Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP 4 ), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-␣. Binding specificity was determined using displacement of 1-O- Receptor-stimulated phosphoinositide (PI) 1 metabolism generates numerous inositol polyphosphates (InsP n s) and inositol phospholipids, many of which may function as potential second messengers (1). Of the possible PI metabolites, Ins(1,4,5)P 3 (InsP 3 ) and diacylglycerol (DAG) are the best characterized second messengers. Generated by receptor-stimulated phospholipase C, hydrolysis of PtdIns(4,5)P 2 (2), Ins(1,4,5)P 3 binds to and gates an InsP 3 receptor calcium channel on the endoplasmic reticulum (2, 3). The lipid DAG remains in the membrane where it activates several protein kinase C isoforms and may regulate other targets (4, 5). In the membrane, DAG is metabolized rapidly to monoacylglycerol and to several phospholipids. In the cytoplasm, Ins(1,4,5)P 3 can be phosphorylated to Ins(1,3,4,5)P 4 by an InsP 3 3-kinase. Other isomers of InsP 4 , InsP 5 , and InsP 6 , some of which are synthesized independently of Ins(1,4,5)P 3 , have been identified (for review, see Ref. 6), and their production may also be regulated by receptors or during cell growth. Information from receptor binding studies, using radioactive InsP 4 and InsP 6 , have demonstrated that a number of important regulatory proteins contain high affinity InsP n binding sites. InsP n s have been implicated in the regulation of clathrin assembly proteins AP-2 (7, 8), AP-3 (9), the non-clathrin-associated coatomer proteins (10), synaptotagmin (11), and the regulation of the small GTPases ras and/or rap via a specific GTPase-activating protein (GAP) activity (12). Inositol phospholipids have also been postulated as messenger molecules. From in vivo, genetic, and permeabilized cell studies, evidence for critical roles for the inositol phospholipids PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P 2 as regulators of membrane vesicle trafficking and cytoskeletal rearrangements is accumulating rapidly (5, 13-15). One inositol phospholipid, PtdIns(3,4,5)P 3 , has emerged as a potential messenger molecule in receptor-stimulated cells (16 -18). Synthesized by receptor-stimulated PI 3-kinase phosphorylation of PtdIns(4,5)P 2 (18), PtdInsP 3 is not a substrate for PI-specific phospholipase * This work was supported in part by National Institutes of Mental Health Grants R29MH50102 and DDRC P50HD32901 (to A. B. T.). Work at Stony Brook was supported by National Institutes of Health Grant NS29632 (to G. D. P.). The first two authors contributed equally to this study. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank § §...
We tested for the presence of high-affinity phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3 binding sites in four phospholipase C (PLC) isozymes (delta1, beta1, beta2, and beta3), by probing these proteins with analogs of inositol phosphates, D-Ins(1,4,5)P3, D-Ins(1,3,4,5)P4, and InsP6, and polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3, which contain a photoactivatable benzoyldihydrocinnamide moiety. Only PLC-delta1 was specifically radiolabeled. More than 90% of the label was found in tryptic and chymotryptic fragments which reacted with antisera against the pleckstrin homology (PH) domain, whereas less than 5% was recovered in fragments that encompassed the catalytic core. In separate experiments, the isolated delta1-PH domain was also specifically labeled. Equilibrium binding of D-Ins(1,4,5)P3 to PLC-delta1 indicated the presence of a single, high-affinity binding site; binding of D-Ins(1,4,5)P3 to PLC-beta1, -beta2, or -beta3 was not detected. The catalytic activity of PLC-delta1 was inhibited by the product D-Ins(1,4,5)P3, whereas no inhibition of PLC-beta1, -beta2, or -beta3 activity was observed. These results demonstrate that the PH domain is the sole high-affinity PI(4,5)P2 binding site of PLC-delta1 and that a similar site is not present in PLC-beta1, -beta2, or -beta3. The data are consistent with the idea that the PH domain of PLC-delta1, but not the beta isozymes, directs the catalytic core to membranes enriched in PI(4,5)P2 and is subject to product inhibition.
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