DA) receptor function. We found that PSD-95 physically associates with either D1 or D2 DA receptors in co-transfected HEK-293 cells. Stimulation of DA receptors altered the association between D1 receptor and PSD-95 in a time-dependent manner. Functional assays indicated that PSD-95 co-expression did not affect D1 receptor-stimulated cAMP production, Gs-protein activation or receptor desensitization. However, PSD-95 accelerated the recovery of internalized membrane receptors by promoting receptor recycling, thus resulting in enhanced resensitization of internalized D1 receptors. Our results provide a novel mechanism for regulating DA receptor recycling that may play an important role in postsynaptic DA functional modulation and synaptic neuroplasticity.

The present study provides the first evidence that PSD-95 is essential in D1R-regulated NR1a/NR2B receptor function.

Aim: To examine whether (-)-stepholidine (SPD) has a direct effect on the Nmethyl-D-aspartic acid receptors (NMDAR) containing the NMDA receptor subunits NR2A or NR2B and to compare its effect with those of haloperidol (Hal) and clozapine (Cloz). Methods: NMDAR was transiently expressed in human embryonic kidney 293 (HEK293) cells. Changes in intracellular calcium concentration ([Ca 2+ ] i ) induced by NMDAR activation were monitored with Fura-2 ratio imaging techniques. Results: SPD had no significant effects on either subunit of NMDAR at a concentration of less than 100 µmol/L. Hal selectively inhibited NMDAR containing the NR2B subunit, whereas Cloz inhibited both subunits of NMDAR. Although both Hal and Cloz inhibited NR1a/NR2B receptor-mediated Ca 2+ influx, their effects were different. Hal was more potent and had a faster peak effect than Cloz. Conclusion: Both Hal and Cloz inhibit NMDAR-mediated function, whereas SPD produced only a little inhibition at a high concentration. Based on our other studies, the modulation of SPD on NMDAR function may be via D 1 receptor action underlying an indirect mechanism.

JAK2 kinase mutation V617F is prevalent in myeloproliferative diseases (MPD), including polycythemia vera (PV, 81-99%), essential thrombocytosis (ET, 41-72%) and primary myelofibrosis (PMF, 39-57%). This point mutation constitutively activates the JAK kinase and leads to oncogenic potential of host cells, and thus making JAK2 a promising molecular target for MPD therapy. HM-018 is a small molecule inhibitor against JAK kinase and the compound's preclinical anti-MPD effects from signal transduction to biological consequences were investigated. HM-018 was found to inhibit JAK kinase 1, 2, 3 and TYK with IC50 of 0.010, 0.006, 0.040 and 0.047 μM, respectively. The compound demonstrated >100 folds selectivity against a panel of 63 kinases. In accordance with enzymatic activity, HM-018 suppressed ligand dependent or constitutive JAK activation in multiple cell lines as evidenced by the decrease of STAT3/5 phosphorylation. As a result, JAK dependent cell proliferation was significantly inhibited by HM-018. EPO-mediated mouse PV model was utilized to evaluate the in vivo efficacy of the compound. HM-018 could shrink enlarged mouse spleen, a typical symptom of PV, in a dose dependent manner accompanied with decreased STAT5 phosphorylation both in animal spleen and in bone marrow after oral dosing for 7 days. To better mimic MPD development in a more clinically-relevant manner, JAK2-V617F-tranfected 32D cells were injected into mice intravenously, and it was observed that oral treatment of HM-018 not only prolonged the animal's life span, but also reduced MPD-related symptoms, such as spleen weight increase and organ invasion by malignant cells. Meanwhile, HM-018 exhibited a favorable pharmacokinetic profile and acceptable safety window in rats. Based on the preclinical data, HM-018 demonstrated anti-MPD potency both in vitro and in vivo, and the studies have provided rationale to further develop this compound as possible MPD therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1797. doi:1538-7445.AM2012-1797