Pann-Ghill Suh

Phospholipase C isozymes selectively couple to specific neurotransmitter receptors

A variety of extracellular signals are transduced across the cell membrane by the enzyme phosphoinositide-specific phospholipase C-β (PLC-β) coupled with guanine-nucleotide-binding G proteins. There are four isoenzymes of PLC-β, β1–β4, but their functions in vivo are not known. Here we investigate the role of PLC-β1 and PLC-β4 in the brain by generating null mutations in mice: we found that PLCβ1−/− mice developed epilepsy and PLCβ4−/− mice showed ataxia. We determined the molecular basis of these phenotypes and show that PLC-β1 is involved in signal transduction in the cerebral cortex and hippocampus by coupling predominantly to the muscarinic acetylcholine receptor, whereas PLC-β4 works through the metabotropic glutamate receptor in the cerebellum, illustrating how PLC-β isoenzymes are used to generate different functions in the brain.

Cloning and sequence of multiple forms of phospholipase C

Three phospholipase C isozymes (PLC-I, II, and III) have been purified from bovine brain. Here, phospholipase C-related cDNA clones corresponding to PLC-I and PLC-III were isolated from a rat brain lambda gt11 expression cDNA library using specific monoclonal antibodies and sequenced. Each of them encodes a distinct polypeptide with a calculated molecular mass of 138,225 (PLC-I) and 85,840 (PLC-III). Comparison of these two with the sequence of another isozyme PLC-II (Mr = 148,431) that we have previously characterized revealed a low overall sequence homology. Nevertheless, a significant amino acid sequence similarity between the three enzymes was found in two regions, one of about 150 amino acids and the other of about 120 amino acids. The two conserved domains were separated by a variable region. The variable region sequence of PLC-II is relatively long and has recently been shown to contain regions homologous to the noncatalytic domain of the nonreceptor tyrosine kinases. Those of PLC-I and III were short and appeared to be unrelated to these tyrosine kinases. The physiological implications of the multiple species of PLC enzymes are discussed.

Characterization of the Shank Family of Synaptic Proteins MULTIPLE GENES, ALTERNATIVE SPLICING, AND DIFFERENTIAL EXPRESSION IN BRAIN AND DEVELOPMENT

Shank1, Shank2, and Shank3 constitute a family of proteins that may function as molecular scaffolds in the postsynaptic density (PSD). Shank directly interacts with GKAP and Homer, thus potentially bridging the N-methyl-d-aspartate receptor-PSD-95-GKAP complex and the mGluR-Homer complex in synapses (Naisbitt, S., Kim, E., Tu, J. C., Xiao, B., Sala, S., Valtschanoff, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999) Neuron 23, 569–582; Tu, J. C., Xiao, B., Naisbitt, S., Yuan, J. P., Petralia, R. S., Brakeman, P., Doan, A., Aakalu, V. K., Lanahan, A. A., Sheng, M., and Worley, P. F. (1999) Neuron 23, 583–592). Shank contains multiple domains for protein-protein interaction including ankyrin repeats, an SH3 domain, a PSD-95/Dlg/ZO-1 domain, a sterile α motif domain, and a proline-rich region. By characterizingShank cDNA clones and RT-PCR products, we found that there are four sites for alternative splicing in Shank1 and another four sites in Shank2, some of which result in deletion of specific domains of the Shank protein. In addition, the expression of the splice variants is differentially regulated in different regions of rat brain during development. Immunoblot analysis of Shank proteins in rat brain using five different Shank antibodies reveals marked heterogeneity in size (120–240 kDa) and differential spatiotemporal expression. Shank1 immunoreactivity is concentrated at excitatory synaptic sites in adult brain, and the punctate staining of Shank1 is seen in developing rat brains as early as postnatal day 7. These results suggest that alternative splicing in the Shank family may be a mechanism that regulates the molecular structure of Shank and the spectrum of Shank-interacting proteins in the PSDs of adult and developing brain.

Crystal structures of human DJ-1 and Escherichia coli Hsp31, which share an evolutionarily conserved domain.

Human DJ-1 and Escherichia coli Hsp31 belong to ThiJ/PfpI family, whose members contain a conserved domain. DJ-1 is associated with autosomal recessive early onset parkinsonism and Hsp31 is a molecular chaperone. Structural comparisons between DJ-1, Hsp31, and an Archaea protease, a member of ThiJ/PfpI family, lead to the identification of the chaperone activity of DJ-1 and the proteolytic activity of Hsp31. Moreover, the comparisons provide insights into how the functional diversity is realized in proteins that share an evolutionarily conserved domain. On the basis of the chaperone activity the possible role of DJ-1 in the pathogenesis of Parkinsons disease is discussed.

Novel Compound 2-Methyl-2H-pyrazole-3-carboxylic Acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191) Prevents 2,3,7,8-TCDD-Induced Toxicity by Antagonizing the Aryl Hydrocarbon Receptor

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental pollutant with many toxic effects, including endocrine disruption, reproductive dysfunction, immunotoxicity, liver damage, and cancer. These are mediated by TCDD binding to and activating the aryl hydrocarbon receptor (AhR), a basic helix-loop-helix transcription factor. In this regard, targeting the AhR using novel small molecule inhibitors is an attractive strategy for the development of potential preventive agents. In this study, by screening a chemical library composed of approximately 10,000 compounds, we identified a novel compound, 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191), that potently inhibits TCDD-induced AhR-dependent transcription. In addition, CH-223191 blocked the binding of TCDD to AhR and inhibited TCDD-mediated nuclear translocation and DNA binding of AhR. These inhibitory effects of CH-223191 prevented the expression of cytochrome P450 enzymes, target genes of the AhR. Unlike many known antagonists of AhR, CH-223191 did not have detectable AhR agonist-like activity or estrogenic potency, suggesting that CH-223191 is a specific antagonist of AhR. It is noteworthy that CH-223191 potently prevented TCDD-elicited cytochrome P450 induction, liver toxicity, and wasting syndrome in mice. Taken together, these results demonstrate that this novel compound, CH-223191, may be a useful agent for the study of AhR-mediated signal transduction and the prevention of TCDD-associated pathology.

Overexpression of phospholipase D1 in human breast cancer tissues

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) to produce phosphatidic acid (PA) and choline. PLD is a major enzyme implicated in important cellular processes, such as cell proliferation. We designed this study to investigate the expression of PLD in human breast carcinomas and non-malignant tissues using RT-PCR, Western blot analysis, immunohistochemistry and an Arf-dependent PLD activity assay. We examined about 550 bp of PCR product and 120 kDa of PLD protein. Our results showed that PLD protein and mRNA levels were overexpressed in 14 of 17 breast cancer tissues. We also observed increased expression by immunohistochemistry and Arf-dependent PLD activity in microsomes of human breast tumors, which correlated well with PLD expression. PLD expression was elevated in human breast tumors compared with normal breast tissues. These results implicate a possible role of PLD in human breast tumorigenesis and suggest that PLD may be useful as a marker for malignant disease in the breast.

Phospholipase C-δ1 Is Activated by Capacitative Calcium Entry That Follows Phospholipase C-β Activation upon Bradykinin Stimulation

To characterize the regulatory mechanism of phospholipase C-δ1 (PLC-δ1) in the bradykinin (BK) receptor-mediated signaling pathway, we used a clone of PC12 cells, which stably overexpress PLC-δ1 (PC12-D1). Stimulation with BK induced a significantly higher Ca2+ elevation and inositol 1,4,5-trisphosphate (IP3) production with a much lower half-maximal effective concentration (EC50) of BK in PC12-D1 cells than in wild type (PC12-W) or vector-transfected (PC12-V) cells. However, BK-induced intracellular Ca2+ release and IP3 generation was similar between PC12-V and PC12-D1 cells in the absence of extracellular Ca2+, suggesting that the availability of extracellular Ca2+ is essential to the activation of PLC-δ1. When PC12-D1 cells were treated with agents that induce Ca2+ influx, more IP3 was produced, suggesting that the Ca2+ entry induces IP3production in PC12-D1 cells. Furthermore, the additional IP3 production after BK-induced capacitative calcium entry was detected in PC12-D1 cells, suggesting that PLC-δ1 is mainly activated by capacitative calcium entry. When cells were stimulated with BK in the presence of extracellular Ca2+, [3H]norepinephrine secretion was much greater from PC12-D1 cells than from PC12-V cells. Our results suggest that PLC-δ1 is activated by capacitative calcium entry following the activation of PLC-β, additively inducing IP3 production and Ca2+ rise in BK-stimulated PC12 cells.

Requirement for the L-type Ca2+ channel α1D subunit in postnatal pancreatic β cell generation

Pancreatic β cells are the source of insulin, which directly lowers blood glucose levels in the body. Our analyses of α1D gene-knockout (α1D–/–) mice show that the L-type calcium channel, α1D, is required for proper β cell generation in the postnatal pancreas. Knockout mice were characteristically slightly smaller than their littermates and exhibited hypoinsulinemia and glucose intolerance. However, isolated α1D–/– islets persisted in glucose sensing and insulin secretion, with compensatory overexpression of another L-type channel gene, α1C. Histologically, newborn α1D–/– mice had an equivalent number of islets to wild-type mice. In contrast, adult α1D–/– mice showed a decrease in the number and size of islets, compared with littermate wild-type mice due to a decrease in β cell generation. TUNEL staining showed that there was no increase in cell death in α1D–/– islets, and a 5-bromo-2′ deoxyuridine-labeling (BrdU-labeling) assay illustrated significant reduction in the proliferation rate of β cells in α1D–/– islets.

Endothelial progenitor cell homing: prominent role of the IGF2-IGF2R-PLCβ2 axis

Homing of endothelial progenitor cells (EPCs) to the neovascular zone is now considered to be an essential step in the formation of vascular networks during embryonic development and also for neovascularization in postnatal life. We report here the prominent role of the insulin-like growth factor 2 (IGF2)/IGF2 receptor (IGF2R) system in promoting EPC homing. With high-level expression of IGF2R in EPCs, IGF2-induced hypoxic conditions stimulated multiple steps of EPC homing in vitro and promoted both EPC recruitment and incorporation into the neovascular area, resulting in enhanced angiogenesis in vivo. Remarkably, all IGF2 actions were exerted predominantly through IGF2R-linked G(i) protein signaling and required intracellular Ca(2+) mobilization induced by the beta2 isoform of phospholipase C. Together, these findings indicate that locally generated IGF2 at either ischemic or tumor sites may contribute to postnatal vasculogenesis by augmenting the recruitment of EPCs. The utilization of the IGF2/IGF2R system may therefore be useful for the development of novel means to treat angiogenesis-dependent diseases.

The phox homology domain of phospholipase D activates dynamin GTPase activity and accelerates EGFR endocytosis

Dynamin is a large GTP-binding protein that mediates endocytosis by hydrolyzing GTP. Previously, we reported that phospholipase D2 (PLD2) interacts with dynamin in a GTP-dependent manner. This implies that PLD may regulate the GTPase cycle of dynamin. Here, we show that PLD functions as a GTPase activating protein (GAP) through its phox homology domain (PX), which directly activates the GTPase domain of dynamin, and that the arginine residues in the PLD–PX are vital for this GAP function. Moreover, wild-type PLD–PX, but not mutated PLD–PXs defective for GAP function in vitro, increased epidermal growth factor receptor (EGFR) endocytosis at physiological EGF concentrations. In addition, the silencing of PLDs was shown to retard EGFR endocytosis and the addition of wild-type PLDs or lipase-inactive PLDs, but not PLD1 mutants with defective GAP activity for dynamin in vitro, resulted in the recovery of EGFR endocytosis. These findings suggest that PLD, functioning as an intermolecular GAP for dynamin, accelerates EGFR endocytosis. Moreover, we determined that the phox homology domain itself had GAP activity — a novel function in addition to its role as a binding motif for proteins or lipids.