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.

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.

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.

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.

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.

Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin.

Human tumour necrosis factor has about 30% homology in its amino acid sequence with lymphotoxin, a lymphokine that has similar biological properties. Recombinant tumour necrosis factor can be obtained by expression of its complementary DNA in Escherichia coli and induces the haemorrhagic necrosis of transplanted methylcholanthrene-induced sarcomas in syngeneic mice.

Insulin-like growth factor II precursor gene organization in relation to insulin gene family.

The insulin gene family, comprised of insulin, relaxin, insulin-like growth factors I and II (IGF-I and IGF-II) and possibly the beta-subunit of 7S nerve growth factor, represents a group of structurally related polypeptides whose biological functions have diverged. The IGFs, or somatomedins, constitute a class of polypeptides that have a key role in pre-adolescent mammalian growth (see ref. 4 for review). IGF-I expression is regulated by growth hormone and mediates postnatal growth, while IGF-II appears to be induced by placental lactogen during prenatal development. The primary structures of both human IGFs have been determined and are closely related. A polypeptide highly homologous to human IGF-II is secreted by the rat liver cell line, BRL-3A. As this polypeptide, termed multiplication stimulating activity (MSA), differs from human IGF-II by only five amino acid residues, MSA probably represents the rat IGF-II protein. Using molecular cloning techniques, we have isolated cDNA and chromosomal genes coding for the MSA and human IGF-II precursors, respectively. Our data, presented here, indicate that both MSA and human IGF-II are synthesized initially as larger precursor molecules. The deduced preprohormones both have molecular weights (MWs) of 20,100 and contain C-terminal propeptides of 89 amino acid residues, which we have named E-peptides. The organization of the IGF-II precursor gene is discussed in relation to that of other insulin gene family members.

Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells.

The complete 1,210-amino acid sequence of the human epidermal growth factor (EGF) receptor precursor, deduced from cDNA clones derived from placental and A431 carcinoma cells, reveals close similarity between the entire predicted v-erb-B mRNA oncogene product and the receptor transmembrane and cytoplasmic domains. A single transmembrane region of 23 amino acids separates the extracellular EGF binding and cytoplasmic domains. The receptor gene is amplified and apparently rearranged in A431 cells, generating a truncated 2.8-kilobase mRNA which encodes only the extracellular EGF binding domain.

The amino acid sequence of murine p53 determined from a c-DNA clone.

A c-DNA clone containing the complete sequence information for the murine p53 protein, from embryonal carcinoma cells, has been isolated. The nucleotide sequence of this clone reveals an open reading frame encoding a protein of 390 amino acids with a molecular weight of 43,364 Da. The NH2-terminal domain of this protein is acidic whereas the carboxyl terminus is rich in basic amino acid residues. These terminal domains are separated by a proline-rich, hydrophobic run of amino acids. Proline comprises approximately 10% of the total amino acid residues. Two tryptic peptides, derived from p53 protein radiolabeled with either methionine or proline, were purified and the position of these labeled residues in the peptide was determined. The positions of three methionine and five proline residues in these two peptides matched the amino acid sequence of the predicted open reading frame determined from the c-DNA clone.

Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations.

Kirsten (Ki)-ras cDNA clones were prepared from human lung and colon carcinoma cell lines expressing an activated c-Ki-ras2 gene. DNA sequence analysis and transfection studies indicate that different point mutations at the same codon can activate the gene; that most human c-Ki-ras2 mRNA uses sequences from a fourth coding exon distinct from that of its viral counterpart; and that at least one cell line is functionally homozygous for the activated gene.