Disrupted in Schizophrenia 1 Interactome: evidence for the close connectivity of risk genes and a potential synaptic basis for schizophrenia.

Disrupted in Schizophrenia 1 (DISC1) is a schizophrenia risk gene associated with cognitive deficits in both schizophrenics and the normal ageing population. In this study, we have generated a network of protein-protein interactions (PPIs) around DISC1. This has been achieved by utilising iterative yeast-two hybrid (Y2H) screens, combined with detailed pathway and functional analysis. This so-called 'DISC1 interactome' contains many novel PPIs and provides a molecular framework to explore the function of DISC1. The network implicates DISC1 in processes of cytoskeletal stability and organisation, intracellular transport and cell-cycle/division. In particular, DISC1 looks to have a PPI profile consistent with that of an essential synaptic protein, which fits well with the underlying molecular pathology observed at the synaptic level and the cognitive deficits seen behaviourally in schizophrenics. Utilising a similar approach with dysbindin (DTNBP1), a second schizophrenia risk gene, we show that dysbindin and DISC1 share common PPIs suggesting they may affect common biological processes and that the function of schizophrenia risk genes may converge.

Overexpression of the GABA(A) receptor epsilon subunit results in insensitivity to anaesthetics.

The epsilon -subunit of the GABA(A) receptor was independently cloned and functionally characterised in recombinant expression systems by two groups (Davies, P.A. et al., Nature 385 (1997) 820; Whiting, P.J. et al., Journal of Neuroscience 17 (1997) 5027). Both groups showed that co-expression of alphabeta epsilon -subunits produced functional receptors, however the sensitivity of these receptors to the potentiating effects of general anaesthetic agents differed. Co-expression of the two epsilon -constructs (hereafter referred to as epsilon (MRK) from Whiting, P.J. et al., Journal of Neuroscience 17 (1997) 5027) and epsilon (TIGR) from Davies et al., Nature 385 (1997) 820) with alpha1beta1 in Xenopus oocytes produced receptors that were sensitive (alpha1beta1 epsilon (MRK)) and insensitive (alpha1beta1 epsilon (TIGR)) to the potentiating effects of pentobarbitone, 5alpha-pregnan-3alpha-ol-20-one and etomidate. Both alpha1beta1 epsilon (MRK) and alpha1beta1 epsilon (TIGR) receptors were directly activated by these agents, however for pentobarbitone and 5alpha-pregnan-3alpha-ol-20-one this effect was greater on alpha1beta1 epsilon (TIGR) than alpha1beta1 epsilon (MRK). alpha1beta1 epsilon (TIGR) receptors were more sensitive to GABA and had a larger degree of constitutive activity than alpha1beta1 epsilon (MRK). Insensitivity to the potentiating effects of anaesthetics was not due to the single amino acid difference between the two constructs nor to differences in the 5' and 3' untranslated regions. Transfer of epsilon (TIGR) from its original vector, pCDM8, into pcDNA1.1Amp and reduction in the amount of epsilon (TIGR) in pCDM8 relative to the amount of alpha1 and beta1 injected into the oocyte restored potentiation by pentobarbitone. Increased expression of epsilon (TIGR) protein compared to epsilon (MRK) was confirmed by Western blotting. We conclude that the differences in the potentiating effects of anaesthetic agents on alpha1beta1 epsilon (MRK/TIGR) receptors is due to overexpression of epsilon (TIGR) in the pCDM8 vector, relative to the alpha1 and beta1-subunits, which may lead to an altered stoichiometry.

Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA(A) receptors.

1: The pharmacology of the stable cell line expressing human alpha(4)beta(3)delta GABA(A) receptor was investigated using whole-cell patch-clamp techniques. 2: alpha(4)beta(3)delta receptors exhibited increased sensitivity to GABA when compared to alpha(4)beta(3)gamma(2) receptors, with EC(50)'s of 0.50 (0.46, 0.53) microM and 2.6 (2.5, 2.6) microM respectively. Additionally, the GABA partial agonists piperidine-4-sulphonate (P4S) and 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) displayed markedly higher efficacy at alpha(4)beta(3)delta receptors, indeed THIP demonstrated greater efficacy than GABA at these receptors. 3: The delta subunit conferred slow desensitization to GABA, with rate constants of 4.8+/-0.5 s for alpha(4)beta(3)delta and 2.5+/-0.2 s for alpha(4)beta(3)gamma(2). However, both P4S and THIP demonstrated similar levels of desensitization on both receptor subtypes suggesting this effect is agonist specific. 4: alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2) demonstrated equal sensitivity to inhibition by the cation zinc (2-3 microM IC(50)). However, alpha(4)beta(3)delta receptors demonstrated greater sensitivity to inhibition by lanthanum. The IC(50) for GABA antagonists SR-95531 and picrotoxin, was similar for alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2). Likewise, inhibition was observed on both subtypes at high and low pH. 5: alpha(4)beta(3)delta receptors were insensitive to modulation by benzodiazepine ligands. In contrast Ro15-4513 and bretazenil potentiated GABA responses on alpha(4)beta(3)gamma(2) cells, and the inverse agonist DMCM showed allosteric inhibition of alpha(4)beta(3)gamma(2) receptors. 6: The efficacy of neurosteroids at alpha(4)beta(3)delta receptors was greatly enhanced over that observed at alpha(4)beta(3)gamma(2) receptors. The greatest effect was observed using THDOC with 524+/-71.6% potentiation at alpha(4)beta(3)delta and 297.9+/-49.7% at alpha(4)beta(3)gamma(2) receptors. Inhibition by the steroid pregnenolone sulphate however, showed no subtype selectivity. The efficacy of both pentobarbitone and propofol was slightly augmented and etomidate greatly enhanced at alpha(4)beta(3)delta receptors versus alpha(4)beta(3)gamma(2) receptors. 7: We show that the alpha(4)beta(3)delta receptor has a distinct pharmacology and kinetic profile. With its restricted distribution within the brain and unique pharmacology this receptor may play an important role in the action of neurosteroids and anaesthetics. British Journal of Pharmacology (2002) 136, 965-974

alpha4beta3delta GABA(A) receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential.

Selective modulators of gamma-aminobutyric acid, type A (GABA(A)) receptors containing alpha(4) subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(-tk) cells, we stably expressed the native GABA(A) receptor subunit combinations alpha(3)beta(3)gamma(2,) alpha(4)beta(3)gamma(2), and, for the first time, alpha(4)beta(3)delta and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABA(A) receptor antagonists and, for alpha(3)beta(3)gamma(2) and alpha(4)beta(3)gamma(2) receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with alpha(4) and beta(3), delta subunits, compared with gamma(2), conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. alpha(4)beta(3)delta responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3-7-fold more efficacious at alpha(4)beta(3)delta compared with alpha(4)beta(3)gamma(2.) The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABA(A) receptor. For GABA(A) receptors containing alpha(4) subunits, our experiments reveal that inclusion of delta instead of gamma(2) subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of alpha(4)beta(3)delta receptors, consistent with a central role for this subunit combination in premenstrual syndrome.

Identification of two major sites in the type I interleukin-1 receptor cytoplasmic region responsible for coupling to pro-inflammatory signaling pathways.

Type I interleukin-1 receptor is the prototype for a family of proteins, which play a central role in early responses to injury and infection. The similarity of function across the family is reflected in similarity in signaling: all members tested couple to activation of NFkappaB and stress kinases. The coupling to these pathways is mediated by a 200-residue intracellular domain (the Toll/interleukin-1 receptor domain), in which sequence conservation is primarily confined to three short motifs (boxes 1, 2, and 3) located at amino acid residue positions 10 (box 1), 60 (box 2), and 170 (box 3). We have analyzed the contribution of these motifs to function by alanine scanning mutagenesis of the human interleukin-1 receptor type I. Mutant receptors were tested for expression, ligand binding, activation of receptor-associated kinase(s), NFkappaB, stress kinases, and transcription. Mutations in all three motifs led to low cell surface expression. Mutants in box 3 were, however, wild type for signaling, whereas mutants in boxes 1 and 2 were defective. We conclude that the conserved motifs box 1 and box 2 mediate the coupling of molecules in the family to inflammation signaling pathways.

theta, a novel gamma-aminobutyric acid type A receptor subunit.

gamma-Aminobutyric acid type A (GABA-A) receptors are a major mediator of inhibitory neurotransmission in the mammalian central nervous system, and the site of action of a number of clinically important drugs. These receptors exist as a family of subtypes with distinct temporal and spatial patterns of expression and distinct properties that presumably underlie a precise role for each subtype. The newest member of this gene family is the theta subunit. The deduced polypeptide sequence is 627 amino acids long and has highest sequence identity (50.5%) with the beta1 subunit. Within the rat striatum, this subunit coassembles with alpha2, beta1, and gamma1, suggesting that gamma-aminobutyric acid type A receptors consisting of arrangements other than alpha beta + gamma, delta, or epsilon do exist. Expression of alpha2beta1gamma1theta in transfected mammalian cells leads to the formation of receptors with a 4-fold decrease in the affinity for gamma-aminobutyric acid compared with alpha2beta1gamma1. This subunit has a unique distribution, with studies so far suggesting significant expression within monoaminergic neurons of both human and monkey brain.

Molecular and functional diversity of the expanding GABA-A receptor gene family.

Fast inhibitory neurotransmission in the mammalian CNS is mediated primarily by the neurotransmitter gamma-aminobutyric acid (GABA), which, upon binding to its receptor, leads to opening of the intrinsic ion channel, allowing chloride to enter the cell. Over the past 10 years it has become clear that a family of GABA-A receptor subtypes exists, generated through the coassembly of polypeptides selected from alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta, epsilon, and pie to form what is most likely a pentomeric macromolecule. The gene transcripts, and indeed the polypeptides, show distinct patterns of temporal and spatial expression, such that the GABA-A receptor subtypes have a defined localization that presumably reflects their physiological role. A picture is beginning to emerge of the properties conferred to receptor subtypes by the different subunits; these include different functional properties, differential modulation by protein kinases, and the targeting to different membrane compartments. These properties presumably underlie the different physiological roles of the various receptor subtypes. Recently we have identified a further member of the GABA-A receptor gene family, which we have termed theta, which appears to be most closely related to the beta subunits. The structure, function, and distribution of theta-containing receptors, and receptors containing the recently reported epsilon subunit, are described.

Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer.

We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged alpha1-, beta2-, or gamma2-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the alpha1(c-Myc)beta2gamma2 and the alpha1beta2(c-Myc)gamma2 receptors was twice that obtained with alpha1beta2gamma2(c-Myc), suggesting that there are 2x alpha-, 2x beta-, and 1x gamma-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining alpha1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with alpha1(c-Myc)beta2gamma2 and alpha1beta2(c-Myc)gamma2, but no significant energy transfer was observed with alpha1beta2gamma2(c-Myc) receptors, indicating the absence of a second gamma-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, alpha1beta2gamma2, is composed of two copies each of the alpha- and beta-subunits and one copy of the gamma-subunit (i.e. (alpha1)2(beta2)2(gamma2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins.

Functional characteristics of recombinant human GABA(A) receptors containing the epsilon-subunit.

(1) This paper further examines the functional characteristics of recombinant human GABA(A) receptors containing the epsilon-subunit expressed in Xenopus oocytes. (2) Alpha1beta1epsilon receptors are not modulated by benzodiazepine ligands or by a number of hypothalamic hormones. (3) The intravenous anaesthetic agents pentobarbital, propofol and etomidate all potentiate sub-maximal GABA currents (EC20) to a similar degree in alpha1beta1epsilon and alpha1beta1gamma2s receptors. (4) Direct activation by pentobarbital produced a similar maximum response on alpha1beta1epsilon and alpha1beta1gamma2s, however, both the EC50 and slope were lower on alpha1beta1epsilon compared to alpha1beta1gamma2s. (5) These results describe a novel pharmacology for recombinant alpha1beta1epsilon receptors.

Neuronally restricted RNA splicing regulates the expression of a novel GABAA receptor subunit conferring atypical functional properties [corrected; erratum to be published].

We report the isolation and characterization of a cDNA encoding a novel member of the GABA receptor gene family, epsilon. This polypeptide is 506 amino acids in length and exhibits its greatest amino acid sequence identity with the GABAA receptor gamma3 subunit (47%), although this degree of homology is not sufficient for it to be classified as a fourth gamma subunit. The epsilon subunit coassembles with GABAA receptor alpha and beta subunits in Xenopus laevis oocytes and transfected mammalian cells to form functional GABA-gated channels. alpha1beta1epsilon GABAA receptors, like alpha1beta1gamma2s receptors, are modulated by pentobarbital and the steroid 5alpha-pregnan-3alpha-ol-20-one but, unlike alpha1beta1gamma2s receptors, are insensitive to flunitrazepam. Additionally, alpha1beta1epsilon receptors exhibit rapid desensitization kinetics, as compared with alpha1beta1 or alpha1beta1gamma2s. Northern analysis demonstrates widespread expression of a large epsilon subunit transcript in a variety of non-neuronal tissues and expression of a smaller transcript in brain and spinal cord. Sequence analysis demonstrated that the large transcript contained an unspliced intron, whereas the small transcript represents the mature mRNA, suggesting regulation of expression of the epsilon subunit via neuronally restricted RNA splicing. In situ hybridization and immunocytochemistry reveal a pattern of expression in the brain restricted primarily to the hypothalamus, suggesting a role in neuroendocrine regulation, and also to subfields of the hippocampus, suggesting a role in the modulation of long term potentiation and memory.

Structure-function studies of interleukin 15 using site-specific mutagenesis, polyethylene glycol conjugation, and homology modeling.

Interleukin (IL)-15 is a multifunctional cytokine that shares many biological activities with IL-2. This functional overlap, as well as receptor binding subunits shared by IL-15 and IL-2, suggests tertiary structural similarities between these two cytokines. In this study, recombinant human IL-15 was PEGylated via lysine-specific conjugation chemistry in order to extend the circulation half-life of this cytokine. Although PEGylation did extend the beta-elimination circulation half-life of IL-15 by greater than 50-fold, the biological activity of polyethylene glycol (PEG)-IL-15 was significantly altered. Specifically, PEG-IL-15 lost its ability to stimulate the proliferation of CTLL but took on the properties of a specific IL-15 antagonist in vitro. In comparing sequence alignments and molecular models for IL-2 and IL-15, it was noted that lysine residues resided in regions of IL-15 that may have selectively disrupted receptor subunit binding. We hypothesized that PEGylation of IL-15 interferes with beta but not alpha receptor subunit binding, resulting in the IL-15 antagonist activity observed in vitro. The validity of this hypothesis was tested by engineering site-specific mutants of human IL-15 as suggested by the IL-15 model (IL-15D8S and IL-15Q108S block beta and gamma receptor subunit binding, respectively). As with PEG-IL-15, these mutants were unable to stimulate CTLL proliferation but were able to specifically inhibit the proliferation of CTLL in response to unmodified IL-15. These results supported our model of IL-15 and confirmed that interference of beta receptor subunit binding by adjacent PEGylation could be responsible for the altered biological activity observed for PEG-IL-15.

The cloning and characterization of human MyD88: a member of an IL-1 receptor related family.

Murine MyD88, an RNA with homology both to the interleukin-1 receptor signaling domain and to 'death-domains', is rapidly upregulated during differentiation of the myeloleukemic cell line M1. We have cloned the human homologue of murine MyD88 and re-evaluated the murine sequence. The open reading frame for both species encodes a 296 amino acid protein, which for murine MyD88 is 53 amino acids longer than originally published. Human MyD88 cDNA is encoded by 5 exons, and maps to chromosome 3p21.3-p22 by fluorescence in situ hybridization (FISH). Overexpression of the death domain region leads to transcriptional activation of the IL-8 promoter.

T1/ST2 signaling establishes it as a member of an expanding interleukin-1 receptor family.

Through data base searches, we have discovered new proteins that share homology with the signaling domain of the type I interleukin-1 receptor (IL-1RI): human "randomly sequenced cDNA 786" (rsc786), murine MyD88, and two partial Drosophila open reading frames, MstProx and STSDm2245. Comparisons between these new proteins and known IL-1RI homologous proteins such as Toll, 18-Wheeler, and T1/ST2 revealed six clusters of amino acid similarity. We tested the hypothesis that sequence similarity between the signaling domain of IL-1RI and the three mammalian family members might indicate functional similarity. Chimeric IL-1RI receptors expressing the putative signaling domains of T1/ST2, MyD88, and rsc786 were assayed by three separate IL-1 responsive assays, NF-kappaB, phosphorylation of an epidermal growth factor receptor peptide, and an interleukin 8 promoter-controlled reporter construct, for their ability to transduce an IL-1-stimulated signal. All three assays were positive in response to the T1/ST2 chimera, while the MyD88 and rsc786 chimeras failed to respond. These data indicate that the sequence homology between IL-1RI and T1/ST2 indicates a functional homology as well.

Cloning of a putative ligand for the T1/ST2 receptor.

T1/ST2 is a receptor-like molecule homologous to the type I interleukin-1 receptor. Despite this sequence similarity, we have been unable to demonstrate binding of T1/ST2 to any of the three interleukin-1 species. In searching for a ligand for T1/ST2, we have cloned a cell surface protein to which it binds. This protein is unable to initiate signal transduction by the T1/ST2 receptor in several in vitro assays.

IL-1Rrp is a novel receptor-like molecule similar to the type I interleukin-1 receptor and its homologues T1/ST2 and IL-1R AcP.

A novel member of the interleukin-1 receptor family has been cloned by polymerase chain reaction using degenerate oligonucleotide primers derived from regions of sequence conservation, using as template a yeast artificial chromosome known to contain both interleukin-1 (IL-1) receptors and T1/ST2. The new receptor, called IL-1 receptor-related protein or IL-1Rrp, fails to bind any of the known IL-1 ligands. A chimeric receptor, in which the IL-1Rrp cytoplasmic domain is fused to the extracellular and transmembrane regions of the IL-1 receptor, responds to IL-1 following transfection into COS cells by activation of NFkappaB and induction of IL-8 promoter function.

A single chain antibody fragment expressed in bacteria neutralizes tick-borne flaviviruses.

A recombinant single chain antibody fragment (scFv) that identifies a neutralizing epitope on the envelope glycoprotein of louping iII (LI) and tick-borne encephalitis (TBE) virus has been developed using a bacteriophage expression system. The mRNA was extracted from a cloned hybridoma cell culture that produces a mouse monoclonal antibody (MAb 4.2) known to map to amino acids 308-311 of LI and TBE virus, corresponding to domain B on the proposed two-dimensional model of the tick-borne encephalitis virus envelope protein. The V-genes encoding the antigen-binding site of MAb 4.2 were amplified and cloned for expression as a fusion protein to the pIII coat protein of filamentous phage. Solid phase selection of these phage against the LI virus antigen, was necessary to isolate the correct MAb 4.2 scFv fragment which was subsequently produced in soluble form in bacteria and harvested from the culture supernatant medium. The characteristics of this expressed single chain antibody were compared with MAb 4.2. The expressed antibody portrayed the antigenic specificity of MAb 4.2 and also neutralized the infectivity of louping iII and some other tick-borne flaviviruses. The potential of this technique for studying antigen-antibody interactions and for the development of prophylactic reagents are discussed.

By-passing immunization. Human antibodies from V-gene libraries displayed on phage.

We have mimicked features of immune selection to make human antibodies in bacteria. Diverse libraries of immunoglobulin heavy (VH) and light (V kappa and V lambda) chain variable (V) genes were prepared from peripheral blood lymphocytes (PBLs) of unimmunized donors by polymerase chain reaction (PCR) amplification. Genes encoding single chain Fv fragments were made by randomly combining heavy and light chain V-genes using PCR, and the combinatorial library (greater than 10(7) members) cloned for display on the surface of a phage. Rare phage with "antigen-binding" activities were selected by four rounds of growth and panning with "antigen" (turkey egg-white lysozyme (TEL) or bovine serum albumin) or "hapten" (2-phenyloxazol-5-one (phOx], and the encoding heavy and light chain genes were sequenced. The V-genes were human with some nearly identical to known germ-line V-genes, while others were more heavily mutated. Soluble antibody fragments were prepared and shown to bind specifically to antigen or hapten and with good affinities, Ka (TEL) = 10(7) M-1; Ka (phOx) = 2 x 10(6) M-1. Isolation of higher-affinity fragments may require the use of larger primary libraries or the construction of secondary libraries from the binders. Nevertheless, our results suggest that a single large phage display library can be used to isolate human antibodies against any antigen, by-passing both hybridoma technology and immunization.