Disruption of nNOS-PSD95 protein-protein interaction inhibits acute thermal hyperalgesia and chronic mechanical allodynia in rodents.

BACKGROUND AND PURPOSE: Post-synaptic density protein 95 (PSD95) contains three PSD95/Dosophilia disc large/ZO-1 homology domains and links neuronal nitric oxide synthase (nNOS) with the N-methyl-D-aspartic acid (NMDA) receptor. This report assesses the effects of disruption of the protein-protein interaction between nNOS and PSD95 on pain sensitivity in rodent models of hyperalgesia and neuropathic pain. EXPERIMENTAL APPROACH: We generated two molecules that interfered with the nNOS-PSD95 interaction: IC87201, a small molecule inhibitor; and tat-nNOS (residues 1-299), a cell permeable fusion protein containing the PSD95 binding domain of nNOS. We then characterized these inhibitors using in vitro and in vivo models of acute hyperalgesia and chronic allodynia, both of which are thought to require nNOS activation. KEY RESULTS: IC87201 and tat-nNOS (1-299) inhibited the in vitro binding of nNOS with PSD95, without inhibiting nNOS catalytic activity. Both inhibitors also blocked NMDA-induced 3',5'-cyclic guanosine monophosphate (cGMP) production in primary hippocampal cultures. Intrathecal administration of either inhibitor potently reversed NMDA-induced thermal hyperalgesia in mice. At anti-hyperalgesic doses, there was no effect on acute pain thresholds or motor coordination. Intrathecal administration of IC87201 and tat-nNOS also reversed mechanical allodynia induced by chronic constriction of the sciatic nerve. CONCLUSIONS AND IMPLICATIONS: nNOS-PSD95 interaction is important in maintaining hypersensitivity in acute and chronic pain. Disruption of the nNOS-PSD95 interaction provides a novel approach to obtain selective anti-hyperalgesic compounds.

A family of heptahelical receptors with adhesion-like domains: a marriage between two super families.

Some G protein-coupled receptors (GPCRs) are regulators of cell adhesion via inside-out effector signaling pathways. Such is the case with leukocyte chemokine receptors which stimulate intracellular second messenger pathways resulting in upregulation of integrin adhesion to ligands present in the extracellular matrix or on opposing cells resulting in chemotaxis and extravasation during immune surveillance. Remarkably, a family of GPCRs has recently been discovered that may themselves be triggered by cell-cell or cell-matrix interactions. Along with a canonical heptahelical membrane-spanning region, these intriguing proteins contain putative cell adhesion-like modules. The evidence to date suggests that they are involved in lymphocyte activation, macrophage biology, synaptic exocytosis and planar polarization during organogenesis.

Antibody engagement of intercellular adhesion molecule 3 triggers apoptosis of normal and leukaemic myeloid marrow cells.

Intercellular adhesion molecule 3 (ICAM-3, CD50) is an immunoglobulin (Ig) domain-containing cell-cell adhesion receptor that binds to the lymphocyte function antigen 1 (LFA-1; CD11a/CD18) integrin. It is constitutively expressed on haematopoietic precursors and differentiated leucocytes, as well as on most leukaemic cells. ICAM-3/LFA-1 binding during a lymphocyte-mediated cellular immune response has been well established; however, its role in the marrow compartment is unclear. In this study, marrow cells from normal and acute leukaemic donors, as well as leukaemic cell lines, were cultured in the presence of various monoclonal antibodies (mAbs) to ICAM-3, and apoptosis was subsequently measured by annexin V binding. Anti-ICAM-3 mAb ICR 1.1 engagement triggered increased percentages of apoptosis among normal and leukaemic marrow myeloid cells. Fab fragments of ICR 1.1 mimicked the intact mAb, suggesting that the apoptotic signal was independent of Fc receptor interactions and did not require bivalent epitope engagement. In addition, the apoptotic signal was found to be independent of ICAM-1/LFA-1 binding interactions, as well as Fas/FasL and tumour necrosis factor alpha (TNF-alpha)/TNF receptor-activated pathways, as neutralizing antibodies to CD11a/CD18, Fas and TNF-alpha failed to abrogate the response.

The intercellular adhesion molecule (ICAM) family of proteins. New members and novel functions.

Macromolecular adhesive associations between cells are important for transmitting spatial and temporal information that is critical for immune system function. One such group of proteins, the intercellular adhesion molecules (ICAMs), has grown as newly identified members are revealed. In addition, the functions of the ICAMs, in general, have begun to be better understood, including intracellular signaling events. This information has led to the design of novel therapeutic agents that may prove effective in a variety of disease states.

Coengagement of ICAM-3 and Fc receptors induces chemokine secretion and spreading by myeloid leukocytes.

ICAM-3 is expressed at high levels on myeloid leukocytes, but its function on these cells is unknown. We tested the hypothesis that it transduces outside-in proinflammatory signals using immobilized mAbs to engage ICAM-3 on freshly isolated human monocytes and neutrophils. Two immobilized Abs that recognize epitopes in the extracellular domain 1 of ICAM-3, which is critical for recognition by the alphaL/beta2 integrin, potently induced secretion of MIP-1alpha, IL-8, and MCP-1 by monocytes and triggered IL-8 secretion by neutrophils. These chemokines are products of immediate-early genes that are induced when myeloid cells are activated. Chemokine secretion induced by "triggering" Abs was greater than that induced by isotype-matched immobilized Abs against ICAM-1, ICAM-2, PECAM-1, control Igs, or immobilized control proteins. Coengagement of ICAM-3 and Fc receptors (FcgammaRI or FcgammaRII) was required for maximal chemokine secretion by monocytes. Microscopy documented that there is also dramatic spreading of monocytes when surface ICAM-3 is engaged by immobilized Abs. Spreading was induced by Fab and F(ab')2 fragments of triggering anti-ICAM-3 mAb, demonstrating direct outside-in signaling, but was not required for chemokine secretion. These experiments indicate that ICAM-3 may transmit outside-in signals when it is engaged by beta2 integrins during myeloid cell-cell interactions in inflammatory lesions. Binding of Fc receptors by Ig in the local environment can amplify the responses.

Functional mapping of the cytoplasmic region of intercellular adhesion molecule-3 reveals important roles for serine residues.

Intercellular adhesion molecule-3 (ICAM-3), a ligand for beta2 integrins, elicits a variety of activation responses in lymphocytes. We describe a functional mapping study that focuses on the 37-residue cytoplasmic region of ICAM-3. Carboxyl-terminal truncations delineated portions involved in T cell antigen receptor costimulation, homotypic aggregation, and cellular spreading. Truncation of the membrane distal 25 residues resulted in loss of T cell antigen receptor costimulation as determined by interleukin 2 secretion. Aggregation and cell spreading were sensitive to truncation of the membrane distal and proximal thirds of the cytoplasmic portion. Phosphoamino acid analysis revealed that ICAM-3 from activated cells contained phosphoserine and phosphopeptide mapping identified Ser489 as a site of phosphorylation in vivo. Mutation of Ser489 or Ser515 to alanine blocked interleukin 2 secretion, aggregation and cell spreading, while mutation of other serine residues affected only a subset of functions. Ser489 was a phosphorylation site in vitro for recombinant protein kinase Ctheta. Finally, treatment of Jurkat cells with chelerythrine chloride, a protein kinase C inhibitor, prevented ICAM-3-triggered spreading. This study delineates separable regions and amino acid residues within the cytoplasmic portion of ICAM-3 that are important for T cell function.

LFA-1 binding site in ICAM-3 contains a conserved motif and non-contiguous amino acids.

The intercellular adhesion molecule-3 (ICAM-3) is a counter receptor for the integrin LFA-1 that supports cell-cell adhesion dependent functions. ICAM-3 is a member of the immunoglobulin superfamily possessing five immunoglobulin-like domains. Here, we characterize the overall shape of ICAM-3 and the amino acid residues involved in binding LFA-1 and monoclonal antibodies (Mab). Electron microscopic observations show that ICAM-3 is predominantly a straight rod of 15 nm in length, suggesting a head to tail arrangement of the immunoglobulin-like domains. Six out of nine ICAM-3 Mab described blocked the interaction with LFA-1 to varying degrees. Domain assignment of blocking Mab epitopes and characterization of LFA-1-dependent cell adhesion to ICAM-3 mutants demonstrate that the amino-terminal domain of ICAM-3 interacts with LFA-1. A conserved amino acid motif including residues E37 and T38 form an integrin binding site (IBS) in ICAM-3. This motif has also been shown to function as an IBS in ICAM-3 and VCAM-1 and hence many form a common site of contact in all CAMs of this type. Other ICAM-3 residues critical to adhesive interactions, such as Q75, conserved in ICAM-1 and ICAM-2, but not VCAM-1, may confer specificity to LFA-1 binding. This residue, Q75, is predicted to locate in a model of ICAM-3 to the same site as RGD in the immunoglobulin-like domain of fibronectin that binds several integrins. This suggest an evolutionary relationship between ICAMs and fibronectin interactions with integrins.

Inhibition of HIV-1 gp160-dependent membrane fusion by a furin-directed alpha 1-antitrypsin variant.

Furin is a membrane-associated calcium-dependent serine endoprotease that cleaves proproteins on the carboxyl side of the consensus sequence -Arg-X-Lys/Arg-Arg-. Using site-directed mutagenesis, a variant alpha 1-antitrypsin (alpha 1-AT) was constructed which contains in its reactive site -Arg-X-X-Arg-, the minimal sequence required for efficient processing by furin (Molloy, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R., and Thomas, G. (1992) J. Biol. Chem. 267, 16396-16402). This alpha 1-AT variant, [Arg355 Arg358]alpha 1-AT (alpha 1-PDX), is greater than 3,000-fold more effective than [Arg358]alpha 1-AT (alpha 1-AT Pittsburgh, alpha 1-PIT) at inhibiting furin in vitro (K0.5 = 0.03 microgram/ml). Furthermore, the P4 Arg in alpha 1-PDX greatly attenuates the thrombin inhibitory properties of this serpin (> 300-fold) compared with alpha 1-PIT (which contains a P4 Ala), thus increasing the selectivity of alpha 1-PDX for furin. Expression studies show that alpha 1-PDX, and not alpha 1-PIT, blocks the processing of two furin substrates, pro-beta-nerve growth factor and human immunodeficiency virus (HIV)-1 gp160 in transfected cells. In addition, a syncytium assay shows that alpha 1-PDX blocks the membrane fusogenic properties of HIV-1 gp160. The potential use of alpha 1-PDX in manipulating the activation of proproteins in a tissue- and time-specific manner is discussed.

A unique Kex2-like endoprotease from Drosophila melanogaster is expressed in the central nervous system during early embryogenesis.

Complementary DNA sequences were cloned from a Drosophila library encoding a 1,101 amino acid polypeptide that we have named dKLIP-1. The deduced protein is structurally similar to the yeast KEX2 prohormone endoprotease including the conserved Asp, His, and Ser catalytic triad residues characteristic of the subtilisin family. When coexpressed in vivo with pro-beta-NGF, dKLIP-1 greatly enhanced the endoproteolytic conversion of the precursor to mature beta-NGF by cleavage at a -Lys-Arg- doublet. In adults, dKLIP-1 transcripts were detected in cortical regions of the CNS and fat body. Most striking, however, was the high level of maternal transcripts deposited into developing oocytes. The temporal and spatial expression of dKLIP-1 mRNAs during embryonic development indicates a potential role for this novel Kex2p-like endoprotease in early embryogenesis and neurogenesis.

The rat gonadotropin-releasing hormone: SH locus: structure and hypothalamic expression.

The rat GnRH gene as expressed in the central nervous system is comprised of four exons and three introns and spans 4.5 kilobases of genomic DNA. Recently it has been shown that the DNA strand opposite that which is transcribed to produce GnRH mRNA is transcribed in heart to produce a set of transcripts, SH RNAs, which share significant exonic sequences with the GnRH gene. The nucleotide sequence of this locus and approximately 3 kilobases on either side has been determined. Northern analysis of hypothalamic RNA probed with GnRH and SH strand specific probes demonstrate that both GnRH and SH RNAs are present within the preoptic hypothalamus. The cap sites for GnRH and SH transcripts have been localized using polymerase chain reaction technology. Results from these experiments indicate that in the preoptic hypothalamus GnRH transcription initiates from three sites. The majority of GnRH transcripts is spliced efficiently and gives rise to the major class of GnRH mRNA. A second spliced population is present in lower abundance, while a third population is not spliced. The SH gene contains at least two distinct promoters, from which two populations of transcripts are derived containing unique 5'-sequences spliced to a common 3'-region.

Molecular characterization of a new immunoglobulin superfamily protein with potential roles in opioid binding and cell contact.

A purified opioid-binding protein has been characterized by cDNA cloning. The cDNA sequence predicts an extracellularly located glycoprotein of 345 amino acids. This protein does not possess a membrane-spanning domain but contains a C-terminal hydrophobic sequence characteristic of membrane attachment by a phosphatidylinositol linkage. It displays homology to the immunoglobulin protein superfamily, featuring three domains that resemble disulfide-bonded constant regions. More specifically, the protein is most homologous to a subfamily of proteins which includes the neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG) and one subgroup of the tyrosine kinase growth factor receptors comprising the platelet-derived growth factor receptor (PDGF R), the colony-stimulating factor 1 receptor (CSF-1 R) and the c-kit protooncogene. These sequence homologies suggest that the protein could be involved in either cell recognition and adhesion, peptidergic ligand binding or both.

Tissue-specific and developmentally regulated transcription of the insulin-like growth factor 2 gene.

Transcription of the rat and human IGF-2 gene loci is unusually complex. The pattern of expression of these genes varies both between tissues and within a given tissue during different stages of development. Alternative splicing or possibly transcriptional initiation events generate variant IGF-2 mRNAs that contain different 5'-untranslated leader sequences. These leader exon sequences are shared with non-IGF-2 mRNAs. Certain noncoding IGF-2 gene sequence elements are transcribed extensively and are found in multiple copies elsewhere in the human genome. Furthermore, IGF-2 mRNA levels are particularly high in a variety of human malignancies.

A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse.

Hereditary hypogonadism in the hypogonadal (hpg) mouse is caused by a deletional mutation of at least 33.5 kilobases encompassing the distal half of the gene for the common biosynthetic precursor of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP). The partially deleted gene is transcriptionally active as revealed by in situ hybridization histochemistry of hpg hypothalamic tissue sections, but immunocytochemical analysis failed to show the presence of antigen corresponding to any part of the precursor protein.

Biochemical characterization of polypeptides encoded by mutated human Ha-ras1 genes.

We expressed six forms of p21-ras polypeptides in Escherichia coli with differing transformation potentials resulting from amino acid substitutions at position 12. The ability of the encoded p21's to autophosphorylate, bind guanine nucleotides, and hydrolyze GTP was assessed. All versions of p21 bound GTP equivalently; the kinase activity, while dependent upon residue 12, did not correlate with the transforming potential of the polypeptide. All transforming versions exhibited an impaired GTPase activity, while a novel nontransforming derivative [p21(pro-12)] possessed an enhanced GTPase activity. These results provide strong support for the proposal that an impairment of the cellular p21 GTPase activity can unmask its transforming potential.

Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hormone and prolactin release-inhibiting factor in human and rat.

Cloned cDNAs encoding the precursor protein for gonadotropin-releasing hormone (Gn-RH) and prolactin release-inhibiting factor (PIF) were isolated from libraries derived from human and rat hypothalamic mRNA. Nucleotide sequence analyses predict precursor proteins of 92 amino acids for both species and show identity between the human placental and human hypothalamic precursor proteins. Whereas the Gn-RH peptide structure is completely conserved in human and rat, the PIF domain of the precursor displays 70% interspecies homology. Genomic analyses revealed the presence of a single Gn-RH-PIF gene in human and rat containing sequences corresponding to the cDNA distributed across four exons.

Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-beta.

Inhibin, a specific and potent polypeptide inhibitor of the secretion of follicle-stimulating hormone (FSH), of gonadal origin and thus a potential contraceptive, may constitute a missing link in the mechanism controlling the differential secretion of the pituitary gonadotropins. Inhibin-like bioactivity has been reported in various fluids and extracts of testis and in ovarian follicular fluid. Although there have been several attempts to purify inhibin from seminal plasma, purification from follicular fluid has been more successful (refs 14-16; for review see ref. 17). We have previously isolated two forms (A and B) of inhibin from porcine follicular fluid. Each form comprised two dissimilar subunits of relative molecular mass (Mr) 18,000 (18K, referred to here as the alpha-subunit) and 14K (the beta-subunit), crosslinked by one or more disulphide bridge(s). Forms A and B differ in the N-terminal sequence of their 14K subunit. Preliminary structural characterization of porcine and bovine ovarian inhibins shows that they have similar properties. Here, we have used the N-terminal amino-acid sequence data on the subunits of each inhibin to identify cloned complementary DNAs encoding the biosynthetic precursors and report that inhibins are the product of a gene family that also includes transforming growth factor-beta (TGF-beta) and whose structural organization is similar to that of pituitary and placental glycoprotein hormones.

Defining the borders of the chicken proto-fps gene, a precursor of Fujinami sarcoma virus.

The transforming (onc) genes of retroviruses contain specific sequences, derived from as yet poorly defined, normal cellular genes, termed proto-onc genes. Proto-onc genes must be defined to explain their docility compared to the oncogenicity of the viral derivatives. Here we set out to determine the borders of the chicken proto-fps gene from which the onc genes of avian Fujinami (FSV) and PRC sarcoma viruses (PRCSV) are derived. These onc genes are hybrids of an element from the gag gene of retroviruses (delta gag) linked to a 2.8-kb domain from proto-fps. To identify the 5' border of proto-fps we have sequenced 1.5 kb beyond the 5' border of overlap with viral fps utilizing a proto-fps clone derived previously. A possible promoter was identified that maps 736 nucleotides from this border. The 736 nucleotides contain two possible exons with 121 codons, and short regions of homology with the delta gag termini of FSV and PRCII. A translation stop codon and an adjacent polyadenylation signal were identified just prior to the 3' border of overlap with viral fps within a 1.15-kb sequence of a newly isolated proto-fps clone. Comparing four exons within this 1.15 kb proto-fps sequence with known fps equivalents of FSV and PRCSV, we have detected strain-specific, but no common point mutations in each viral genome. A 3.3-kb polyadenylated proto-fps mRNA was detected in chicken liver RNA by gel electrophoresis and hybridization with proto-fps DNA. We conclude that the coding capacity of proto-fps is just over 3 kb, consistent with the size of the putative proto-fps protein of 98 kDa and hence slightly larger than that of viral fps. Thus proto-fps and the viral delta gag-fps genes each contain distinct 5' regulatory and coding sequences and share the 3' terminal fps domains. It is suggested that this difference, rather than scattered point mutations, is responsible for the oncogenic function of the viral genes and the unknown cellular function of proto-fps.

Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor.

A murine tumor necrosis factor (MuTNF) cDNA was isolated from a cDNA library prepared by using mRNA from the murine macrophage-like cell line PU5-1.8 induced with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. The cDNA encodes a polypeptide consisting of a 79 amino acid pre sequence followed by a mature MuTNF sequence of 156 amino acids. The 235 amino acid murine pre-TNF polypeptide is 79% homologous to the human pre-TNF protein. There is one potential N-linked glycosylation site on MuTNF, in contrast to human TNF, which lacks any such site. The MuTNF cDNA, when engineered for expression in Escherichia coli, was found to direct the synthesis of biologically active MuTNF as determined by its cytotoxicity against several transformed cell lines.

Nucleotide sequence of two overlapping myc-related genes in avian carcinoma virus OK10 and their relation to the myc genes of other viruses and the cell.

Avian carcinoma virus OK10 has the genetic structure gag-delta pol-myc-delta env. It shares the transformation-specific myc sequence with three other avian carcinoma viruses (MC29, MH2, CMII) and also with a normal chicken gene proto-myc and the gag, pol, and env elements with non-transforming retroviruses. Unlike the other myc-containing viruses, which synthesize singular myc proteins, OK10 synthesizes two different myc-related proteins of 200 and 57 kDa. Here we have sequenced the myc region of an infectious OK10 provirus to investigate how OK10 synthesizes two different proteins from the same myc domain and to identify characteristic differences between the normal proto-myc gene and the myc-related viral transforming genes. It was found that the 1.6-kilobase myc domain of OK10 is colinear and coterminal with the myc domains of MC29, MH2, and the terminal two exons of proto-myc. It is preceded by the same splice acceptor as the myc sequence of MH2 and as the second proto-myc exon. From this and the known structure of retroviruses, it follows that the OK10 gene encoding the 57-kDa protein is discontinuous with a small 5' exon that includes six gag codons and a large 3' myc exon (delta gag-myc). This gene and the delta gag-myc gene of MH2 are isogenic. The proto-myc-derived intron preceding the myc domain of OK10 is in the same reading frame as the adjacent delta pol and myc domains and, hence, is part of the gag-delta pol-myc gene encoding the 200-kDa protein. Sequence comparisons with proto-myc and MC29 and MH2 indicate that there are no characteristic mutations that set apart the viral myc domains from proto-myc. It is concluded that transforming function of viral myc-related genes correlates with the lack of a viral equivalent of the first proto-myc exon(s) and conjugation of the viral myc domains with large or small retroviral genetic elements rather than with specific point mutations. Because OK10 and MH2 each contain two genes with potential transforming function (namely, delta gag-myc and gag-delta pol-myc or delta gag-mht, respectively), it remains to be determined whether the delta gag-myc genes have transforming function on their own or need helper genes. The possible helper requirement cannot be very specific because the two potential helper genes are very different.