PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40(phox).

The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b(558) (gp91(phox) and p22(phox)) and three soluble factors (GTP-Rac, p47(phox) and p67(phox) (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40(phox) has subsequently been discovered as a binding partner for p67(phox) (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40(phox) to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX domain of p40(phox). These results offer new insights into the roles for PI(3)Ks and p40(phox) in ROS formation and define a cellular ligand for the orphan PX domain.

Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B.

Protein kinase B (PKB) is activated in response to phosphoinositide 3-kinases and their lipid products phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3] and PtdIns(3,4)P2 in the signaling pathways used by a wide variety of growth factors, antigens, and inflammatory stimuli. PKB is a direct target of these lipids, but this regulation is complex. The lipids can bind to the pleckstrin homologous domain of PKB, causing its translocation to the membrane, and also enable upstream, Thr308-directed kinases to phosphorylate and activate PKB. Four isoforms of these PKB kinases were purified from sheep brain. They bound PtdIns(3,4,5)P3 and associated with lipid vesicles containing it. These kinases contain an NH2-terminal catalytic domain and a COOH-terminal pleckstrin homologous domain, and their heterologous expression augments receptor activation of PKB, which suggests they are the primary signal transducers that enable PtdIns(3,4,5)P3 or PtdIns- (3,4)P2 to activate PKB and hence to control signaling pathways regulating cell survival, glucose uptake, and glycogen metabolism.

Translocation of PDK-1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B.

BACKGROUND: Protein kinase B (PKB) is involved in the regulation of apoptosis, protein synthesis and glycogen metabolism in mammalian cells. Phosphoinositide-dependent protein kinase (PDK-1) activates PKB in a manner dependent on phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which is also needed for the translocation of PKB to the plasma membrane. It has been proposed that the amount of PKB activated is determined exclusively as a result of its translocation, and that a constitutively active pool of membrane-associated PDK-1 simply phosphorylates all the PKB made available. Here, we have investigated the effects of membrane localisation of PDK-1 on PKB activation. RESULTS: Ectopically expressed PDK-1 translocated to the plasma membrane in response to platelet-derived growth factor (PDGF) and translocation was sensitive to wortmannin, an inhibitor of phosphoinositide 3-kinase. Translocation of PDK-1 also occurred upon its co-expression with constitutively active phosphoinositide 3-kinase, but not with an inactive form. Overexpression of PDK-1 enhanced the ability of PDGF to activate PKB. PDK-1 disrupted in the pleckstrin homology (PH) domain which did not translocate to the membrane did not increase PKB activity in response to PDGF, whereas membrane-targeted PDK-1 activated PKB to the extent that it could not be activated further by PDGF. CONCLUSIONS: In response to PDGF, binding of Ptdlns (3,4,5)P3 and/or Ptdlns(3,4)P2 to the PH domain of PDK-1 causes its translocation to the plasma membrane where it co-localises with PKB, significantly contributing to the scale of PKB activation.

DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation.

BACKGROUND: Phosphoinositide (PI) 3-kinase and its second messenger products, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), play important roles in signalling processes crucial for cell movement, differentiation and survival. Previously, we isolated a 32kDa PtdIns(3,4,5)P(3)-binding protein from porcine leukocytes. This protein contains an amino-terminal Src homology 2 (SH2) domain and a carboxy-terminal pleckstrin homology (PH) domain, and is identical to the recently described DAPP1 (also known as PHISH or Bam32) protein. Here, we characterised the subcellular distribution of DAPP1 in response to cell stimulation. RESULTS: When expressed transiently in porcine aortic endothelial (PAE) cells, DAPP1 translocated from the cytosol to the plasma membrane in response to platelet-derived growth factor (PDGF). This translocation was dependent on both PI 3-kinase activity and an intact DAPP1 PH domain. Following recruitment to the plasma membrane, DAPP1 entered the cell in vesicles. Similar responses were seen in DT40 chicken B cells following antibody treatment, and Rat-1 fibroblasts following epidermal growth factor (EGF) or PDGF treatment. Colocalisation studies in PAE cells suggested entry of DAPP1 by endocytosis in a population of early endosomes containing internalised PDGF-beta receptors. DAPP1 also underwent PI 3-kinase-dependent phosphorylation on Tyr139 in response to PDGF stimulation, and this event was involved in the vesicular response. CONCLUSIONS: This is the first report of plasma-membrane recruitment and endocytosis of a PI 3-kinase effector protein in response to cell stimulation. The results suggest a novel role for DAPP1 in endosomal trafficking or sorting.

The sites of tryptic cleavage in bovine secretory component: structural and functional implications.

Tryptic fragments from bovine secretory component and sIgA have been separated by HPLC and/or SDS polyacrylamide gel electrophoresis and electroblotting. Their N-terminal amino acid sequences have been determined and their positions in the secretory component molecule deduced by homology with the amino acid sequences of human secretory component and rabbit polyimmunoglobulin receptor. Taken in conjunction with the known binding affinities of the tryptic fragments, the results imply that the three most N-terminal domains of secretory component are directly involved in binding IgM and IgA dimers. The results also favour the concept of an extended 'zig-zag' structure for the secretory component molecule.

Sequence of ovine Ig gamma 2 constant region heavy chain cDNA and molecular modelling of ruminant IgG isotypes.

Ovine mesenteric lymph node mRNA was used for PCR amplification of DNA coding for immunoglobulin gamma 1 and gamma 2 heavy chain constant regions. Primers complementary to regions of CH1 conserved between ruminants were used for upstream priming, with downstream priming on the poly-A segment. PCR products of the appropriate length were cloned and gamma positive clones selected with a CH1 conserved-region probe. Of these, gamma 1 clones were positively selected and gamma 2 clones negatively selected with a gamma 1 hinge-specific probe. Ovine gamma 2 cDNA has 93% identity of nucleotides with ovine gamma 1. Both ovine gamma 1 and gamma 2 CH1 domains encoded two consecutive cysteine residues (Cys-127, -128, Kabat numbering), an arrangement which is deduced to form a pair of disulphide bridges, one to the L chain and one as an intra-chain bridge to the uppermost Cys of the hinge, as in rabbit and goat IgG. The majority of the differences between the isotypes occur in the hinge region and an evolutionary pattern for ruminant IgG hinges can now be identified. IgG1 isotypes are typical, with hinges containing the C-terminal Cys-Pro motif, but deletion and replacement of nucleotides (in the ancestral gene) of ruminant gamma 2 has shortened the IgG2 hinge, removing the Cys-Pro motif and the consensus high affinity Fc gamma RI receptor motif at the start of CH2. An N-terminal glycosylation site and the peptide motif for complement C1q binding are present in CH2 of both isotypes. The hinge regions of gamma 1 and gamma 2 and predicted structures for ovine IgG1 and IgG2 have been modelled. Close apposition of Fab and Fc in IgG2 produces steric hindrance at the normally accessible Fab/hinge/Fc interface; the structural differences between the ruminant isotypes form a basis for understanding some of the differences in their effector properties.

Acetylcholine receptor molecules of the nematode Caenorhabditis elegans.

Receptors for acetylcholine are present in nematodes. Studies using physiological and biochemical methods have revealed the existence of nicotinic acetylcholine receptors with a novel pharmacology. Caenorhabditis elegans provides a particularly suitable organism with which to investigate such receptors using molecular genetic approaches. Mutants resistant to the cholinergic agonist (and anthelmintic drug) levamisole have permitted the isolation of a number of genes, including structural subunits of the nicotinic acetylcholine receptor. The only known viable mutants of nicotinic receptors are those of Caenorhabditis elegans. This organism offers the prospect of studying the developmental and regulatory effects of the loss of a single component of the receptor. Using Caenorhabditis elegans it is possible to select interesting phenotypic mutations by in vivo mutagenesis before determining the causative lesion. Resistance genes other than those encoding structural subunits are of particular interest, as they will encode additional polypeptides closely associated with nicotinic receptor function. Such proteins are often difficult or impossible to identify using conventional biochemical approaches, whereas genetic selection should permit their identification.

Unusual features of the T-cell receptor C domains are revealed by structural comparisons with other members of the immunoglobulin superfamily.

The amino acid sequences of the domains of human, mouse and rabbit T-cell antigen receptors have been aligned with those of immunoglobulin domains of known three-dimensional structure. Computerized secondary structure predictions have been performed on the sequences and putative models of the domains have been constructed. The receptor V alpha and V beta domains are closely related to immunoglobulin VH domains. The receptor C alpha domain shows some major divergences from immunoglobulin C domains and the C beta domain displays an unusual feature. The implications for T-cell receptor function are discussed.

Tertiary structures for the extracellular domains of the epithelial polyimmunoglobulin receptor (secretory component) derived by primary structure comparisons with immunoglobulins.

The amino acid sequences of the rabbit receptor and human secretory component (SC) domains have been compared with those of immunoglobin (Ig) domains. Accessible and inaccessible sites of tryptic cleavage in bovine SC have been located by sequence homology. Computerized secondary structure prediction and three dimensional model building have been carried out. The resulting tertiary structures are extremely Ig-like consisting of two superposed beta-pleated sheets. All carbohydrate sites lie at external positions as do tryptic cleavage sites. Potential sites for tryptic hydrolysis that are not cleaved lie at buried or partially buried positions. 6. Inter-beta-sheet contact between domains appears to be highly unlikely so that the quaternary structure is largely determined by longitudinal contacts.

Some observations on the replacement of the conserved amino acid residues of immunoglobulin domains.

1. Computer graphics have been used to model replacements of conserved residues in immunoglobulin domains. 2. Replacements, even those involving large changes in side chain volume, could be accommodated in the domain but rotation and repacking of surrounding side chains was generally necessary. 3. Repacking often resulted in increases in inter-atomic distances between side chains and loss of some van der Waals' contacts. This would be expected to make the domain slightly less stable. 4. Losses in domain stability might not have a serious affect on antigen binding but could result in circulating antibody becoming more susceptible to biological degradation with considerable reduction in biological half-life.

Some observations on conserved polar side chains in immunoglobulin V-domains.

1. The roles of conserved polar residues have been studied in 12 V-domains for which atomic coordinates are available. 2. In most cases a particular residue had a similar side chain conformation in all V-domains examined and the polar group provided the same hydrogen bonds which helped to stabilize the conformations of the domains. 3. In the case of a conserved glutamine/glutamic acid residue the buried side chain could adopt a variety of conformations and the polar group could form different hydrogen bonds from one domain to another. However, they contributed similarly to domain stability. 4. In the case of a conserved threonine/serine residue its side chain showed relative rotations of up to 180 degrees from one domain to another. The hydroxyl group could be buried or exposed at the domain surface. In some domains it formed hydrogen bonds to two other protein atoms but in other domains there was a single hydrogen bond or none at all. The varied roles of this residue are discussed in the text.

Expression of a testis-specific putative actin-capping protein associated with the developing acrosome during rat spermiogenesis.

Actin-capping proteins are ubiquitous components of mammalian cells. They are known to regulate the polymerization state of actin and hence indirectly control the activity of the cytoskeleton and cell shape. As part of our investigation into the molecular mechanisms that direct differentiation of a round spermatid into an elongating spermatozoa, we report on a testis-specific 1.7-kb transcript from rat testis with sequence similarities to the alpha subunit of actin-capping proteins (ACPs) from somatic cells. The transcript contains a putative cAMP-responsive motif (CREM) upstream of the initiation codon in the DNA sequence and is expressed postmeiotically, first appearing between 20 and 30 days of postnatal development. The primary amino acid sequence is 90% identical to that of a previously identified testis-specific mouse protein, gsg3, both showing approximately 40% homology to the alpha subunit of somatic ACPs. An affinity-purified polyclonal antibody to a synthetic peptide derived from the rat transcript identified a 32-kDa protein on Western blots of testicular extracts. Indirect immunofluorescent localization of the protein on frozen sections of adult rat testis showed that it is intracellular and accumulates asymmetrically in the cytoplasm of round spermatids coincident with the position of the developing acrosome. This spatial expression parallels the distribution of F-actin during sperm differentiation, supporting the hypothesis that testis-specific ACPs have an important role in determining the final shape of mature sperm heads. A disturbance in the expression of these ACPs may underlie many of the abnormalities in sperm morphology observed in infertile semen.

Fatty acylation of phospholipase D1 on cysteine residues 240 and 241 determines localization on intracellular membranes.

We have reported previously that phospholipase D1 (PLD1) is labeled specifically with [(3)H]palmitate following transient expression and immunoprecipitation and that this modification appeared important both for membrane localization and catalytic activity. In this work we identify by mutagenesis that the acylation sites on PLD1 are cysteine residues 240 and 241, with the cysteine at position 241 accounting for most but not all of the modification. Replacement of both cysteine residues with either serines or alanines resulted in a mutant protein that contained undetectable [(3)H]palmitate. In comparison with the wild type protein, the double mutant showed reduced catalytic activity in vivo, whereas its activity in vitro was unchanged. In addition, the localization of the double mutant was altered in comparison with the wild type protein, whereas wild type PLD1 is primarily on intracellular membranes and on punctate structures, the double mutant was on plasma membrane. Because cysteines 240 and 241 lie within a putative pleckstrin homology domain of PLD1, it is likely that fatty acylation on these residues modulates the function of the PLD1 pleckstrin homology domain.

Identification of a binding site for integrin alphaEbeta7 in the N-terminal domain of E-cadherin.

The integrin alphaEbeta7, which is predominantly expressed on mucosal T lymphocytes, has recently been shown to recognize the cell adhesion molecule, E-cadherin, on epithelial cells. We have carried out mutations on E-cadherin, involving domain deletions as well as substitutions of specific amino acids, in order to identify the sites recognized by the integrin. Binding of alphaEbeta7 required the presence of the first two N-terminal domains of E-cadherin. Deletion of extracellular domains 3 and 4 or truncation of the cytoplasmic domain of E-cadherin had no consequence on integrin binding. Substitution of a glutamic acid in the BC loop of the Ig structure of the fist, N-terminal, domain of E-cadherin abrogated binding of alphaEbeta7. This mutation did not appear to affect the conformation of the domain nor the pattern of expression of E-cadherin on the cell surface. Synthetic peptides encompassing the first domain of E-cadherin had very little inhibitory effect on the interaction with alphaEbeta7. Our results highlight structural dissimilarities between recognition of E-cadherin by alphaEbeta7 and recognition of other members of the IgSF by integrins and show that the heterophilic (integrin binding) and homophilic sites in the N-terminal domain of E-cadherin are distinct.

Studies on transcriptional regulation of the mucosal T-cell integrin alphaEbeta7 (CD103).

Integrin alphaEbeta7 is expressed almost exclusively by mucosal T cells and mucosal dendritic antigen-presenting cells (APCs) and is thought to be induced locally by transforming growth factor-beta (TGF-beta). In mice, mRNA for the alphaE subunit was found to be abundant in mucosal T cells but absent from other tissues. Exposure of a T-cell line to TGF-beta strongly up-regulated alphaE mRNA levels within 30 min, and nuclear run-on experiments established that regulation occurred at the level of transcription. The organization of the human alphaE gene and a very closely linked novel gene, ELG, was determined. The alphaE promoter was tested in T cells and fibroblasts and functioned equally well in both cell types and did not confer TGF-beta responsiveness. Regions of the promoter providing enhancer activity and phorbol 12-myristate 13-acetate (PMA) responsiveness were identified by deletion studies. DNAse 1 hypersensitivity analysis of 36 kb of the alphaE gene revealed one hypersensitive site, found only in alphaE+ cells, located near the transcription start points. These results show that, unlike the situation with other integrins, lineage specificity and cytokine responsiveness of alphaE transcription are not conferred by the proximal promoter. Specificity may depend on distant control elements that have not yet been identified.

Structural organization of the mouse phosphatidylinositol 3-kinase p110d gene.

Phosphatidylinositol 3-kinases are a family of dual specificity lipid/protein kinases. The products of PI3K's, phosphatidylinositol(3,4,5) triphosphate and phosphatidylinositol(3,4) bisphosphate, act as second messengers connecting activated transmembrane receptors to signaling pathways that control gene transcription, proliferation, transformation, programmed cell death, adhesion, migration and vesicular transport. There is evidence that different isoforms of PI3K's activate specific signaling pathways and are thus responsible for integrating cellular responses. The elucidation of the genomic structure of the catalytic subunits is a necessary step for the investigation of the function of PI3K isoforms by inactivation of the gene in vivo. The structural organization of p110alpha, beta, and gamma genes has been previously reported. Here we report the cloning, sequencing, and structural organization of the mouse p110delta gene from a murine 129/Sv genomic library. The p110delta gene consists of 22 exons and spans over 13 kb. Comparison of the genomic structure with that of p110alpha, beta, and gamma demonstrates that the p110delta gene shares its exon structure with p110beta, the most closely related PI3K at the amino acid level.

Site-directed mutagenesis of boar proacrosin reveals residues involved in binding of zona pellucida glycoproteins.

Proacrosin, the zymogen form of the serine protease beta-acrosin, is thought to function as a secondary binding molecule between mammalian gametes during fertilization (Jansen et al., 1995: Int J Dev Biol 39, 501-510). The interaction involves strong ionic bonds between positively charged amino acids on proacrosin and negatively charged polysulphate groups on zona pellucida glycoproteins. In this investigation, we identified the basic residues on proacrosin that are important for this binding. Site-directed mutagenesis shows that two groups of amino acids comprising His47, Arg50, and Arg51 together with Arg250, Lys252, and Arg253 are crucial because their deletion or replacement severely reduces affinity for zona glycoproteins. Molecular models of proacrosin reveal that these residues are located along one face of the protein on two exposed surface loops that project over and around the catalytic site. These findings support the hypothesis that polysulphate binding sites on proacrosin are formed by a restricted number of basic amino acids on the surface of the protein, presenting a specific orientation that is complementary to negatively charged sulphate groups on zona glycoproteins. Identification and elucidation of the stereochemistry of these charged moieties will aid design of new kinds of nonsteroidal antifertility agents.

The novel epididymal secretory protein ESP13.2 in Macaca fascicularis.

Newly synthesized mammalian spermatozoa undergo critical modifications as they pass along the epididymis. The modifications endow spermatozoa with fertilizing ability and occur largely as a consequence of epididymal gene expression. With this in mind, we here employed a cDNA cloning strategy designed to identify key epididymal gene products. We describe a novel cynomolgus monkey (Macaca fascicularis) epididymal transcript designated cy-ESP13.2, of 690 nucleotides. The putative human ortholog was cloned and is highly conserved. Both cDNA sequences predict small, secretory proteins with a disulfide-stabilized core. Anti-peptide polyclonal antibodies were raised to a predicted cy-ESP13.2 surface loop. Western blotting with these antibodies revealed high-level, epididymis-specific expression of cy-ESP13.2, consistent with the pattern of cy-ESP13.2 mRNA expression assessed by Northern blotting. cy-ESP13.2 protein was of 30 kDa and was readily detectable in epithelial cells lining the efferent ductules, initial segment, and cauda regions of the epididymis, but not on spermatozoa. Similarities to members of the four-disulfide-core family suggest clues to ESP13.2 function.

Nucleotide sequences and three-dimensional modelling of the VH and VL domains of two human monoclonal antibodies specific for the D antigen of the human Rh-blood-group system.

The nucleotide sequences were determined for the VH and VL domains of two human IgG1 antibodies, Pag-1 and Fog-B, specific for the D antigen of the Rh-blood-group system. The VH-region genes of the two antibodies were derived from separate germ-line genes within the VH-IV gene family, but both antibodies used the same JH6 gene. The D-region genes differed from each other, and no similarity was found to known D regions. The light chain of Fog-B belongs to the V lambda-I subgroup and that of Pag-1 probably belongs to the V lambda-V subgroup; both light chains used the J2/3 gene. Three-dimensional models of the variable domains were made, based on those of known crystallographic structure. The surface contours at the combining sites are clearly different, consistent with the evidence that the antibodies recognize different but overlapping epitopes. Some details of the molecular modelling of hypervariable regions have been deposited as Supplementary Publication SUP 50155 (6 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.

The distribution of Tap2 alleles among laboratory rat RT1 haplotypes.

We are reporting the cDNA sequences of Tap2 from two cima and two cimb rat strains. Comparison of the cDNA sequences shows that these alleles fall into two groups, which we refer to as Tap2-A and Tap2-B. We found that alleles from the Tap2-B group are more closely related to the mouse homologue than are Tap2-A alleles, and among the 48 nucleotides which differ between the Tap2-A and Tap2-B cDNAs, three affect restriction sites. We defined pairs of oligonucleotides which allow amplification of the regions bearing these restriction sites from genomic DNA or cDNA, and this technique has been successful for the genotyping of all of the 56 laboratory strains of Rattus norvegicus tested and for five cell lines tested so far. All 14 known RT1 standard haplotypes were tested, and 7 found to belong to the Tap2-B group, and 7 to Tap2-A. We also found that intron sizes among the alleles of the Tap2-B group fall into two subgroups, providing further insight into the phylogeny of these various haplotypes.