gtp-gamma-4-azidoanilide and Glioma

While the cytoskeleton is known to play several roles in the biology of the cell, one role, which has been revealed only recently, is that of a participant in the signal transduction process. Tubulin binds specifically to the alpha subunits of Gs (stimulatory GTP-binding regulatory protein of adenylyl cyclase), Gi1 (inhibitory protein of adenylyl cyclase), and Gq and transactivates those molecules through direct transfer of GTP. The relevance of this transactivation process to G proteins which are normally activated by a neurotransmitter-occupied receptor is the subject of this study. C6 glioma cells, made permeable with saponin, retained tight coupling between Gs and the beta-adrenergic receptor. Although 5-guanylylimidodiphosphate (GppNHp) was incapable of activating Gs (and subsequently, adenylyl cyclase) in the absence of agonist, tubulin with GppNHp bound (tubulin-GppNHp) activated adenylyl cyclase with an EC(50) of 30 nM. Desensitization of beta-adrenergic receptors by isoproterenol exposure had no effect on the ability of tubulin-GppNHp to activate Gs and adenylyl cyclase. When the photoaffinity GTP analog, azidoanilido GTP (AAGTP; P3(4-azidoanilido)-P1-5'-GTP), was added to C6 membranes or permeable C6 cells, it was only weakly incorporated by G alpha s in the absence of isoproterenol. When the same concentration of dimeric tubulin with AAGTP bound was introduced, AAGTP was transferred from tubulin to G alpha s, activating the latter species. Similar 'preferential' activation of G alpha s by tubulin-AAGTP versus the free nucleotide was seen using purified components. Thus, membrane-associated tubulin may serve to activate G alpha s, independent of signals not normally coupled to that protein. Tubulin may act as an agent to link a variety of membrane-associated signalling systems.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Animals; Azides; Cell Membrane; Cell Membrane Permeability; Enzyme Activation; Glioma; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Precipitin Tests; Rats; Receptors, Adrenergic, beta; Saponins; Signal Transduction; Tubulin; Tumor Cells, Cultured

The G proteins G S and Gi1 appear to be capable of binding to tubulin specifically, and it has been suggested that such binding results in G protein activation via direct transfer of GTP. This study was undertaken to demonstrate that consequences of G protein activation by tubulin, i.e., stimulation or inhibition of adenyl cyclase, were dependent on the G proteins expressed as well as unique aspects of the membrane or cytoskeleton in a given cell type. Membranes from rat C6 glioma cells, which express G s alpha but not G i alpha 1, responded to the addition of tubulin with a stable activation of adenyl cyclase. Conversely, membranes from rat cerebral cortex, which contain both G s and G i 1, responded to exogenous tubulin with a stable inhibition of adenyl cyclase. Unlike C6 membranes, cerebral cortex membranes are richly endowed with tubulin, and antitubulin antibodies immunoprecipitated complexes of tubulin and G i 1 and G s from detergent extracts of these membranes. Nearly 90% of the G s alpha from Triton X-114 extracts coimmunoprecipitated with tubulin, suggesting that these proteins exist as a complex in the synaptic membrane. Such complexes may provide the framework for a G protein-cytoskeleton link that participates in the modulation of cellular signal transduction.

Topics: Adenylyl Cyclase Inhibitors; Affinity Labels; Animals; Azides; Cerebral Cortex; Chickens; Glioma; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Rats; Sheep; Synaptic Membranes; Synaptosomes; Tubulin; Tumor Cells, Cultured

Topics: Affinity Labels; Animals; Autoradiography; Azides; Electrophoresis, Polyacrylamide Gel; Glioma; GTP-Binding Proteins; Guanosine Triphosphate; Indicators and Reagents; Macromolecular Substances; Molecular Structure; Phosphorus Radioisotopes; Recombinant Proteins; Synaptic Membranes; Tubulin; Tumor Cells, Cultured

Topics: Adenylyl Cyclases; Animals; Azides; Calmodulin; Cerebral Cortex; Cytoskeleton; Enzyme Activation; Glioma; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Microtubules; Neurotransmitter Agents; Rats; Signal Transduction; Tubulin