concanavalin-a and monodansylcadaverine

We have reported that the platelet-derived growth factor receptor-beta (PDGFbeta) forms a novel signaling complex with G protein-coupled receptors (GPCR) (e.g. S1P(1) receptor) that enables more efficient activation of p42/p44 mitogen-activated protein kinase (MAPK) in response to PDGF and sphingosine 1-phosphate (S1P). We now demonstrate that c-Src participates in regulating the endocytosis of PDGFbeta receptor-GPCR complexes in response to PDGF. This leads to association of cytoplasmic p42/p44 MAPK with the receptor complex in endocytic vesicles. c-Src is regulated by G protein betagamma subunits and can interact with beta-arrestin. Indeed, the PDGF-dependent activation of p42/p44 MAPK was reduced by over-expression of the C-terminal domain of GRK2 (sequesters Gbetagamma subunits), the clathrin-binding domain of beta-arrestin and by inhibitors of c-Src and clathrin-mediated endocytosis. Moreover, PDGF and S1P induce the recruitment of c-Src to the PDGFbeta receptor-S1P(1) receptor complex. This leads to a G protein/c-Src-dependent tyrosine phosphorylation of Gab1 and accumulation of dynamin II at the plasma membrane, a step required for endocytosis of the PDGFbeta receptor-GPCR complex. These findings provide important information concerning the molecular organisation of novel receptor tyrosine kinase (RTK)-GPCR signal relays in mammalian cells.

Topics: Adaptor Proteins, Signal Transducing; Animals; Arrestins; beta-Adrenergic Receptor Kinases; beta-Arrestins; Cadaverine; Cell Line; Cells, Cultured; Concanavalin A; Cyclic AMP-Dependent Protein Kinases; Dynamin II; Endocytosis; Enzyme Inhibitors; GRB2 Adaptor Protein; Guinea Pigs; Humans; Immunoprecipitation; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Smooth Muscle; Pertussis Toxin; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Platelet-Derived Growth Factor; Proto-Oncogene Proteins pp60(c-src); Pyrimidines; Receptor, Platelet-Derived Growth Factor beta; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Transfection; Transport Vesicles

Two isoforms of the dopamine D2 receptor, D2L (long) and D2S (short), differ by the insertion of a 29-amino acid specific to D2L within the putative third intracellular loop of the receptor. Here, we examined D2 receptor-mediated MAPK activation in association with receptor internalization. Overexpression of beta-arrestin 1 and 2 increased the D2S-mediated activation of MAPK, whereas it did not affect the activation of MAPK by D2L. Expression of a dominant negative beta-arrestin 2 (319-418) mutant and of a dominant negative dynamin I (K44A) mutant inhibited the activation of MAPK by D2S, but not the activation of MAPK by D2L. Treatment with inhibitors of internalization, i.e. concanavalin A and monodansylcadaverin, blocked D2S-mediated MAPK activation but not D2L-mediated activation. By confocal microscopy, we observed beta-arrestin 1 and 2, translocated to the plasma membrane and colocalized with D2L and D2S receptors upon stimulation with dopamine, and this was followed by the translocation of receptors into endocytic vesicles. Moreover, the expression of the beta-arrestin 2 (319-418) mutant blocked the internalization of both D2L and D2S. In addition, although K44A dynamin mutant expression did not alter D2L internalization, it completely blocked the internalization of D2S. The stimulation of D2L induces activation of MAPK via transactivation of the platelet-derived growth factor receptor, whereas D2S does not. Taken together, these data suggest that D2L activates MAPK signaling by mobilizing the growth factor receptor, platelet-derived growth factor receptor, whereas D2S appears to activate MAPK signaling by mobilizing clathrin-mediated endocytosis in a beta-arrestin/dynamin-dependent manner.

Topics: Animals; Arrestins; beta-Arrestin 1; beta-Arrestin 2; beta-Arrestins; Cadaverine; Cells, Cultured; Concanavalin A; Cricetinae; Dopamine; Dynamin I; Enzyme Activation; Enzyme Inhibitors; Genes, Dominant; Green Fluorescent Proteins; Humans; Kidney; Lectins; Luminescent Proteins; Microscopy, Confocal; Mitogen-Activated Protein Kinases; Mutation; Protein Isoforms; Pyrimidines; Receptors, Dopamine D2; Receptors, Platelet-Derived Growth Factor; Red Fluorescent Protein; src-Family Kinases

The existence of an invasive adenylate cyclase in dialyzed urea extracts of the bacterium Bordetella pertussis has been suggested recently. Gel filtration of B. pertussis dialyzed urea extract shows that the invasive enzyme constitutes only a small portion of the total adenylate cyclase activity found in the extract. Its size is different than the size of the two peaks of adenylate cyclase activity identified in the extract. Ca2+ is absolutely required for the penetration of the invasive enzyme, it also controls the rate of intracellular cAMP accumulation in human lymphocytes exposed to dialyzed extract. These characteristics may be attributed to the increase in the size of the invasive enzyme as found by gel filtration chromatography of the extract in the absence of Ca2+. Removal of nonpenetrating adenylate cyclase that adheres to lymphocytes permits a direct assay of the intracellular enzyme. The time course of intracellular accumulation of adenylate cyclase activity is similar to the time course of intracellular accumulation of cAMP, suggesting that the invasive enzyme is rapidly deactivated, but not degraded, since it can be detected upon cell disruption. No appreciable amount of the enzyme is introduced when cells are incubated with extract at 4 degrees C for 120 min, then washed and incubated further at 37 degrees C. Concanavalin A inhibits cAMP accumulation irrespective of the time of its addition, and EGTA prevents penetration of the invasive enzyme even if added 20 min after addition of extract. These findings are different from those observed in other bacterial toxins thought to be internalized by receptor-mediated endocytosis. However, the cellular penetration of B. pertussis adenylate cyclase is cell-selective. It does not occur in human erythrocytes. In addition to human lymphocytes, S49 cyc- murine lymphoma, turkey erythrocytes, and rat oocytes accumulate cAMP in response to B. pertussis extract.

Topics: Adenylyl Cyclases; Bordetella pertussis; Cadaverine; Calcium; Chromatography, Gel; Concanavalin A; Cyclic AMP; Disease Susceptibility; Dose-Response Relationship, Drug; Egtazic Acid; Humans; In Vitro Techniques; Kinetics; Lymphocytes; Time Factors

Transglutaminase activity in human peripheral lymphocytes is enhanced after incubation of the cells with concanavalin A. Streptococcal proliferative factor toxin (erythrogenic toxin) from Streptococcus pyogenes and Toxic shock syndrome toxin from Staphylococcus aureus were purified and tested for their ability to enhance transglutaminase activity. Mononuclear leukocyte transglutaminase activity was enhanced 3-5-fold 30 min after incubation with either toxin. Enhancement occurred only when toxin was incubated with intact cells; addition of toxin to cell lysates was without effect. Transglutaminase was not measurable extracellularly. Histamine and dansyl cadaverine, competitive substrates for transglutaminase, inhibited [3H]putrescine incorporation into casein and [3H]thymidine incorporation into DNA. Incubation of lymphocytes with cycloheximide and either toxin or concanavalin A did not inhibit enzyme activity. These bacterial toxins, like phytomitogens, may perturb the cellular membrane and mediate their effect by transglutaminase-mediated cross-linking of membrane proteins.

Topics: Acyltransferases; Bacterial Proteins; Bacterial Toxins; Cadaverine; Concanavalin A; Cycloheximide; Enterotoxins; Exotoxins; Histamine; Humans; Membrane Proteins; Monocytes; Putrescine; Superantigens; Thymidine; Transglutaminases

The muscarinic agonist, cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide (CD), and histamine desensitize the responses of guinea-pig ileal longitudinal smooth muscle to subsequently administered CD or histamine, respectively. Desensitization by CD, but not by histamine, was inhibited by concanavalin A (Con A) and this protection was prevented by co-administration of succinyl-Con A or by cytochalasin B or colchicine at concentrations which alone did not affect desensitization. Dansylcadaverine and bacitracin, inhibitors of transglutaminase, both protected against CD desensitization but were without effect on desensitization mediated by histamine. Benzilylcholine mustard, which reduced muscarinic receptor density, significantly reduced CD-induced desensitization. The effects of Con A and the transglutaminase inhibitors suggest that agonist-induced changes in muscarinic receptor distribution or receptor-effector coupling, via cytoskeletal and transglutaminase-sensitive mechanisms, are a component of CD-induced desensitization. These processes do not appear to be involved in histamine receptor-mediated desensitization. Although desensitization by CD or histamine is nonspecific in this preparation, both agonists cross-desensitizing, the effects of Con A, dansylcadaverine and bacitracin clearly indicate differences in the desensitization process elicited by these two stimulants.

Topics: Animals; Bacitracin; Cadaverine; Concanavalin A; Diamines; Guinea Pigs; Ileum; Kinetics; Muscle, Smooth; Receptors, Cholinergic; Receptors, Muscarinic

Topics: Animals; Bacitracin; Cadaverine; Chloroquine; Chorionic Gonadotropin; Concanavalin A; Corpus Luteum; Female; gamma-Glutamyltransferase; Kinetics; Methylamines; Progesterone; Receptors, Cell Surface; Receptors, LH; Sheep