SUMMARY N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extra-synaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292–1304 (NR2BCT). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2BCT that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca2+ influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extra-synaptic sites and this interaction acts as a central mediator for stroke damage.

Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (I(crac)). A critical question in cell signalling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac).

Abstract. Although the actin cytoskeleton has been implicated in vesicle trafficking, docking and fusion, its site of action and relation to the Ca2÷-mediated activation of the docking and fusion machinery have not been elucidated. In this study, we examined the role of actin filaments in regulated exocytosis by introducing highly specific actin monomer-binding proteins, the ~-thymosins or a gelsolin fragment, into streptolysin O-permeabilized pancreatic acinar cells. These proteins had stimulatory and inhibitory effects. Low concentrations elicited rapid and robust exocytosis with a profile comparable to the initial phase of regulated exocytosis, but without raising [Ca2+], and even when [Ca 2÷] was clamped at low levels by EGTA. No additional cofactors were required.Direct visualization and quantitation of actin filaments showed that B-thymosin, like agonists, induced actin depolymerization at the apical membrane where exocytosis occurs. Blocking actin depolymerization by phalloidin or neutralizing fl-thymosin by complexing with exogenous actin prevented exocytosis. These findings show that the cortical actin network acts as a dominant negative clamp which blocks constitutive exocytosis. In addition, actin filaments also have a positive role. High concentrations of the actin depolymerizing proteins inhibited all phases of exocytosis. The inhibition overrides stimulation by agonists and all downstream effectors tested, suggesting that exocytosis cannot occur without a minimal actin cytoskeletal structure.T hE final steps of regulated exocytosis involve vesicle docking, triggering, and membrane fusion. There is now increasing evidence that regulated exocytosis employs a constitutively operating fusion machinery shared by many vesicular trafficking processes and specialized clamps to prevent fusion until the appropriate signals are received (4,40,41). The actin network under the plasma membrane has long been proposed as a physical barrier to granule docking because it transiently depolymerizes during exocytosis (3,30,42,43). The cortical actin can therefore be considered as a part of the clamping apparatus. However, in many cell types, drugs which depolymerize actin do not elicit exocytosis but can potentiate agonist-evoked responses (23,25,38). On the basis of such evidence, it was suggested that dissolution of the actin cytoskeleton is a necessary but not sufficient part of regulated exocytosis. Nevertheless, the exact role of actin in exocytosis remains unclear, since contradictory results were obtained in other cells (1) and between intact and permeabilized cells (19). Furthermore, some cells have cytochalasin-insensitive pools of actin filaments (8). A large part of the uncertainty is due to the nonspecific nature of some of the drugs, which precludes unequivocal conclusions.Address all correspondence to Dr. Helen L. Yin, Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, 648-8685. In the present study, we used a different approach to examine th...

Highlights d Ultra-deep rRNA-depleted RNA sequencing of 144 localized prostate tumors d Fusion gene profiles differentiate localized from metastatic disease d Widespread RNA circularization events define clinically distinct tumor subtypes d Functional screening reveals pervasive circular isoformspecific essentiality