Fig 2: Functional (immunoprecipitation) studies demonstrate that phospholipase D (PLD)1 expression tracks 5-hydroxytryptamine (5-HT) 2C receptor (5-HT2CR) expression in crude synaptosomal fractions of the amygdala and hippocampus in the paired group of animals in Day-14 drug-free state. (A, C) Synaptosomal fractions were immunoprecipitated (IP) with 5-HT2CR antibody and immunoblotted (IB) with PLD1, and the converse was also performed (C-D) in both the amygdala (A-B) and the hippocampus (C-D). Uniform loading was verified by measuring ß-actin. Each experiment was repeated twice. These studies establish that increased association in the amygdala and decreased association in the hippocampus of 5-HT2CR-PLD at the crude synaptosomal levels are involved with the expression of conditioned hyperactivity.

Fig 3: PKCa-dependent phosphorylation of GRA7 was essential for interaction with ASC and PLD1.(A) THP-1 cells were stimulated with rGRA7 (5 µg/ml) for the indicated times (left) or 30 min. (right), followed by IP with aHis-agarose bead (left) or aPKCa (right) and IB with aPKCa, aPKCßI, aPKC?, aHis, and aActin. (B) Phos-tag and SDS-PAGE analyses of GST-GRA7, GRA7-IWT, GRA7-IS52A, GRA7-IIIWT, GRA7-IIIT121A, or GRA7-IIIS135A expressed together with Flag-tagged PKCa in 293T cells left untreated (CIP-) or treated with calf intestinal alkaline phosphatase (CIP+), and subjected to GST pulldown, followed by IB with aGST. WCLs were used for IB with aFlag or aActin. (C) Mapping of PKCa phosphorylation sites on GRA7 by tiled peptide array analysis using purified recombinant PKCa. Phosphorylation intensity of 15-amino acid peptides that span full-length GRA7 and are each shifted by 3 amino acids was detected using MultiGauge version 3.0. The serines in the two peptides that showed a phosphorylation signal stronger than 100 PSL/mm2 are indicated above the corresponding peaks. (D) 293T cells were co-transfected with GST or GST-GRA7-I and truncated mutant constructs together with V5-ASC (left), or GST or GST-GRA7-III and truncated mutant constructs together with AU1-PLD1 (right), and subjected to GST pulldown, followed by IB with aV5 or aAU1. WCLs were used for IB with aGST, aV5, aAU1, aPKCa, or aActin. (E) BMDMs from PKCa+/+ and PKCa-/- were stimulated with rGRA7 for 30 min., followed by IP with aHis-agarose bead and IB with aASC, aPLD1, aPKCa, and aActin. The data are representative of four independent experiments with similar results (A to E).

Fig 4: Dorsal hippocampus (dHC) crude synaptosomal membrane phospholipase D (PLD)1/PLD2 isoforms show reduced phosphorylation states in the Day-1, while either decreased (PLD2) or unchanged (PLD1) phosphorylation and decreased PLD1/PLD2 expression in the Day-14 drug-free states in the paired group following conditioned hyperactivity (CH). 5-Hydroxytryptamine (5-HT) 2C receptor (5-HT2CR) expression remains unchanged in Day-1 and decreases in the Day-14 drug-free state in the paired group following CH. Results represent the percent change in expression of the unpaired and paired compared to control group (±SEM). 5-HT2CR expression in the paired group (A, hatched bars) is diminished by 50%, only in the Day-14 (dark background), not in the Day-1 (clear background) drug-free state compared with the unpaired group (slanted bars). Decreased phosphorylation states were observed in the paired group of animals (cross-hatch bars) compared with unpaired (slanted bars) at (B) pPLD1T147 (middle panel), pPLD1S561 (last panel) in PLD1 isoform, and (C) pPLD2Y169 (last panel) in the PLD2 isoform Day-1 (clear background) drug-free state. Phosphorylation states of the PLD1 isoform (B, middle and last panel) did not show any significant increase or decrease in paired group (cross hatched bars) in the Day-14 (dark background) drug-free state. However, the overall dHC crude synaptosomal expression for PLD1 (B, first panel) and PLD2 (C, first panel) levels were decreased by 50% in the paired (cross-hatched bars) compared with the unpaired (slanted bars) animals in the Day-14 (dark background) drug-free state. No changes in the paired (cross hatched bars) compared with unpaired (slanted bars) groups were observed in either PLD1 (B, first panel) or PLD2 (C, first panel) in the Day-1 (clear background) drug-free state. Diminished phosphorylation state of the PLD2 isoform Y169 (C, last panel) was observed in both the Day-1 (clear background) and Day-14 (dark background) drug-free states. # P<.05 vs paired; *P<.05 vs unpaired; n=8 animals/group.

Fig 5: GRA7-III-induced activation of PLD1 was dependent on interaction with PLD1.(A) Bacterially purified 6xHis-GRA7-III and its mutants were analyzed by Coomassie blue staining (left) or IB with aHis (right). (B) BMDMs from PKCa+/+ and PKCa-/- mice were stimulated with rGRA7-III (5 µg/ml) and its mutants for the indicated times, followed by IB to detect phosphorylated and total forms of PLD1 and PLD2. Actin was used as a loading control. (C) BMDMs were stimulated with rGRA7-III and its mutants for 30 min. and analyzed for PLD activity assay in vitro. Data shown are the means ± SD of five experiments. (D) Fluorescence confocal images in BMDMs were stimulated with rGRA7 and its mutants for 30 min., fixed, immunostained with antibodies for His (Alexa 488) and PLD1 (Alexa 568). Scale bar, 20 µm. The data are representative of three independent experiments with similar results (A, B, and D).