Inositol (1,4,5)-trisphosphate [Ins(1,4,5) P 3 ] is one of the key intracellular second messengers in cells and mobilizes Ca 2+ stores in the ER (endoplasmic reticulum). Ins(1,4,5) P 3 has a short half-life within the cell, and is rapidly metabolized through one of two pathways, one of which involves further phosphorylation of the inositol ring: Ins(1,4,5) P 3 3-kinase (IP 3 -3K) phosphorylates Ins(1,4,5) P 3 , resulting in the formation of inositol (1,3,4,5)-tetrakisphosphate [Ins(1,3,4,5) P 4 ]. There are three known isoforms of IP 3 -3K, designated IP 3 -3KA, IP 3 -3KB and IP 3 -3KC. These have differing N-termini, but highly conserved C-termini harbouring the catalytic domain. The three IP 3 -3K isoforms have different subcellular locations and the B-kinase is uniquely present in both cytosolic and membrane-bound pools. As it is the N-terminus of the B-kinase that differs most from the A- and C-kinases, we have hypothesized that this portion of the protein may be responsible for membrane localization. Although there are no known membrane-targeting protein motifs within the sequence of IP 3 -3KB, it is found to be tightly associated with the ER membrane. Here, we show that specific regions of the N-terminus of IP 3 -3KB are necessary and sufficient for efficient membrane localization of the protein. We also report that, in the presence of Ca 2+ , the kinase domain of IP 3 -3KB is cleaved from the membrane-anchoring region by calpain.

Ins(1,4,5) P 3 3-kinase (IP3K) phosphorylates the Ca 2+ -mobilizing second messenger Ins(1,4,5) P 3 to yield the putative second messenger Ins(1,3,4,5) P 4 . A HeLa cell line was established expressing the rat B isoform of IP3K under the control of an inducible promoter. The IP3KB-transfected cell line possessed 23-fold greater IP3K activity than untransfected cells after induction of IP3KB expression, but only 0.23-fold greater activity when IP3KB expression was not induced. Elevating IP3KB expression significantly reduced levels of Ins(1,4,5) P 3 and increased levels of Ins(1,3,4,5) P 4 after stimulation of cells with histamine, but had no effect on basal levels. Histamine- and ATP-evoked cytosolic Ca 2+ responses were dramatically reduced upon elevation of IP3KB expression. On stimulation with a supramaximal dose of histamine, 67% of cells induced to express IP3KB gave no detectable elevation in cytosolic Ca 2+ , compared with 3% of uninduced cells. The quantity of Ca 2+ within thapsigargin-sensitive and -insensitive stores was unaffected by elevation of IP3KB expression, as was capacitative Ca 2+ entry. These data suggest that IP3KB may play a significant role in the regulation of Ins(1,4,5) P 3 levels, and consequently in Ca 2+ responses following stimulation of cells with Ins(1,4,5) P 3 -elevating agonists.

Neurabin-II (spinophilin) is a ubiquitously expressed F-actin-binding protein containing an N-terminal actin-binding domain, a PDZ (PSD95/discs large/ZO-1) domain and a C-terminal domain predicted to form a coiled-coil structure. We have stably expressed a green fluorescent protein (GFP)-tagged version of neurabin-II in PC12 cells, and characterized the in vivo dynamics of this actin-binding protein using confocal fluorescence microscopy. We show that GFP-neurabin-II localizes to actin filaments, especially at cortical sites and areas underlying sites of active membrane remodelling. GFP-neurabin-II labels only a subset of F-actin within these cells, as indicated by rhodamine-phalloidin staining. Both actin filaments and small, highly motile structures within the cell body are seen. Photobleaching experiments show that GFP-neurabin-II also exhibits highly dynamic behaviour when bound to actin filaments. Latrunculin B treatment results in rapid relocalization of GFP-neurabin-II to the cytosol, whereas cytochalasin D treatment causes the collapse of GFP-neurabin-II fluorescence to intensely fluorescent foci of F-actin within the cell body. This collapse is reversed on cytochalasin D removal, recovery from which is greatly accelerated by stimulation of cells with epidermal growth factor (EGF). Furthermore, we show that this EGF-induced relocalization of GFP-neurabin-II is dependent on the activity of the small GTPase Rac1 but not the activity of ADP-ribosylation factor 6.

Multiple sorting steps within eukaryotic cells are mediated by tyrosine-based sorting motifs. Motifs conforming to the consensus -YXX∅- (where ∅ indicates a bulky hydrophobic residue) have been shown to specify high-efficiency internalization from the plasma membrane, targeting from the plasma membrane to the trans -Golgi network and targeting to lysosomal compartments as well as being involved in basolateral sorting in polarized cells. These motifs are recognized by the medium-chain subunits of heterotetrameric adaptor complexes. Whereas these motifs have been shown to be sufficient to mediate interaction with the µ -chains, we and others have shown that their context is important in determining the affinity of interaction. In this study we have investigated the interaction between the tyrosine motifs of the type-1 integral membrane proteins TGN38 and lgp120 with medium-chain subunits using the yeast two-hybrid system. Whereas the wild-type version of the cytosolic domain of TGN38 interacts with highest affinity with µ 2, we show that the cytosolic domain of lgp120 interacts almost exclusively with µ 3A. The specificity of binding of tyrosine-based sorting motifs to µ -chains is shown to be highly sensitive to the context in which the motif lies. For example, the -YQTI- motif of lgp120 is effectively non-functional with regard to µ -chain binding when placed in the context of the TGN38 cytosolic domain. Deletion of four amino acids (NLKL) at the extreme C-terminus of TGN38, leaving the YXX∅ motif as the C-terminus, greatly enhances the affinity of interaction with µ 2. Furthermore, addition of these same residues to the extreme C-terminus of lgp120 effectively abolishes the interaction of the cytosolic domain of lgp120 with µ -chains. We also show that the newly identified µ -adaptin-related protein 2 ( µ 4) only interacts weakly with tyrosine-based sorting motifs.

The generation of superoxide by the NADPH oxidase of neutrophils is accompanied by the efflux of H + ions through a H + channel. gp91- phox , a protein component of the oxidase, has been shown previously to function as a H + channel [Henderson, Banting and Chappell (1995) J. Biol. Chem. 270 , 5909–5916]. We have constructed a CHO cell line (CHO-N) that expresses an N-terminal fragment of gp91- phox containing the predicted multiple transmembrane domains of the protein. These cells exhibit H + fluxes in response to an imposed proton motive force and in the presence of arachidonate (to open the channel). The H + fluxes were indistinguishable from those observed in cells expressing full-length gp91- phox . Therefore the N-terminal 230 amino acids of gp91- phox contain all that is required to function as the NADPH oxidase-associated H + channel.

We previously reported the isolation of a rat cDNA clone encoding a protein with significant sequence homology to the B isoform of human myo -inositol 1,4,5-trisphosphate 3-kinase (IP 3 3-kinase B); this protein was thus designated rat IP 3 3-kinase B [Thomas, Brake, Luzio, Stanley and Banting (1994) Biochim. Biophys. Acta 1220 , 219–222]. However, no IP 3 kinase isoform had been shown to generate the physiologically important isoform of inositol tetrakisphosphate, i.e. inositol 1,3,4,5-tetrakisphosphate. We now present direct evidence that the putative rat IP 3 3-kinase B is genuinely an IP 3 3-kinase. We also show that the enzyme exists both as a peripheral membrane protein tightly associated with the cytosolic face of the extended endoplasmic reticulum network, and as a cytosolic protein. Association of the IP 3 3-kinase with membranes is not affected by treatment with brefeldin A, Na 2 CO 3 (pH 11.5), 2 M NaCl, or alteration of [Ca 2+ ]. However, treatment of isolated membranes with 4 M urea leads to dissociation of the kinase from the membrane, implying that membrane association involves specific, conformation-dependent protein–protein interactions. The fact that IP 3 3-kinase B is localized exclusively to membranes of Ca 2+ stores, is consistent with a model where this kinase plays a role in IP 3 -dependent Ca 2+ release.

We have expressed rat myo -inositol 1,4,5-trisphosphate (IP 3 ) 3-kinase B as both a full-length, recombinant, non-fusion protein and a full-length, recombinant, fusion protein with maltose-binding protein (MBP) in Escherichia coli . The fusion protein with MBP is soluble, binds calmodulin and is enzymically active whereas the non-fusion protein is insoluble and does not bind calmodulin unless co-expressed with bacterial chaperone proteins (either GroES and GroEL, or DnaK, DnaJ and GrpE). However, soluble, calmodulin-binding non-fusion IP 3 3-kinase B is enzymically inactive. The catalytic domain of the enzyme has previously been shown to reside near the C-terminus; the results we present suggest an auto-regulatory role for the N-terminus.