Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Glucocorticoid enhancement of memory storage involves noradrenergic activation in the basolateral amygdala

Evidence indicates that the modulatory effects of the adrenergic stress hormone epinephrine as well as several other neuromodulatory systems on memory storage are mediated by activation of β-adrenergic mechanisms in the amygdala. In view of our recent findings indicating that the amygdala is involved in mediating the effects of glucocorticoids on memory storage, the present study examined whether the glucocorticoid-induced effects on memory storage depend on β-adrenergic activation within the amygdala. Microinfusions (0.5 μg in 0.2 μl) of either propranolol (a nonspecific β-adrenergic antagonist), atenolol (a β1-adrenergic antagonist), or zinterol (a β2-adrenergic antagonist) administered bilaterally into the basolateral nucleus of the amygdala (BLA) of male Sprague–Dawley rats 10 min before training blocked the enhancing effect of posttraining systemic injections of dexamethasone (0.3 mg/kg) on 48-h memory for inhibitory avoidance training. Infusions of these β-adrenergic antagonists into the central nucleus of the amygdala did not block the dexamethasone-induced memory enhancement. Furthermore, atenolol (0.5 μg) blocked the memory-enhancing effects of the specific glucocorticoid receptor (GR or type II) agonist RU 28362 infused concurrently into the BLA immediately posttraining. These results strongly suggest that β-adrenergic activation is an essential step in mediating glucocorticoid effects on memory storage and that the BLA is a locus of interaction for these two systems.

Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.

Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.

Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction.

Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains.

The human cytomegalovirus (HCMV) early glycoprotein products of the US11 and US2 open reading frames cause increased turnover of major histocompatibility complex (MHC) class I heavy chains. Since US2 is homologous to another HCMV gene (US3), we hypothesized that the US3 gene product also may affect MHC class I expression. In cells constitutively expressing the HCMV US3 gene, MHC class I heavy chains formed a stable complex with beta 2-microglobulin. However, maturation of the N-linked glycan of MHC class I heavy chains was impaired in US3+ cells. The glycoprotein product of US3 (gpUS3) occurs mostly in a high-mannose form and coimmunoprecipitates with beta 2-microglobulin associated class I heavy chains. Mature class I molecules were detected at steady state on the surface of US3+ cells, as in control cells. Substantial perinuclear accumulation of heavy chains was observed in US3+ cells. The data suggest that gpUS3 impairs egress of MHC class I heavy chains from the endoplasmic reticulum.

The natural killer cell receptor Ly-49A recognizes a peptide-induced conformational determinant on its major histocompatibility complex class I ligand.

Natural killer (NK) cells are inhibited from killing cellular targets by major histocompatibility complex (MHC) class I molecules. In the mouse, this can be mediated by the Ly-49A NK cell receptor that specifically binds the H-2Dd MHC class I molecule, then inhibits NK cell activity. Previous experiments have indicated that Ly-49A recognizes the alpha 1/alpha 2 domains of MHC class I and that no specific MHC-bound peptide appeared to be involved. We demonstrate here that alanine-substituted peptides, having only the minimal anchor motifs, stabilized H-2Dd expression and provided resistance to H-2Dd-transfected, transporter associated with processing (TAP)-deficient cells from lysis by Ly-49A+ NK cells. Peptide-induced resistance was blocked only by an mAb that binds a conformational determinant on H-2Dd. Moreover, stabilization of "empty" H-2Dd heavy chains by exogenous beta 2-microglobulin did not confer resistance. In contrast to data for MHC class I-restricted T cells that are specific for peptides displayed MHC molecules, these data indicate that NK cells are specific for a peptide-induced conformational determinant, independent of specific peptide. This fundamental distinction between NK cells and T cells further implies that NK cells are sensitive only to global changes in MHC class I conformation or expression, rather than to specific pathogen-encoded peptides. This is consistent with the "missing self" hypothesis, which postulates that NK cells survey tissues for normal expression of MHC class I.

Induction of insulin and islet amyloid polypeptide production in pancreatic islet glucagonoma cells by insulin promoter factor 1.

Insulin promoter factor 1 (IPF1), a member of the homeodomain protein family, serves an early role in pancreas formation, as evidenced by the lack of pancreas formation in mice carrying a targeted disruption of the IPF1 gene [Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. (1994) Nature (London) 371, 606-609]. In adults, IPF1 expression is restricted to the beta-cells in the islets of Langerhans. We report here that IPF1 induces expression of a subset of beta-cell-specific genes (insulin and islet amyloid polypeptide) when ectopically expressed in clones of transformed pancreatic islet alpha-cells. In contrast, expression of IPF1 in rat embryo fibroblasts factor failed to induce insulin and islet amyloid polypeptide expression. This is most likely due to the lack of at least one other essential insulin gene transcription factor, the basic helix-loop-helix protein Beta 2/NeuroD, which is expressed in both alpha- and beta-cells. We conclude that IPF1 is a potent transcriptional activator of endogenous insulin genes in non-beta islet cells, which suggests an important role of IPF1 in beta-cell maturation.

A monoclonal IgG anticardiolipin antibody from a patient with the antiphospholipid syndrome is thrombogenic in mice.

Antiphospholipid antibodies, including anticardiolipin antibodies (ACA), are strongly associated with recurrent thrombosis in patients with the antiphospholipid syndrome (APS). To date, reports about the binding specificities of ACA and their role(s) in causing and/or sustaining thrombosis in APS are conflicting and controversial. The plasmas of patients with APS, usually containing a mixture of autoantibodies, vary in binding specificity for different phospholipids/cofactors and vary in in vitro lupus anticoagulant activity. Although in vivo assays that allow assessment of the pathogenic procoagulant activity of patient autoantibodies have recently been developed, the complex nature of the mixed species prevented determination of the particular species responsible for in vivo thrombosis. We have generated two human IgG monoclonal ACA from an APS patient with recurrent thrombosis. Both bound to cardiolipin in the presence of 10% bovine serum, but not in its absence, and both were reactive against phosphatidic acid, but were nonreactive against purified human beta-2 glycoprotein 1, DNA, heparan sulfate, or four other test antigens. Both monoclonal autoantibodies lacked lupus anticoagulant activity and did not inhibit prothrombinase activity. Remarkably, one of the monoclonal antibodies has thrombogenic properties when tested in an in vivo mouse model. This finding provides the first direct evidence that a particular antiphospholipid antibody specificity may contribute to in vivo thrombosis.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Heterometallic hybrids of homometallic human hemoglobins.

Hybridization experiments between normal Hb tetramers (Fe2+ Hb) and those with four metal-substituted hemes (i.e., replacement of Fe2+ by Co2+, Mg2+, Mn2+, Mn3+, Ni2+, or Zn2+) have revealed unexpected behavior. These homometallic Hbs have previously served as models that mimic the deoxy or oxy properties of normal Fe2+ Hb. In this study, hybrids were composed of one alpha 1 beta 1 dimer that is metal-substituted at both hemes, in association with a second dimer alpha 2 beta 2 that has normal Fe2+ hemes. Both metal-substituted subunits are unligated, whereas the two Fe2+ subunits either are both unligated or both ligated with O2, CO, or CN. It was found that four of the metal-substituted Hbs (Mg2+ Hb, Mn2+ Hb, Ni2+ Hb, and Zn2+ Hb) did not form detectable amounts of heterometallic hybrids with normal Fe2+ Hb even though (i) their homometallic parents formed tight tetrameric complexes with stabilities similar to that of Fe2+ Hb and (ii) hybrids with metal substitution at both alpha sites or both beta sites are known to form readily. This striking positional effect was independent of whether the normal Fe2+ hemes were ligated and of which ligand was used. These findings indicate that surprisingly large changes in tetramer behavior can arise from small and subtle perturbations at the heme sites. Possible origins of these effects are considered.

HLA-G expression during preimplantation human embryo development.

HLA-G is a nonclassical class I major histocompatibility complex molecule with a restricted pattern of expression that includes the placental extravillus cytotrophoblast cells in direct contact with maternal tissues. Circumstantial evidence suggests that HLA-G may play a role in protection of the semiallogeneic human fetus. We examined whether HLA-G is expressed during the critical period of preimplantation human development and whether expression of this molecule could be correlated with the cleavage rate of embryos. Using reverse transcription PCR on surplus human embryos and unfertilized oocytes from patients undergoing in vitro fertilization we detected HLA-G heavy chain mRNA in 40% of 148 of blastocysts tested. The presence of HLA-G mRNA was also detected in unfertilized oocytes and in early embryos, but not in control cumulus oophorus cells. beta 2-Microglobulin mRNA was also found in those embryos expressing HLA-G. In concordance with our mRNA data, a similar proportion of embryos stained positive for HLA-G utilizing a specific monoclonal antibody. Interestingly, expression of HLA-G mRNA was associated with an increased cleavage rate, as compared to embryos lacking HLA-G transcript. Thus, HLA-G could be a functional homologue of the mouse Qa-2 antigen, which has been implicated in differences in the rate of preimplantation embryo development. To our knowledge, the presence of HLA-G mRNA and protein in human preimplantation embryos and oocytes has not been reported previously. The correlation of HLA-G mRNA expression with cleavage rate suggests that this molecule may play an important role in human pre-embryo development.

Expression of a single-chain HLA class I molecule in a human cell line: presentation of exogenous peptide and processed antigen to cytotoxic T lymphocytes.

We have synthesized a recombinant gene encoding a single-chain HLA-A2/beta 2-microglobulin (beta 2m) molecule by linking beta 2m through its carboxyl terminus via a short peptide spacer to HLA-A2 (A*0201). This gene has been expressed in the beta 2m-deficient colorectal tumor cell line DLD-1. Transfection of this cell with the single-chain construct was associated with conformationally correct cell surface expression of a class I molecule of appropriate molecular mass. The single-chain HLA class I molecule presented either exogenously added peptide or (after interferon-gamma treatment) endogenously processed antigen to an influenza A matrix-specific, HLA-A2-restricted cytotoxic T-lymphocyte line. The need for interferon gamma for the processing and presentation of endogenous antigen suggests that DLD-1 has an antigen-processing defect that can be up-regulated, a feature that may be found in other carcinomas. Our data indicate that single-chain HLA class I constructs can form functional class I molecules capable of presenting endogenously processed antigens. Such molecules should be of use for functional studies, as well as providing potential anticancer immunotherapeutic agents or vaccines.

Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley.

Fructans play an important role in assimilate partitioning and possibly in stress tolerance in many plant families. Sucrose:fructan 6-fructosyltransferase (6-SFT), an enzyme catalyzing the formation and extension of beta-2,6-linked fructans typical of grasses, was purified from barley (Hordeum vulgare L.). It occurred in two closely similar isoforms with indistinguishable catalytic properties, both consisting of two subunits with apparent masses of 49 and 23 kDa. Oligonucleotides, designed according to the sequences of tryptic peptides from the large subunit, were used to amplify corresponding sequences from barley cDNA. The main fragment generated was cloned and used to screen a barley cDNA expression library. The longest cDNA obtained was transiently expressed in Nicotiana plumbaginifolia protoplasts and shown to encode a functional 6-SFT. The deduced amino acid sequence of the cDNA comprises both subunits of 6-SFT. It has high similarity to plant invertases and other beta-fructosyl hydrolases but only little to bacterial fructosyltransferases catalyzing the same type of reaction as 6-SFT.

Molecular heterogeneity of progenitors and radial migration in the developing cerebral cortex revealed by transgene expression.

We have analyzed the developmental pattern of beta-galactosidase (beta-gal) expression in the cerebral cortex of the beta 2nZ3'1 transgenic mouse line, which was generated using regulatory elements of the beta 2-microglobulin gene and shows ectopic expression in nervous tissue. From embryonic day 10 onward, beta-gal was expressed in the medial and dorsal cortices, including the hippocampal region, whereas lateral cortical areas were devoid of labeling. During the period of cortical neurogenesis (embryonic days 11-17), beta-gal was expressed by selective precursors in the proliferative ventricular zone of the neocortex and hippocampus, as well as by a number of migrating and postmigratory neurons arranged into narrow radial stripes above the labeled progenitors. Thus, the transgene labels a subset of cortical progenitors and their progeny. Postnatally, radial clusters of beta-gal-positive neurons were discernible until postpartum day 10. At this age, the clusters were 250 to 500 microns wide, composed of neurons spanning all the cortical layers and exhibiting several neuronal phenotypes. These data suggest molecular heterogeneity of cortical progenitors and of the cohorts of postmitotic neurons originating from them, which implies intrinsic molecular mosaicism in both cortical progenitors and developing neurons. Furthermore, the data show that neurons committed to the expression of the transgene migrate along very narrow, radial stripes.

Cloning of a gamma-aminobutyric acid type C receptor subunit in rat retina with a methionine residue critical for picrotoxinin channel block.

Ionotropic receptors for gamma-aminobutyric acid (GABA) are important to inhibitory neurotransmission in the mammalian retina, mediating GABAA and GABAC responses. In many species, these responses are blocked by the convulsant picrotoxinin (PTX), although the mechanism of block is not fully understood. In contrast, GABAC responses in the rat retina are extremely resistant to PTX. We hypothesized that this difference could be explained by molecular characterization of the receptors underlying the GABAC response. Here we report the cloning of two rat GABA receptor subunits, designated r rho 1 and r rho 2 after their previously identified human homologues. When coexpressed in Xenopus oocytes, r rho 1/r rho 2 heteromeric receptors mimicked PTX-resistant GABAC responses of the rat retina. PTX resistance is apparently conferred in native heteromeric receptors by r rho 2 subunits since homomeric r rho 1 receptors were sensitive to PTX; r rho 2 subunits alone were unable to form functional homomeric receptors. Site-directed mutagenesis confirmed that a single amino acid residue in the second membrane-spanning region (a methionine in r rho 2 in place of a threonine in r rho 1) is the predominant determinant of PTX resistance in the rat receptor. This study reveals not only the molecular mechanism underlying PTX blockade of GABA receptors but also the heteromeric nature of native receptors in the rat retina that underlie the PTX-resistant GABAC response.