Crystal structure of the HLA-Cw3 allotype-specific killer cell inhibitory receptor KIR2DL2

Killer cell inhibitory receptors (KIR) protect class I HLAs expressing target cells from natural killer (NK) cell-mediated lysis. To understand the molecular basis of this receptor-ligand recognition, we have crystallized the extracellular ligand-binding domains of KIR2DL2, a member of the Ig superfamily receptors that recognize HLA-Cw1, 3, 7, and 8 allotypes. The structure was determined in two different crystal forms, an orthorhombic P212121 and a trigonal P3221 space group, to resolutions of 3.0 and 2.9 Å, respectively. The overall fold of this structure, like KIR2DL1, exhibits K-type Ig topology with cis-proline residues in both domains that define β-strand switching, which sets KIR apart from the C2-type hematopoietic growth hormone receptor fold. The hinge angle of KIR2DL2 is approximately 80°, 14° larger than that observed in KIR2DL1 despite the existence of conserved hydrophobic residues near the hinge region. There is also a 5° difference in the observed hinge angles in two crystal forms of 2DL2, suggesting that the interdomain hinge angle is not fixed. The putative ligand-binding site is formed by residues from several variable loops with charge distribution apparently complementary to that of HLA-C. The packing of the receptors in the orthorhombic crystal form offers an intriguing model for receptor aggregation on the cell surface.

Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4

The receptor 2B4 belongs to the Ig superfamily and is found on the surface of all murine natural killer (NK) cells as well as T cells displaying non-MHC-restricted cytotoxicity. Previous studies have suggested that 2B4 is an activating molecule because cross-linking of this receptor results in increased cytotoxicity and γ-interferon secretion as well as granule exocytosis. However, it was recently shown that the gene for 2B4 encodes two different products that arise by alternative splicing. These gene products differ solely in their cytoplasmic domains. One form has a cytoplasmic tail of 150 amino acids (2B4L) and the other has a tail of 93 amino acids (2B4S). To determine the function of each receptor, cDNAs for 2B4S and 2B4L were transfected into the rat NK cell line RNK-16. Interestingly, the two forms of 2B4 had opposing functions. 2B4S was able to mediate redirected lysis of P815 tumor targets, suggesting that this form represents an activating receptor. However, 2B4L expression led to an inhibition of redirected lysis of P815 targets when the mAb 3.2.3 (specific for rat NKRP1) was used. In addition, 2B4L constitutively inhibits lysis of YAC-1 tumor targets. 2B4L is a tyrosine phosphoprotein, and removal of domains containing these residues abrogates its inhibitory function. Like other inhibitory receptors, 2B4L associates with the tyrosine phosphatase SHP-2. Thus, 2B4L is an inhibitory receptor belonging to the Ig superfamily.

The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase

Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.

Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: The Fab is directed against an intermediate in the helix-coil dynamics of the enzyme

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a γ turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.

Nucleus reticularis neurons mediate diverse inhibitory effects in thalamus

Detailed information regarding the contribution of individual γ-aminobutyric acid (GABA)-containing inhibitory neurons to the overall synaptic activity of single postsynaptic cells is essential to our understanding of fundamental elements of synaptic integration and operation of neuronal circuits. For example, GABA-containing cells in the thalamic reticular nucleus (nRt) provide major inhibitory innervation of thalamic relay nuclei that is critical to thalamocortical rhythm generation. To investigate the contribution of individual nRt neurons to the strength of this internuclear inhibition, we obtained whole-cell recordings of unitary inhibitory postsynaptic currents (IPSCs) evoked in ventrobasal thalamocortical (VB) neurons by stimulation of single nRt cells in rat thalamic slices, in conjunction with intracellular biocytin labeling. Two types of monosynaptic IPSCs could be distinguished. “Weak” inhibitory connections were characterized by a significant number of postsynaptic failures in response to presynaptic nRt action potentials and relatively small IPSCs. In contrast, “strong” inhibition was characterized by the absence of postsynaptic failures and significantly larger unitary IPSCs. By using miniature IPSC amplitudes to infer quantal size, we estimated that unitary IPSCs associated with weak inhibition resulted from activation of 1–3 release sites, whereas stronger inhibition would require simultaneous activation of 5–70 release sites. The inhibitory strengths were positively correlated with the density of axonal swellings of the presynaptic nRt neurons, an indicator that characterizes different nRt axonal arborization patterns. These results demonstrate that there is a heterogeneity of inhibitory interactions between nRt and VB neurons, and that variations in gross morphological features of axonal arbors in the central nervous system can be associated with significant differences in postsynaptic response characteristics.

Expression of HLA class I-specific inhibitory natural killer cell receptors in HIV-specific cytolytic T lymphocytes: Impairment of specific cytolytic functions

Human T lymphocytes have been shown to express inhibitory natural killer cell receptors (NKR), which can down-regulate T cell antigen receptor-mediated T cell function, including cytolytic activity. In the present study, we demonstrate that CD3+NKR+ cells can be identified in HIV-infected patients. HIV-specific cytolytic activity was analyzed in five patients in whom autologous lymphoblastoid B cell lines could be derived as a source of autologous target cells. Phytohemagglutinin-activated T cell populations that had been cultured in interleukin 2 displayed HIV-specific cytotoxic T lymphocyte (CTL) activity against HIV env, gag, pol, and nef in 3 of 5 patients. Addition of anti-NKR mAb of IgM isotype could increase the specific CTL activity. Moreover, in one additional patient, HIV-specific CTL activity was undetectable; however, after addition of anti-NKR mAb such CTL activity appeared de novo. Similar results were obtained by analysis of CD3+NKR+ clones derived from two patients. These data provide direct evidence that CD3+NKR+ cells may include antigen (HIV)-specific CTLs and that mAb-mediated masking of inhibitory NKR may revert the down-regulation of CTL function.

MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-β superfamily

Macrophages play a key role in both normal and pathological processes involving immune and inflammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor β (TGF-β) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1β, tumor necrosis factor α (TNF-α), interleukin 2, and macrophage colony-stimulating factor but not interferon γ, or lipopolysaccharide (LPS). Its expression is also increased by TGF-β. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein of Mr 25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-α production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinflammatory cytokines and TGF-β may serve to limit the later phases of macrophage activation.

Induction of inhibitory factor κBα mRNA in the central nervous system after peripheral lipopolysaccharide administration: An in situ hybridization histochemistry study in the rat

In this study we investigate the mRNA expression of inhibitory factor κBα (IκBα) in cells of the rat brain induced by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). IκB controls the activity of nuclear factor κB, which regulates the transcription of many immune signal molecules. The detection of IκB induction, therefore, would reveal the extent and the cellular location of brain-derived immune molecules in response to peripheral immune challenges. Low levels of IκBα mRNA were found in the large blood vessels and in circumventricular organs (CVOs) of saline-injected control animals. After an i.p. LPS injection (2.5 mg/kg), dramatic induction of IκBα mRNA occurred in four spatio-temporal patterns. Induced signals were first detected at 0.5 hr in the lumen of large blood vessels and in blood vessels of the choroid plexus and CVOs. Second, at 1–2 hr, labeling dramatically increased in the CVOs and choroid plexus and spread to small vascular and glial cells throughout the entire brain; these responses peaked at 2 hr and declined thereafter. Third, cells of the meninges became activated at 2 hr and persisted until 12 hr after the LPS injection. Finally, only at 12 hr, induced signals were present in ventricular ependyma. Thus, IκBα mRNA is induced in brain after peripheral LPS injection, beginning in cells lining the blood side of the blood–brain barrier and progressing to cells inside brain. The spatiotemporal patterns suggest that cells of the blood–brain barrier synthesize immune signal molecules to activate cells inside the central nervous system in response to peripheral LPS. The cerebrospinal fluid appears to be a conduit for these signal molecules.

Removal of LIF (leukemia inhibitory factor) results in increased vitamin A (retinol) metabolism to 4-oxoretinol in embryonic stem cells

Retinoids, vitamin A (retinol) and its metabolic derivatives, are required for normal vertebrate development. In murine embryonic stem (ES) cells, which remain undifferentiated when cultured in the presence of LIF (leukemia inhibitory factor), little metabolism of exogenously added retinol takes place. After LIF removal, ES cells metabolize exogenously added retinol to 4-hydroxyretinol and 4-oxoretinol and concomitantly differentiate. The conversion of retinol to 4-oxoretinol is a high-capacity reaction because most of the exogenous retinol is metabolized rapidly, even when cells are exposed to physiological (≈1 μM) concentrations of retinol in the medium. No retinoic acid or 4-oxoRA synthesis from retinol was detected in ES cells cultured with or without LIF. The cytochrome P450 enzyme CYP26 (retinoic acid hydroxylase) is responsible for the metabolism of retinol to 4-oxoretinol, and CYP26 mRNA is greatly induced (>15-fold) after LIF removal. Concomitant with the expression of CYP26, differentiating ES cells grown in the absence of LIF activate the expression of the differentiation marker gene FGF-5 whereas the expression of the stem cell marker gene FGF-4 decreases. The strong correlation between the production of polar metabolites of retinol and the differentiation of ES cells upon removal of LIF suggests that one important action of LIF in these cells is to prevent retinol metabolism to biologically active, polar metabolites such as 4-oxoretinol.

Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors

Natural killer (NK) cell cytotoxicity is regulated in large part by the expression of NK cell receptors able to bind class I major histocompatibility complex glycoproteins. The receptors associated with recognition of HLA-C allospecificities are the two-domain Ig-like molecules, p50 and p58 proteins, with highly homologous extracellular domains but differing in that they have either an activating or inhibitory function, respectively, depending on the transmembrane domain and cytoplasmic tails that they possess. We have compared the binding to HLA-Cw7 of an inhibitory p58 molecule, NKAT2, the highly homologous activating p50 molecule, clone 49, and a second activating p50 molecule, clone 39, which has homologies to both NKAT1 and NKAT2. NKAT2 binds to HLA-Cw7 with very rapid association and dissociation rates. However, the p50 receptors bind only very weakly, if at all, to HLA-C. The molecular basis of this difference is analyzed, and the functional significance of these observations is discussed.

The α1 domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: Is HLA-G the public ligand for natural killer cell inhibitory receptors?

We have investigated the protective role of the membrane-bound HLA-G1 and HLA-G2 isoforms against natural killer (NK) cell cytotoxicity. For this purpose, HLA-G1 and HLA-G2 cDNAs were transfected into the HLA class I-negative human K562 cell line, a known reference target for NK lysis. The HLA-G1 protein, encoded by a full-length mRNA, presents a structure similar to that of classical HLA class I antigens. The HLA-G2 protein, deduced from an alternatively spliced transcript, consists of the α1 domain linked to the α3 domain. In this study we demonstrate that (i) HLA-G2 is present at the cell surface as a truncated class I molecule associated with β2-microglobulin; (ii) NK cytolysis, observed in peripheral blood mononuclear cells and in polyclonal CD3− CD16+ CD56+ NK cells obtained from 20 donors, is inhibited by both HLA-G1 and HLA-G2; this HLA-G-mediated inhibition is reversed by blocking HLA-G with a specific mAb; this led us to the conjecture that HLA-G is the public ligand for NK inhibitory receptors (NKIR) present in all individuals; (iii) the α1 domain common to HLA-G1 and HLA-G2 could mediate this protection from NK lysis; and (iv) when transfected into the K562 cell line, both HLA-G1 and HLA-G2 abolish lysis by the T cell leukemia NK-like YT2C2 clone due to interaction between the HLA-G isoform on the target cell surface and a membrane receptor on YT2C2. Because NKIR1 and NKIR2, known to interact with HLA-G, were undetectable on YT2C2, we conclude that a yet-unknown specific receptor for HLA-G1 and HLA-G2 is present on these cells.

Glucose intolerance caused by a defect in the entero-insular axis: A study in gastric inhibitory polypeptide receptor knockout mice

Mice with a targeted mutation of the gastric inhibitory polypeptide (GIP) receptor gene (GIPR) were generated to determine the role of GIP as a mediator of signals from the gut to pancreatic β cells. GIPR−/− mice have higher blood glucose levels with impaired initial insulin response after oral glucose load. Although blood glucose levels after meal ingestion are not increased by high-fat diet in GIPR+/+ mice because of compensatory higher insulin secretion, they are significantly increased in GIPR−/− mice because of the lack of such enhancement. Accordingly, early insulin secretion mediated by GIP determines glucose tolerance after oral glucose load in vivo, and because GIP plays an important role in the compensatory enhancement of insulin secretion produced by a high insulin demand, a defect in this entero-insular axis may contribute to the pathogenesis of diabetes.

Constitutive tyrosine phosphorylation of the inhibitory paired Ig-like receptor PIR-B

PIR-A and PIR-B are activating and inhibitory Ig-like receptors on murine B lymphocytes, dendritic cells, and myeloid-lineage cells. The inhibitory function of PIR-B is mediated via its cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, whereas PIR-A pairs with the Fc receptor common γ chain to form an activating receptor complex. In these studies, we observed constitutive tyrosine phosphorylation of PIR-B molecules on macrophages and B lymphocytes, irrespective of the cell activation status. Splenocyte PIR-B molecules were constitutively associated with the SHP-1 protein tyrosine phosphatase and Lyn protein tyrosine kinase. In Lyn-deficient mice, PIR-B tyrosine phosphorylation was greatly reduced. Unexpectedly, tyrosine phosphorylation of PIR-B was not observed in most myeloid and B cell lines but could be induced by ligation of the PIR molecules. Finally, the phosphorylation status of PIR-B was significantly reduced in MHC class I-deficient mice, although not in mice deficient in TAP1 or MHC class II expression. These findings suggest a physiological inhibitory role for PIR-B that is regulated by endogenous MHC class I-like ligands.

Insulin-like growth factor binding protein 2 is a growth inhibitory protein conserved in zebrafish

Fish serum contains several specific binding proteins for insulin-like growth factors (IGFBPs). The structure and physiological function of these fish IGFBPs are unknown. Here we report the complete primary sequence of a zebrafish IGFBP deduced from cDNA clones isolated by library screening and rapid amplification of cDNA ends. The full-length 1,757-bp cDNA encodes a protein of 276 aa, which contains a putative 22-residue signal peptide and a 254-residue mature protein. The mature zebrafish IGFBP has a predicted molecular size of 28,440 Da and shows high sequence identity with human IGFBP-2 (52%). The sequence identities with other human IGFBPs are <37%. Chinese hamster ovary cells stably transfected with the zebrafish IGFBP-2 cDNA secreted a 31-kDa protein, which bound to IGF-I and IGF-II with high affinity, but did not bind to Des(1–3)IGF-I or insulin. Northern blot analyses revealed that the zebrafish IGFBP-2 transcript is a 1.8-kb band expressed in many embryonic and adult tissues. In adult zebrafish, IGFBP-2 mRNA levels were greatly reduced by growth hormone treatment but increased by prolonged fasting. When overexpressed or added to cultured zebrafish and mammalian cells, the zebrafish IGFBP-2 significantly inhibited IGF-I-stimulated cell proliferation and DNA synthesis. These results indicate that zebrafish IGFBP-2 is a negative growth regulator acting downstream in the growth hormone-IGF-I axis.

Isolation and characterization of the chicken m2 acetylcholine receptor promoter region: Induction of gene transcription by leukemia inhibitory factor and ciliary neurotrophic factor

We have isolated the promoter region and determined the start sites of transcription for the gene encoding the chicken m2 (cm2) muscarinic acetylcholine receptor. Transfection experiments, using cm2-luciferase reporter gene constructs, demonstrated that a 789-bp genomic fragment was sufficient to drive high level expression in chicken heart primary cultures, while an additional 1.2-kb region was required for maximal expression in mouse septal/neuroblastoma (SN56) cells. Treatment of SN56 cells with the cytokines ciliary neurotrophic factor and leukemia inhibitory factor increases expression of endogenous muscarinic acetylcholine receptors and results in a 4- to 6-fold induction of cm2 promoter driven luciferase expression. We have mapped a region of the cm2 promoter that is necessary for induction by cytokines.