guanosine-5--o-(3-thiotriphosphate) and Leukemia--Erythroblastic--Acute

The nonhydrolyzable GTP analogue GTP-gamma-S was capable of stimulating in vitro phosphorylation of polyphosphoinositides in isolated nuclei prepared from mouse erythroleukemia cells. On the contrary, GDP-beta-S was ineffective. The stimulation was not detectable when nuclei were prepared from erythroleukemia cells induced to differentiate by exposure to dimethyl sulfoxide. Both nuclear phosphomonoesterase and phospholipase C activities were not influenced by GTP-gamma-S. Our results point to the likelihood that nuclear phosphoinositide kinases might be regulated by a GTP-binding protein.

Topics: Animals; Cell Differentiation; Cell Line; Cell Nucleus; Dimethyl Sulfoxide; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Homeostasis; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Phosphatidylinositol Phosphates; Phosphoric Monoester Hydrolases; Thionucleotides; Tumor Cells, Cultured; Type C Phospholipases

Thromboxane A2 (TXA2) induces activation of platelets and vascular smooth muscle contraction via cell surface receptors. A platelet type TXA2 receptor from the megakaryocyte-like HEL cell was cloned with a deduced amino acid sequenced identical to that previously reported for the human placental TXA2 receptor. Transient expression of the HEL cell TXA2 receptor cDNA and radioligand binding studies with the agonist 125I-BOP showed a single class of binding sites with an affinity comparable to a low affinity platelet TXA2 receptor. Using a series of 13-azapinane TXA2 analogs, which discriminate between TXA2 receptor subtypes in platelets and vascular smooth muscle, we found that the cloned HEL cell TXA2 receptor is characteristic of a platelet type TXA2 receptor and that its binding characteristics are different from those of vascular smooth muscle cells. The affinity of the HEL cell TXA2 receptor for 125I-BOP was significantly (P < .05) increased upon co-transfection with G alpha 13 alone, or with G alpha q alone and with G alpha 13 and G alpha 12 together (n = 4-6). GTP gamma S significantly (P < .05) decreased the affinity of the receptor for 125I-BOP in COS-7 cell membranes coexpressing HEL-TXR and G alpha 13 to a value comparable to HEL-TXA2 receptor alone. We conclude that 1) the cloned HEL cell TXA2 receptor has pharmacological characteristics of a low affinity platelet type receptor and 2) that the affinity state of this receptor may be influenced by interaction with G alpha 13 and G alpha q.

Topics: Base Sequence; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Fatty Acids, Unsaturated; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Polymerase Chain Reaction; Receptors, Thromboxane; Transfection; Tumor Cells, Cultured

The post-receptor events which follow the binding of interleukin 1 (IL1) to cells are unclear. The present studies provide evidence for the activation of a guanine nucleotide binding protein (G protein) by IL1 in the membranes of an IL1 receptor-rich strain (NOB-1) of the EL4 murine thymoma line. IL1 alpha and beta increased the binding of the GTP analogue [35S]guanosine 5'-[gamma-thiol]trisphosphate (GTP gamma S) to membranes prepared from these cells. By 1 min after addition of IL1 there was a 2-fold enhancement in binding which was dose dependent in the range 0.1-100 ng/ml. A qualitatively similar result was obtained with IL1 beta although it was 10 times less potent. Specific neutralizing antisera to IL1 alpha and IL1 beta abolished the response. Experiments in which the concentration of [35S]GTP gamma S was varied revealed that IL1 increased the affinity of the binding sites for [35S]GTP gamma S and not their number. IL1 alpha was shown to stimulate GTPase activity in the membranes, the time and concentration dependence of this was similar to that observed for increased [35S]GTP gamma S binding. Half-maximal enhancement of [35S]GTP gamma S binding by IL1 alpha, measured after 4 min, occurred at 5% IL1 receptor occupancy. Maximal stimulation was achieved when 30% of receptors were occupied. Experiments with pertussis and cholera toxins revealed that pretreating membranes with pertussis toxin (100 ng/ml) inhibited by 50% the IL1-induced [35S]GTP gamma S binding and [gamma-32P]GTP hydrolysis. Cholera toxin (100 ng/ml) was without effect. However, both pertussis and cholera toxins at concentrations of 100 ng/ml inhibited IL1-induced IL2 secretion in EL4 NOB-1 cells. These results show that the IL1 receptor of a responsive thymoma line activates, and may be coupled to, a G protein(s). This is a possible mechanism of IL1 signal transduction.

Topics: Alprostadil; Animals; Cell Line; Cell Membrane; Cholera Toxin; Epinephrine; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hydrolysis; Immune Sera; Interleukin-1; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Pertussis Toxin; Phosphoric Monoester Hydrolases; Receptors, Immunologic; Receptors, Interleukin-1; Recombinant Proteins; Signal Transduction; Thionucleotides; Virulence Factors, Bordetella