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The pleckstrin homology (PH) domain is a newly recognized protein module believed to play an important role in signal transduction. While the tertiary structures of several PH domains have been determined, some cocomplexed with ligands, the function of this domain remains elusive. In this report, the PH domain located in the N terminus of human phospholipase C-␦1 (PLC␦1) was found to regulate enzyme activity. The hydrolysis of phosphatidylinositol (PI) was stimulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in a dose-dependent manner with an EC 50 ‫؍‬ 1 M (0.3 mol%), up to 9-fold higher when 5 M (1.5 mol%) of PIP 2 was incorporated into the PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 M of PI with a molar ratio of PI:PS:PC ‫؍‬ 1:5:5). Stimulation was specific for PIP 2 , since other anionic phospholipids including phosphatidylinositol 4-phosphate had no stimulatory effect. PIP 2 -mediated stimulation was, however, inhibited by inositol 1,4,5-triphosphate (IP 3 ) in a dose-dependent manner, suggesting a modulatory role for this inositol. When a nested set of PH domain deletions up to 70 amino acids from the N terminus of PLC␦1 were constructed, the deletion mutant enzymes all catalyzed the hydrolysis of the micelle forms of PI and PIP 2 with specific activities comparable with those of the wild type enzyme. However, the stimulatory effect of PIP 2 was greatly diminished when more than 20 amino acid residues were deleted from the N terminus. To identify the specific residues involved in PIP 2 -mediated enzyme activation, amino acids with functional side chains between residues 20 and 40 were individually changed to glycine. While all these mutations had little effect on the ability of the enzyme to catalyze the hydrolysis of PI or PIP 2 micelles, the catalytic activity of mutants K24G, K30G, K32G, R38G, or W36G was markedly unresponsive to PIP 2 . Analysis of PIP 2 -stimulated PI hydrolysis by a dual substrate binding model of catalysis revealed that the micellar dissociation constant (K s ) of PLC␦1 for the PI/ PS/PC vesicles was reduced from 558 M to 53 M, and the interfacial Michaelis constant (K m ) was reduced from 0.21 to 0.06 by PIP 2 . The maximum rate of PI hydrolysis (V max ) was not affected by PIP 2 . These results demonstrate that a major function of the PH domain of PLC␦1 is to modulate enzyme activity. Further, our results identify PIP 2 as a functional ligand for a PH domain and suggest a general mechanism for the regulation of other proteins by PIP 2 .

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Understanding the intracellular transport of the ␤-amyloid precursor protein (APP) is a major key to elucidate the regulation of APP processing and thus ␤-amyloid peptide generation in Alzheimer disease pathogenesis. APP and its two paralogues, APLP1 and APLP2 (APLPs), are processed in a very similar manner by the same protease activities. A putative candidate involved in APP transport is protein interacting with APP tail 1 (PAT1), which was reported to interact with the APP intracellular domain. We show that PAT1a, which is 99.0% identical to PAT1, binds to APP, APLP1, and APLP2 in vivo and describe their co-localization in trans-Golgi network vesicles or endosomes in primary neurons. We further demonstrate a direct interaction of PAT1a with the basolateral sorting signal of APP/APLPs. Moreover, we provide evidence for a direct role of PAT1a in APP/APLP transport as overexpression or RNA interference-mediated knockdown of PAT1a modulates APP/APLPs levels at the cell surface. Finally, we show that PAT1a promotes APP/APLPs processing, resulting in increased secretion of ␤-amyloid peptide. Taken together, our data establish PAT1a as a functional link between APP/APLPs transport and their processing.Amyloid plaques, the major hallmark of Alzheimer disease, are mainly composed of the ␤-amyloid peptide (A␤), 6 which is proteolytically derived from the ␤-amyloid precursor protein (APP) (1). APP belongs to a protein family with two mammalian paralogues, the ␤-amyloid precursor-like proteins (APLP) 1 and 2 (2-5). APP/APLPs share highly conserved protein domain organization (6), form homo-and heterotypic interactions (7), and are proteolytically processed in a similar manner (8). The extracellular domain of APP/APLPs can be cleaved by ␣-secretases or, alternatively, by the ␤-secretase ␤-site APP cleaving enzyme 1 (BACE 1) (8 -12). The resulting membraneretained C-terminal fragments (CTFs) are subsequently processed by cleavage within the transmembrane domain by the ␥-secretase complex (13,14). Consecutive ␤-and ␥-cleavage of APP/APLPs results in the release of A␤/A␤-like peptides, whereas ␣-and ␥-cleavage generate p3/p3-like fragments, respectively. Concomitantly, both processing pathways liberate the corresponding intracellular domains (ICDs) (8,15,16). A function in nuclear signaling was proposed for the APP/APLP ICDs (16 -18), suggesting that processing of APP/APLPs is a crucial step in the pathology of Alzheimer disease and central to the physiological function of APP/APLPs.For APP, a number of intracellular interaction partners, such as Fe65 (19), Fe65L1 (20), or X11␣ and X11␤ (21), are known to affect its processing. All of these proteins interact with the NPTY motif in the intracellular domain of APP, APLP1, and APLP2 via their phosphotyrosine binding (PTB) domain (22). Protein interacting with APP tail 1 (PAT1) binds to the basolateral sorting sequence (BaSS) of APP and is associated with microtubules. Further, an influence on APP cleavage at the cell surface has been proposed (23). Therefore, a kinesin light cha...

Painful sensations are some of the most frequent complaints of patients who are admitted to local medical clinics. Persistent pain varies according to its causes, often resulting from local tissue damage or inflammation. Central somatosensory pathway lesions that are not adequately relieved can consequently cause central pain syndrome or central neuropathic pain. Research on the molecular mechanisms that underlie this pathogenesis is important for treating such pain. To date, evidence suggests the involvement of ion channels, including adenosine triphosphate (ATP)-gated cation channel P2X receptors, in central nervous system pain transmission and persistent modulation upon and following the occurrence of neuropathic pain. Several P2X receptor subtypes, including P2X2, P2X3, P2X4, and P2X7, have been shown to play diverse roles in the pathogenesis of central pain including the mediation of fast transmission in the peripheral nervous system and modulation of neuronal activity in the central nervous system. This review article highlights the role of the P2X family of ATP receptors in the pathogenesis of central neuropathic pain and pain transmission. We discuss basic research that may be translated to clinical application, suggesting that P2X receptors may be treatment targets for central pain syndrome.

Supplemental Digital Content is Available in the Text.Postthalamic hemorrhagic alterations in neuronal plasticity in the medial thalamus induced by elevations in brain-derived neurotrophic factor levels are a key factor in central poststroke pain.

Point mutagenesis, phosphatidylinositol (PI), and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolysis assays and equilibrium centrifugation PIP 2 assays were used to study the functional roles of four highly conserved arginine residues in the Y region of human phospholipase C ␦1 (PLC␦1) (Arg-527, -549, -556, -701). Most of the mutant enzymes were either partially defective or fully active in their abilities to catalyze the hydrolysis of PI or PIP 2 . However, upon substitution of Arg-549 by glycine or histidine, the mutant enzyme was defective in its ability to catalyze the hydrolysis of PIP 2 , but it is still able to hydrolyze PI. Replacing Arg-549 with lysine had little effect on the level of PI and PIP 2 hydrolytic activities of the mutant enzyme. The residual PIP 2 hydrolyzing activity of R549H is highly dependent on pH. R549H showed 5-10% of the PIP 2 -hydrolyzing activity of the native enzyme between pH 5 and 7 and nondetectable PIP 2 -hydrolyzing activity at pH 8. The PIP 2 -hydrolyzing activity of R549G was not detectable at all pH values. Kinetic analysis of PLC␦1-catalyzed PIP 2 hydrolysis revealed that the micellar dissociation constant K s and interfacial Michaelis constant K m were similar in the native, R549K, and R549H enzymes; but the specific activity at the saturated substrate mole fraction and infinite level of substrate (V max ) of the R549H mutant were reduced by a factor of 15. PIP 2 competitively inhibits the native enzyme to hydrolyze PI at both pH 7 and 8. However, PIP 2 inhibits R549H only at pH 7.0 and does not inhibit R549G at either pH. Taken together, these results suggest that positive charge at position 549 of PLC␦1 protein is essential for the enzyme to recognize and catalyze the hydrolysis of PIP 2 but not PI.Phosphatidylinositide-specific phospholipase C (PI-PLC) 1 hydrolyzes inositol phospholipids into diacylglycerol and inositol 1,4,5-trisphosphate, which function as important second messengers to activate protein kinase C and mobilize intracellular Ca 2ϩ