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Biomedical subjects

G Banting

Publications and source records attributed to G Banting.

At least 19 recordsLinked to original sources
PubMed2000

GAP1IP4BP contains a novel group I pleckstrin homology domain that directs constitutive plasma membrane association.

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).

Amino Acid Substitution↗
PubMed2000

A study of the coregulation and tissue specificity of XG and MIC2 gene expression in eukaryotic cells.

CD99, the product of the MIC2 gene, exhibits an erythroid-specific quantitative polymorphism coregulated with the polymorphism of the XG blood group gene. As a preliminary study of this phenomenon, human XG and CD99 recombinant proteins were expressed in murine RAG cells and analyzed by flow cytometry. Both proteins were expressed independently and at a similar level in single and double transfectants. Immunoprecipitation and Western blot analysis, using the murine monoclonal antibodies NBL-1 and 12E7, revealed species of 26 kd (XG) and 32 kd (CD99), respectively. A putative 28-kd intracellular precursor of CD99 was also detected, as was a 26-kd species after neuraminidase treatment of CD99-expressing cells. No evidence of association or complex formation between XG and CD99 proteins could be proven, either on transfected RAG cells or on human erythrocytes. These results were confirmed using somatic hybrids between single transfectants. These findings suggest that the phenotypic relationship between XG and CD99 is mostly regulated at the transcriptional level, but they do not formally exclude some posttranscriptional effect. Studies on the tissue specificity of XG expression showed that surface expression of the XG protein could not be restored in somatic hybrids between B-lymphoblastoid cell lines from Xg(a+) persons and fibroblasts (RAG) or erythroid (MEL) cells. RT-PCR analysis of the transcripts revealed the existence of an XG mRNA in each cell line, suggesting that the tissue-specific regulation of cell surface XG expression occurs either at a quantitative transcriptional level or is a posttranscriptional event. By Northern blot analysis, XG transcripts were detected in erythroid tissues and several nonerythroid tissues. (Blood. 2000;95:1819-1826)

12E7 Antigen↗
PubMed2000

In vivo dynamics of the F-actin-binding protein neurabin-II.

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.

ADP-Ribosylation Factor 6↗
PubMed2000

Effective formation of major histocompatibility complex class II-peptide complexes from endogenous antigen by thyroid epithelial cells.

In autoimmune thyroid disease, thyroid epithelial cells (TEC) express major histocompatibility complex (MHC) class II molecules, potentially enabling them to present thyroid self-antigens to CD4-positive T cells. However, despite this, TEC may fail to present endogenous antigen as a result of limited processing or MHC class II loading capacity, or inadequate MHC class II levels. We addressed these issues using the cloned rat TEC line, Fischer rat thyroid cell line (FRTL5), which was transfected using an adenoviral expression vector that expressed ovalbumin (OVA) as an integral membrane protein. OVA-expressing FRTL5 cells very efficiently activated a panel of OVA-specific, class II-restricted T-cell hybridomas. This response was dependent on induction of MHC class II molecules by interferon-gamma (IFN-gamma) and was blocked by anti-MHC class II antibodies. Poor responses were seen to exogenously added OVA or OVA peptides. These results provide the most direct evidence to date that TEC can form MHC class II-peptide complexes derived from self-antigen in sufficient quantities to activate T cells.

Animals↗
PubMed1999

Direct interaction of the trans-Golgi network membrane protein, TGN38, with the F-actin binding protein, neurabin.

TGN38 is a type I integral membrane protein that constitutively cycles between the trans-Golgi network (TGN) and plasma membrane. The cytosolic domain of TGN38 interacts with AP2 clathrin adaptor complexes via the tyrosine-containing motif (-SDYQRL-) to direct internalization from the plasma membrane. This motif has previously been shown to direct both internalization and subsequent TGN targeting of TGN38. We have used the cytosolic domain of TGN38 in a two-hybrid screen, and we have identified the brain-specific F-actin binding protein neurabin-I as a TGN38-binding protein. We demonstrate a direct interaction between TGN38 and the ubiquitous homologue of neurabin-I, neurabin-II (also called spinophilin). We have used a combination of yeast two-hybrid and in vitro protein interaction assays to show that this interaction is dependent on the serine (but not tyrosine) residue of the known TGN38 trafficking motif. We show that TGN38 interacts with the coiled coil region of neurabin in vitro and binds preferentially with the dimeric form of neurabin. TGN38 and neurabin also interact in vivo as demonstrated by coimmunoprecipitation from stably transfected PC12 cells. These data suggest that neurabin provides a direct physical link between TGN38-containing membranes and the actin cytoskeleton.

Actins↗
PubMed1999

Phosphorylation of the medium chain subunit of the AP-2 adaptor complex does not influence its interaction with the tyrosine based internalisation motif of TGN38.

Tyrosine based motifs conforming to the consensus YXXphi (where phi represents a bulky hydrophobic residue) have been shown to interact with the medium chain subunit of clathrin adaptor complexes. These medium chains are targets for phosphorylation by a kinase activity associated with clathrin coated vesicles. We have used the clathrin coated vesicle associated kinase activity to specifically phosphorylate a soluble recombinant fusion protein of mu2, the medium chain subunit of the plasma membrane associated adaptor protein complex AP-2. We have tested whether this phosphorylation has any effect on the interaction of mu2 with the tyrosine based motif containing protein, TGN38, that has previously been shown to interact with mu2. Phosphorylation of mu2 was shown to have no significant effect on the in vitro interaction of mu2 with the cytosolic domain of TGN38, indicating that reversible phosphorylation of mu2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs. In addition, although a casein kinase II-like activity has been shown to be associated with clathrin coated vesicles, we show that mu2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coated vesicles. Furthermore the kinase activity associated with clathrin coated vesicles was shown to be capable of phosphorylating dynamin 1. Phosphorylation of dynamin 1 has previously been shown to regulate its interaction with other proteins involved in clathrin mediated endocytosis.

Adaptor Protein Complex 1↗
PubMed1998

Specificity of interaction between adaptor-complex medium chains and the tyrosine-based sorting motifs of TGN38 and lgp120.

Multiple sorting steps within eukaryotic cells are mediated by tyrosine-based sorting motifs. Motifs conforming to the consensus -YXXO- (where O 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 mu-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 mu2, we show that the cytosolic domain of lgp120 interacts almost exclusively with mu3A. The specificity of binding of tyrosine-based sorting motifs to mu-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 mu-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 YXXO motif as the C-terminus, greatly enhances the affinity of interaction with mu2. Furthermore, addition of these same residues to the extreme C-terminus of lgp120 effectively abolishes the interaction of the cytosolic domain of lgp120 with mu-chains. We also show that the newly identified mu-adaptin-related protein 2 (mu4) only interacts weakly with tyrosine-based sorting motifs.

Amino Acid Sequence↗
PubMed1998

Inhibition of the interaction between tyrosine-based motifs and the medium chain subunit of the AP-2 adaptor complex by specific tyrphostins.

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs found within the cytosolic domains of certain integral membrane proteins. Many of these tyrosine motifs conform to the consensus YXXPhi (where Phi represents a bulky hydrophobic residue). This YXXPhi motif has been shown to interact with the medium chain subunits of adaptor complexes that generally link relevant integral membrane protein cytosolic domains to the clathrin coat involved in vesicle formation. The motif YXXPhi is also very similar to motifs that are targets for phosphorylation by tyrosine kinases. Tyrosine kinase inhibitors known as tyrphostins are structural analogues of tyrosine, and so it is possible that tyrphostins could also inhibit interactions between medium chains and YXXPhi motifs. TGN38 is a type I integral membrane protein containing a tyrosine motif, YQRL, within the cytosolic domain. We have previously shown that this motif interacts directly with the medium chain subunit of the plasma membrane localized AP-2 adaptor complex (mu2). We have investigated a range of tyrphostins and demonstrated a specific inhibition of the interaction between mu2 and the TGN38 cytosolic domain by tyrphostin A23 through in vitro analysis and the yeast two-hybrid system. These data raise the exciting possibility that different membrane traffic events could be inhibited by specific tyrphostins.

Adaptor Protein Complex 1↗
PubMed1998

Salmonella typhi uses CFTR to enter intestinal epithelial cells.

Homozygous mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). In the heterozygous state, increased resistance to infectious diseases may maintain mutant CFTR alleles at high levels in selected populations. Here we investigate whether typhoid fever could be one such disease. The disease is initiated when Salmonella typhi enters gastrointestinal epithelial cells for submucosal translocation. We found that S. typhi, but not the related murine pathogen S. typhimurium, uses CFTR for entry into epithelial cells. Cells expressing wild-type CFTR internalized more S. typhi than isogenic cells expressing the most common CFTR mutation, a phenylalanine deleted at residue 508 (delta508). Monoclonal antibodies and synthetic peptides containing a sequence corresponding to the first predicted extracellular domain of CFTR inhibited uptake of S. typhi. Heterozygous deltaF508 Cftr mice translocated 86% fewer S. typhi into the gastrointestinal submucosa than wild-type Cftr mice; no translocation occurred in deltaF508 Cftr homozygous mice. The Cftr genotype had no effect on the translocation of S. typhimurium. Immunoelectron microscopy revealed that more CFTR bound to S. typhi in the submucosa of Cftr wild-type mice than in deltaF508 heterozygous mice. We conclude that diminished levels of CFTR in heterozygotes may decrease susceptibility to typhoid fever.

Animals↗
PubMed1998

Lumenal and transmembrane domains play a role in sorting type I membrane proteins on endocytic pathways.

Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network.

Amino Acid Sequence↗
PubMed1998

Efficient trafficking of TGN38 from the endosome to the trans-Golgi network requires a free hydroxyl group at position 331 in the cytosolic domain.

TGN38 is one of the few known resident integral membrane proteins of the trans-Golgi network (TGN). Since it cycles constitutively between the TGN and the plasma membrane, TGN38 is ideally suited as a model protein for the identification of post-Golgi trafficking motifs. Several studies, employing chimeric constructs to detect such motifs within the cytosolic domain of TGN38, have identified the sequence 333YQRL336 as an autonomous signal capable of localizing reporter proteins to the TGN. In addition, one group has found that an upstream serine residue, S331, may also play a role in TGN38 localization. However, the nature and degree of participation of S331 in the localization of TGN38 remain uncertain, and the effect has been studied in chimeric constructs only. Here we investigate the role of S331 in the context of full-length TGN38. Mutations that abolish the hydroxyl moiety at position 331 (A, D, and E) lead to missorting of endocytosed TGN38 to the lysosome. Conversely, mutation of S331 to T has little effect on the endocytic trafficking of TGN38. Together, these findings indicate that the S331 hydroxyl group has a direct or indirect effect on the ability of the cytosolic tail of TGN38 to interact with trafficking and/or sorting machinery at the level of the early endosome. In addition, mutation of S331 to either A or D results in increased levels of TGN38 at the cell surface. The results confirm that S331 plays a critical role in the intracellular trafficking of TGN38 and further reveal that TGN38 undergoes a signal-mediated trafficking step at the level of the endosome.

Amino Acid Sequence↗
PubMed1998

The steady state distribution of humTGN46 is not significantly altered in cells defective in clathrin-mediated endocytosis.

It has been shown previously that whilst the rat type I integral membrane protein TGN38 (ratTGN38) is predominantly localised to the trans-Golgi network this protein does reach the cell surface from where it is internalised and delivered back to the trans-Golgi network. This protein thus provides a suitable tool for the investigation of trafficking pathways between the trans-Golgi network and the cell surface and back again. The human orthologue of ratTGN38, humTGN46, behaves in a similar fashion. These proteins are internalised from the cell surface via clathrin mediated endocytosis, a process which is dependent upon the GTPase activity of dynamin. We thus reasoned that humTGN46 would accumulate at the surface of cells rendered defective in clathrin mediated endocytosis by virtue of the fact that they express a GTPase defective mutant of dynamin I. It did not. In fact, expression of a dominant negative GTPase defective mutant of dynamin I had no detectable effect on the steady state distribution of humTGN46. One explanation for this observation is that humTGN46 does not travel directly to the cell surface from the trans-Golgi network. Further studies on cells expressing the dominant negative GTPase defective mutant of dynamin I indicate that the major recycling pathway for humTGN46 is in fact between the trans-Golgi network and the early endosome.

Animals↗
PubMed1997

Insulin dependent tyrosine phosphorylation of the tyrosine internalisation motif of TGN38 creates a specific SH2 domain binding site.

Tyrosine-based motifs are involved in both protein targeting and, via SH2 domain binding, intracellular signalling. To date there has only been one example of such a motif acting as both an intracellular sorting signal and SH2 binding determinant, namely that of the T cell costimulation receptor, CTLA-4. We show that insulin stimulation of cultured rat hepatoma cells results in increased cell surface expression of TGN38. Furthermore, the cytosolic domain of TGN38 can be phosphorylated by the insulin receptor in vitro and tyrosine phosphorylated TGN38 can specifically bind to the SH2 domains of the spleen tyrosine kinase Syk. These data imply that tyrosine-based motifs may play a broader role than has previously been accepted and could help to integrate trafficking and signalling events.

Amino Acid Sequence↗
PubMed1997

The arachidonate-activatable, NADPH oxidase-associated H+ channel is contained within the multi-membrane-spanning N-terminal region of gp91-phox.

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.

Animals↗
PubMed1997

Membrane association, localization and topology of rat inositol 1,4,5-trisphosphate 3-kinase B: implications for membrane traffic and Ca2+ homoeostasis.

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 (IP3 3-kinase B); this protein was thus designated rat IP3 3-kinase B [Thomas, Brake, Luzio, Stanley and Banting (1994) Biochim. Biophys. Acta 1220, 219-222]. However, no IP3 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 IP3 3-kinase B is genuinely an IP3 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 IP3 3-kinase with membranes is not affected by treatment with brefeldin A, Na2CO3 (pH 11.5), 2 M NaCl, or alteration of [Ca2+]. 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 IP3 3-kinase B is localized exclusively to membranes of Ca2+ stores, is consistent with a model where this kinase plays a role in IP3-dependent Ca2+ release.

Amino Acid Sequence↗
PubMed1997

Properties of chloride-conductive pathways in rat kidney cortical and outer-medulla brush-border membranes--inhibition by anti-(cystic fibrosis transmembrane regulator) mAbs.

The activity of the Cl(-)-conductive pathways, their regulation by protein kinase A (PKA) and their relationship to the cystic fibrosis transmembrane regulator (CFTR) protein were assessed in rat kidney cortical brush-border-membrane vesicles (cBBMV) and outer medullary vesicles (OMV) by measuring the rate of valinomycin-induced microsomal swelling by light scattering in the presence of an inward Cl- gradient. Valinomycin increased the rate of swelling of cBBMV and OMV, which is consistent with the presence of a Cl(-)-conductive pathway. PKA further increased these rates. This effect was blocked by the inhibitor of protein kinase A, suggesting that phosphorylation by PKA activates these pathways. Four anion-transport inhibitors were tested ¿N-phenylanthranilic acid (PhNHPhCOOH), 5-nitro-2-(3-phenylpropylamino)benzoic acid [N(PhPrNH2)BzOH], glybenclamide and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid¿. Ph2COOH and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid inhibited the basal Cl(-)-conductive pathways, while PKA-treated microsomes were sensitive also to N(PhPrNH2)BzOH and glybenclamide, suggesting that additional Cl- pathways were activated by phosphorylation. The pharmacological properties of these pathways were similar to those of the CFTR Cl- channel. Two anti-CFTR mAbs inhibited PKA-activated valinomycin-induced swelling in cBBMV and OMV, while immunoblot analysis of the corresponding proteins with the same antibodies indicated the presence of a 170-kDa protein. The results thus indicate the presence of a PKA-activated Cl(-)-conductive pathway in cBBMV and OMV, and suggest that CFTR protein is involved in PKA-activated Cl- fluxes in these vesicles.

Animals↗
PubMed1997

Serine 331 and tyrosine 333 are both involved in the interaction between the cytosolic domain of TGN38 and the mu2 subunit of the AP2 clathrin adaptor complex.

TGN38 is a type I integral membrane protein that cycles between the trans-Golgi network and the plasma membrane. Internalization at the cell surface and targeting back to the trans-Golgi network is dependent on a hexapeptide motif, SDYQRL, in the cytosolic tail of the protein. It was recently demonstrated that this motif specifically interacts with the mu2 subunit of the AP2 adaptor complex. We have studied the interaction between the entire cytosolic domain of TGN38 and mu2 using the yeast two hybrid system, in vitro binding of recombinant fusion proteins and IAsys optical biosensor technology. A specific interaction has been demonstrated in each of the systems we have employed. We have shown an absolute requirement for Tyr-333 of TGN38 in binding to mu2. In addition we found that mutation of Ser-331 to alanine reduces the affinity of the interaction. By measuring tryptophan fluorescence at equilibrium, we have also determined the dissociation constant for the interaction between the entire cytosolic tail of TGN38 and mu2 as 58 nM. In contrast to previously published work, our data suggest that not only Tyr-333 but also its context is important in determining the specificity of binding of TGN38 to mu2.

Adaptor Protein Complex 1↗