Dominant effector genetics in mammalian cells.

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.

The control of microvascular permeability and blood pressure by neutral endopeptidase.

Plasma extravasation from postcapillary venules is one of the earliest steps of inflammation. Substance P (SP) and bradykinin (BK) mediate extravasation and cause hypotension. The cell-surface enzyme neutral endopeptidase (NEP) inactivates both peptides. Thus, absence of NEP may predispose development of inflammation and hypotension. We examined these possibilities in mice in which the NEP gene was deleted by homologous recombination. There was widespread basal plasma extravasation in postcapillary venular endothelia in NEP-/- mice, which was reversed by recombinant NEP and antagonists of SP (NK1) and BK (B2) receptors. Mean arterial blood pressure was 20% lower in NEP-/- animals, but this was unaffected by reintroduction of recombinant NEP and the kinin receptor antagonists. The hypotension was also independent of nitric oxide (NO), because NEP-/- mice treated with a NO synthase inhibitor remained hypotensive relative to the wild type. Thus, NEP has important roles in regulating basal microvascular permeability by degrading SP and BK, and may regulate blood pressure set point through a mechanism that is independent of SP, BK and NO. The use of NEP antagonists as candidate drugs in cardiovascular disease is suggested by the blood pressure data reported herein.

Modulation of human lymphocyte function by C3a and C3a(70-77).

Human C3a and the synthetic octapeptide C3a (70-77), which retains the activities of an anaphylatoxin, inhibit in a concentration-dependent manner the generation of leukocyte inhibitory factor (LIF) activity by human mononuclear leukocytes and T lymphocytes cultured with the mitogens phytohemagglutinin (PHA) or concanavalin A (Con A) or the antigen streptokinase-streptodornase (SK-SD). The generation of LIF activity was inhibited by 50% by 10(-8) M C3a or C3a(70-77) with PHA or Con A as the stimulus, whereas a more than 10-fold higher concentration of C3a(70-77) than C3a was required to achieve the same level of suppression with SK-SD as the stimulus. Similar concentrations of C3a(70-77) inhibited to the same extent the migration of T lymphocytes stimulated by alpha-thioglycerol of Con A. Neither C3a nor C3a(70-77) altered significantly the uptake of [3H]thymidine by human mononuclear cells exposed to PHA, Con A, or SK-SD. The capacity of C3a(70-77)-Sepharose,m but not Sepharose alone, to adsorb or inactivate mononuclear leukocytes required for the generation of LIF activity established a direct interaction. Analysis of the lymphocytes in the effluent from C3a(70-77)-Sepharose columns, using monoclonal antibodies to surface antigens, showed a selective depletion of the helper/inducer population of lymphocytes. C3a might represent an important mediator of the functionally selective regulation of human T lymphocyte activities by the complement system.

Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

Structure and expression of a rat agrin.

Agrin is a component of the basal lamina that causes the aggregation of acetylcholine receptors on cultured muscle fibers. An agrin cDNA clone isolated from electromotor neurons of a marine ray was used to characterize the corresponding cDNAs from a rat embryonic spinal cord library. Analysis of a set of clones predicts a 1940 amino acid protein containing 141 cysteine residues. The predicted protein has nine domains homologous to protease inhibitors, a region similar to domain III of laminin, and four epidermal growth factor repeats. The agrin gene is expressed in rat embryonic nervous system and muscle. The rat agrin protein is concentrated at synapses, where it may play a role in development and regeneration.

Identification of RIP3, a RIP-like kinase that activates apoptosis and NFkappaB.

The tumor necrosis factor receptor 1 (TNFR1) and the Fas receptor recruit complexes formed by the interactions between RIP kinase, TRADD, FADD and RAIDD - adaptor proteins that contain death domains - which in turn recruit other proteins to initiate signaling [1][2][3][4][5]. To identify proteins associated with the TNF signaling pathway, we performed a yeast two-hybrid interaction screen using RIP as bait. We isolated a kinase, RIP3, which shares homology with the kinase domain of RIP and RIP2 (also known as Rick or CARDIAK). RIP3 could be co-immunoprecipitated with RIP, TRAF2 and TNFR1 in mammalian cells. The carboxy-terminal domain of RIP3, like that of RIP, could activate the transcription factor NFkappaB and induce apoptosis when expressed in mammalian cells. Interestingly, this region shares no significant sequence homology to the death domain of RIP, the caspase-recruiting domain (CARD) of RIP2 [6][7][8] or any other apoptosis-inducing domain. As with RIP and RIP2, the kinase domain of RIP3 was not required for either NFkappaB activation or apoptosis induction. Overexpression of a dominant-negative mutant of RIP3 strongly inhibited the caspase activation but not the NFkappaB activation induced by TNFalpha. Therefore, RIP3 appears to function as an intermediary in TNFalpha-induced apoptosis.

Measles virus-substance P receptor interactions. Possible novel mechanism of viral fusion.

Measles virus (MV) encodes the fusion protein (F) that mediates cell fusion and intercellular spread of the virus, and is homologous to the carboxy terminus of the neuropeptide substance P (SP). In addition, the oligopeptide Z-D-Phe-L-Phe-Gly, also homologous to F and SP, inhibits MV fusion with target cells. These observations raise the question of whether MV uses the SP receptor (SPR) during a specific phase of its infectious cycle. In this report, we examine the structural and functional consequences of this interaction and show, using cross-linking studies, that MV and SP specifically bind to a 52-58-kD protein, previously reported to comprise the SPR on human IM-9 lymphoblasts. Moreover, bound MV and SP are shown to reciprocally displace each other from these cells. In addition, we demonstrate that anti-SP antisera inhibits the cell-to-cell spread of MV, and that SP blocks MV fusion with target cells. These results indicate the presence of MV-SPR interactions during viral fusion, and suggest possible novel mechanisms for viral entry into cells.

Substance P recognition by a subset of human T lymphocytes.

The interaction of substance P with human blood T-lymphocytes, which stimulates T-lymphocyte proliferation, was quantified by both flow cytometric and direct binding assays. Fluorescence-detection flow cytometry recorded the binding of dichlorotriazinylamino-fluorescein-labeled substance P to 21 +/- 10% (mean +/- SD, n = 6) and 35 +/- 8% (n = 2) of human blood T-lymphocytes before and after stimulation with 10 micrograms/ml of phytohemagglutinin, respectively. The suppressor-cytotoxic (leu 2a) and helper-inducer (leu 3a) subsets identified by phycoerythrin-labeled monoclonal antibodies contained substance P-reactive T-lymphocytes at respective mean frequencies of 10 and 18%. [3H]substance P bound rapidly and reversibly to a mean of 7035 +/- 2850 sites/T-lymphocyte, which exhibited a dissociation constant (KD) of 1.85 +/- 0.70 X 10(-7) M (mean +/- SD, n = 5). [D-Pro2,D-Phe7,D-Trp9]substance P inhibited the binding of dichlorotriazinylamino-fluorescein-labeled substance P and [3H]substance P to T-lymphocytes at concentrations that suppressed the proliferative response to substance P. Substance P, eledoisin, and substance K (alpha-neurokinin), which all share with substance P the carboxy-terminal substituent -Gly-Leu-Met-NH2, were more potent than substance P in inhibiting the binding of [3H]substance P to T-lymphocytes, suggesting the importance of this sequence in the interaction. Purified human blood B-lymphocytes, monocytes, polymorphonuclear leukocytes, and platelets, and cultured Hut 78 cutaneous lymphoma T-cells, Jurkat cells, Molt-4 lymphoblasts, and HL-60 and U-937 monocyte-like cells all showed only minimal specific binding of [3H]substance P. The recognition of substance P by T-lymphocytes provides one mechanism for selective modulation of immunity by sensory nerves.

Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2.

Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin-like enzymes. PAR-2 is highly expressed by small intestinal enterocytes where it is activated by luminal trypsin. The location, mechanism of activation, and biological functions of PAR-2 in the colon, however, are unknown. We localized PAR-2 to the muscularis externa of the rat colon by immunofluorescence. Myocytes in primary culture also expressed PAR-2, assessed by immunofluorescence and RT-PCR. Trypsin, SLIGRL-NH2 (corresponding to the PAR-2 tethered ligand), mast cell tryptase, and a filtrate of degranulated mast cells stimulated a prompt increase in [Ca2+]i in myocytes. The response to tryptase and the mast cell filtrate was inhibited by the tryptase inhibitor BABIM, and abolished by desensitization of PAR-2 with trypsin. PAR-2 activation inhibited the amplitude of rhythmic contractions of strips of rat colon. This response was unaffected by indomethacin, l-NG-nitroarginine methyl ester, a bradykinin B2 receptor antagonist and tetrodotoxin. Thus, PAR-2 is highly expressed by colonic myocytes where it may be cleaved and activated by mast cell tryptase. This may contribute to motility disturbances of the colon during conditions associated with mast cell degranulation.

Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor.

Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.

Potentiation of mitogen-induced human T-lymphocyte activation by retinoic acid.

The capacity of retinoic acid to modulate human T-lymphocyte and B-lymphocyte activation by mitogens was examined. T-lymphocyte proliferation stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) or phytohemagglutinin was enhanced by 5 nM to 5 microM retinoic acid in a dose-dependent manner with a 65 +/- 35% (SD) increase (n = 6, P less than 0.01) in TPA-stimulated proliferation induced by 5 microM retinoic acid. Retinoic acid enhanced T-lymphocyte proliferation over a wide range of background proliferation induced by different TPA concentrations. Retinoic acid alone did not stimulate T-lymphocyte proliferation. In contrast retinoic acid inhibited B-lymphocyte proliferation stimulated by TPA or phytohemagglutinin with 26.7 +/- 23.4% inhibition of TPA-stimulated proliferation induced by 5 microM retinoic acid (P less than 0.02). Retinoic acid had intermediate effects on the proliferation of different mixtures of T- and B-lymphocytes stimulated by TPA or phytohemagglutinin. The recognition that retinoic acid has opposing effects on human T- and B-lymphocyte activation by mitogens may account for the conflicting reports of the effects of retinoids on the immune response of unpurified human lymphocyte preparations.

p15(PAF), a novel PCNA associated factor with increased expression in tumor tissues.

Proliferating cell nuclear antigen (PCNA) is an essential protein in both DNA replication and DNA damage repair. A novel 15 kD protein, p15(PAF), was identified as a PCNA-associated factor in a yeast two-hybrid screen using PCNA as the bait. p15(PAF) is localized primarily in the nucleus. p15(PAF) shares the conserved PCNA binding motif with several other PCNA binding proteins including CDK inhibitor p21. Overexpression of p15(PAF) competes with p21-PCNA binding. Mutation of this motif in p15(PAF) abolished its PCNA-binding activity. Notably, p15(PAF) expression in several types of tumor tissues was significantly increased, especially in esophageal tumors. Like PCNA, p15(PAF) may possess prognostic significance in a broad array of human cancers.

A novel artificial loop scaffold for the noncovalent constraint of peptides.

BACKGROUND: Few examples exist of peptides of < 35 residues that form a stable tertiary structure without disulfide bonds. A method for stabilization and noncovalent constraint of relatively short peptides may allow the construction and use of intracellular peptide libraries containing protein minidomains. RESULTS: We have examined a novel method for the noncovalent constraint of peptides by attaching the peptide EFLIVKS (single-letter amino acid code), which forms dimers, to the amino and carboxyl termini of different peptide inserts. An 18 residue random coil taken from the inhibitor loop of barley chymotrypsin inhibitor 2 was inserted between the peptides to produce a 32-mer minidomain that is attacked only slowly by elastase, has numerous slowly exchanging protons, contains a high beta-structure content and has a T(m) above 37 degrees C. A point mutation disrupting the hydrophobic interior in both dimerizing peptides causes a loss of all slowly exchanging protons and of secondary structure. Adding specific charged residues to each terminus substantially increased the T(m), as did point mutants designed to add interdimerizer ion pairs. Three flexible epitope tag inserts and a nonamer insert do not appear to be folded in a stable structure by EFLIVKS. The properties of two peptides selected for expression in HeLa cells suggest they do form a stable tertiary structure. CONCLUSIONS: Attaching short dimerizing peptides to both the amino and carboxyl termini of several 18-mer peptides appears to create stable monomeric tertiary structures. Mutations in the dimerizers can either destabilize or significantly stabilize a standard 18-mer insert. Dimerizing peptides flanking random insert sequences could be used as a strategy to generate heterogeneous peptide libraries with both extended and folded members.

Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells.

BACKGROUND: Mammalian cell screens of peptide libraries for changes in cellular phenotype may identify novel functional peptides and their cognate binding partners, and allow identification of signal transduction network members or proteins important in disease processes. RESULTS: Green fluorescent protein (GFP) peptide libraries with different structural biases were tested by retroviral expression in A549 carcinoma cells, HUVEC and other cell types. Three different loop replacement libraries, containing 12 or 18 random residues, were compatible with enhanced GFP (EGFP) folding, as was a C-terminally fused random 20-mer library. Library concentrations in A549 cells ranged from ca. 1 to 54 microM. Replacement of loop 3 with known nuclear localization sequence (NLS) peptides, but not with inactive mutants, directed EGFP to the nucleus. Microscopy-based screens of three different libraries for non-uniform localization revealed novel NLS peptides, novel variants of a peroxisomal localization motif, a variety of partial NLS peptides, peptides localized to the nucleolus, and nuclear-excluded peptides. CONCLUSIONS: Peptides can be presented by EGFP in conformations that can functionally interact with cellular constituents in mammalian cells. A phenotypic screen resulting in the discovery of novel localization peptides that were not cell type-specific suggests that this methodology may be applied to other screens in cells derived from diseased organisms, and illustrates the use of intracellular combinatorial peptide chemistry in mammalian cells.

The use of retroviruses as pharmaceutical tools for target discovery and validation in the field of functional genomics.

Retrovirally mediated functional genomics enables identification of physiologically relevant cellular therapeutic targets. Unique properties of retroviruses make them ideal tools for the introduction of large and diverse libraries of potential genetic effectors to a variety of cell types. The identification and recovery of intracellular library elements responsible for altered disease responses establishes a direct basis for pharmaceutical development. Recent innovations in retroviral infection efficiency and expression control have broadened application of the methodology to include libraries of mutagenized cDNAs, peptides and ribozyme genetic effectors.

Functional diversity of histamine and histamine receptors.

In order to analyze the mechanisms by which a single biogenic amine like histamine is capable of inducing a wide variety of both physiologic and pathologic functions in various tissues/cells, histamine responses were dissected in detail from a biochemical and pharmacologic point of view. Histamine is synthesized by multiple isozymes of histidine decarboxylase, and catabolized by either diamine oxidase or histamine-N-methyltransferase. Synthesized intracellular histamine may play a role in cell proliferation, whereas released histamine binds to at least three different histamine-specific receptors, then activates various intracellular components, such as Ca++, cAMP, protein kinase, and ion channels. These second messenger pathways interact differentially with each other in various tissues/cells. Moreover, histamine not only activates its own receptors, but also activates other related receptors such as the serotonin 1c receptor. Therefore, to understand the complex actions of histamine, new approaches should be established, in which multiple phenomena can be monitored simultaneously.

Rab37 is a novel mast cell specific GTPase localized to secretory granules.

GTPases regulate a myriad of cellular functions including signal transduction, cytoskeletal organization and membrane trafficking. Rab GTPases act to coordinate the membrane dynamics of cells by organizing and regulating the activity of effector proteins important in vesicle trafficking. Rab37 is a novel Rab GTPase specifically expressed in the MC-9 mast cell line and bone marrow mast cells. Rab37 is 74% identical to Rab26 and 47% identical to Rab8, a GTPase important in Golgi to plasma membrane vesicle trafficking in mammalian cells. When green fluorescent protein tagged Rab37 is expressed in bone marrow mast cells, the secretory granules are labeled. These data suggest that Rab37 may play an important role in mast cell degranulation making this protein a potentially important target for therapeutic intervention in the treatment of allergy.

Neuro-endocrine interaction on lymphocytes. Testosterone-induced modulation of the lymphocyte substance P receptor.

Human IM-9 B-lymphoblasts have been shown to express the receptor for the neuropeptide substance P (SP). The present study was undertaken to evaluate the effect of sex hormones on this receptor. Testosterone inhibited [125I]SP binding in a dose-dependent manner with an IC50 of approximately 100 nM, while both estradiol and progesterone failed to inhibit SP binding even at concentrations as high as 1 microM. Furthermore, Scatchard analysis indicated that the dissociation constant (Kd) of the SP receptor was markedly increased from 0.25 nM to 2.2 nM when cells were incubated with testosterone. These data indicate a unique neuro-endocrine interaction on lymphocytes involving the substance P receptor.

Endocytosis and recycling of neurokinin 1 receptors in enteric neurons.

Neurotransmission depends on the availability of transmitter and on the presence of functional, high-affinity receptors at the plasma membrane that are capable of binding ligand. The pathway, mechanism and function of endocytosis and recycling of the substance P or neurokinin 1 receptor in enteric neurons were studied using fluorescent substance P, receptor antibodies and confocal microscopy. In both the soma and neurites, substance P induced rapid, clathrin-mediated internalization of the neurokinin 1 receptor into early endosomes, which also contained the transferrin receptor. After 4-8 h, there was a return in surface neurokinin 1 receptor immunoreactivity in the soma, which was not prevented by cycloheximide, and was thus independent of new protein synthesis. This return was prevented by acidotropic agents, therefore required endosomal acidification. This suggests that the neurokinin 1 receptor recycles in the soma. In contrast, in neurites, substance P and the neurokinin 1 receptor remained in endosomes and recycling was not detected. Neurons of the myenteric plexus were heavily innervated by substance P-containing nerve fibers, and K(+)-stimulated release of endogenous substance P from cultured neurons induced internalization of the neurokinin 1-receptor. Therefore, endogenous substance P may induce endocytosis of the neurokinin 1 receptor. In the soma, endocytosis and recycling correlated with loss and recovery of functional binding sites for substance P. suggesting that this process contributes to the regulation of peptidergic neurotransmission. Thus, ligand-induced endocytosis of the neurokinin 1 receptor in myenteric neurons is associated with a loss of surface receptors and functional binding sites. Since release of endogenous substance P induces neurokinin 1 receptor internalization, and neurokinin 1 receptor neurons are innervated by substance P-containing fibers, endocytosis of neuropeptide receptors may regulate neurotransmission.

The use of 33P-labeled oligonucleotides for in situ hybridization of vertebrate embryo frozen sections.

In situ hybridization was performed on frozen sections of whole E16 rat embryos or whole E7 chick embryos using oligonucleotide probes tagged with a 33P-poly A tail. We used two antisense oligonucleotide probes: a 57-mer rat agrin oligo and a 60-mer chicken B-cadherin oligo. After exposure to Hyperfilm beta max for three days, mRNA was detected in distinct organs and tissues of the embryo. After dipping the slides in liquid emulsion and exposing them for two weeks, the grains could be localized to a specific cellular layer, for example, the ganglion layer of the retina or the luminal epithelium of the gizzard. It is concluded that 33P autoradiography is excellent for defining gross areas or cellular layers of mRNA in a vertebrate embryo with a resolution of about 20 microns.