The cytoskeletal protein, tubulin, has been shown to regulate adenylyl cyclase activity through its interaction with the specific G protein ␣ subunits, G␣ s or G␣ i1 . Tubulin activates these G proteins by transferring GTP and stabilizing the active nucleotide-bound G␣ conformation. To study the possibility of tubulin involvement in G␣ q -mediated phospholipase C 1 (PLC 1 ) signaling, the m 1 muscarinic receptor, G␣ q , and PLC 1 were expressed in Sf9 cells. A unique ability of tubulin to regulate PLC 1 was observed. Low concentrations of tubulin, with guanine nucleotide bound, activated PLC 1 , whereas higher concentrations inhibited the enzyme. Interaction of tubulin with both G␣ q and PLC 1 , accompanied by guanine nucleotide transfer from tubulin to G␣ q , is suggested as a mechanism for the enzyme activation. The PLC 1 substrate, phosphatidylinositol 4,5-bisphosphate, bound to tubulin and prevented microtubule assembly. This observation suggested a mechanism for the inhibition of PLC 1 by tubulin, since high tubulin concentrations might prevent the access of PLC 1 to its substrate. Activation of m 1 muscarinic receptors by carbachol relaxed this inhibition, probably by increasing the affinity of G␣ q for tubulin. Involvement of tubulin in the articulation between PLC 1 signaling and microtubule assembly might prove important for the intracellular governing of a broad range of cellular events.
The microtubule protein tubulin regulates adenylyl cyclase and phospholipase Cbeta(1) (PLCbeta(1)) signaling via transactivation of the G-protein subunits Galphas, Galphai1, and Galphaq. Because most tubulin is not membrane associated, this study investigates whether tubulin translocates to the membrane in response to an agonist so that it might regulate G-protein signaling. This was studied in SK-N-SH neuroblastoma cells, which possess a muscarinic receptor-regulated PLCbeta(1)-signaling pathway. Tubulin, at nanomolar concentrations, transactivated Galphaq by the direct transfer of a GTP analog and potentiated carbachol-activated PLCbeta(1). A specific and time-dependent association of tubulin with plasma membranes was observed when SK-N-SH cells were treated with carbachol. The same phenomenon was observed with membranes from Sf9 cells, expressing a recombinant PLCbeta(1) cascade. The time course of this event was concordant both with transactivation of Galphaq by the direct transfer of [(32)P]P(3)(4-azidoanilido)-P(1)-5'-GTP from tubulin as well as with the activation of PLCbeta(1). In SK-N-SH cells, carbachol induced a rapid and transient translocation of tubulin to the plasma membrane, microtubule reorganization, and a change in cell shape as demonstrated by confocal immunofluorescence microscopy. These observations presented a spatial and temporal resolution of the sequence of events underlying receptor-evoked involvement of tubulin in G-protein-mediated signaling. It is suggested that G-protein-coupled receptors might modulate cytoskeletal dynamics, intracellular traffic, and cellular architecture.
The betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interacts with microtubules and is involved in the regulation of the mitotic spindle. In the current study, the anti-microtubular drug nocodazole was employed to investigate the mechanism by which Gbetagamma interacts with tubulin and its possible implications in microtubule assembly in cultured PC12 cells. Nocodazole-induced depolymerization of microtubules drastically inhibited the interaction between Gbetagamma and tubulin. Gbetagamma was preferentially bound to microtubules and treatment with nocodazole suggested that the dissociation of Gbetagamma from microtubules is an early step in the depolymerization process. When microtubules were allowed to recover after removal of nocodazole, the tubulin-Gbetagamma interaction was restored. Unlike Gbetagamma, however, the interaction between tubulin and the alpha subunit of the Gs protein (Gsalpha) was not inhibited by nocodazole, indicating that the inhibition of tubulin-Gbetagamma interactions during microtubule depolymerization is selective. We found that Gbetagamma also interacts with gamma-tubulin, colocalizes with gamma-tubulin in centrosomes, and co-sediments in centrosomal fractions. The interaction between Gbetagamma and gamma-tubulin was unaffected by nocodazole, suggesting that the Gbetagamma-gamma-tubulin interaction is not dependent on assembled microtubules. Taken together, our results suggest that Gbetagamma may play an important and definitive role in microtubule assembly and/or stability. We propose that betagamma-microtubule interaction is an important step for G protein-mediated cell activation. These results may also provide new insights into the mechanism of action of anti-microtubule drugs.
The direct effect of melatonin and related agonists on Li+‐amplified phosphoinositide breakdown was studied in chick brain slices prelabeled with myo‐[2‐3H]‐inositol. The melatonin receptor agonist 6‐chloromelatonin (10–100 µM) increased, in a concentration‐dependent manner, the accumulation of inositol phosphates (IP) in chick brain slices. This effect of 6‐chloromelatonin (10 µM) was rapid as transient increases in IP3/IP4 (maximal increase, 29% at 20 s) and IP2 levels (maximal increase, 36% at 1 min) were observed, followed by a slower but sustained increase in IP1 level (30% at 5 min), when the amount of IP3/IP4 and IP2 had already been decreased to the control level. The phosphoinositide response elicited by 6‐chloromelatonin (10 µM) was dependent on the presence of extracellular calcium. Direct stimulation of membrane phospholipase C by 6‐chloromelatonin (10 µM) in isolated myo‐[2‐3H]inositol‐prelabeled optic tectum membranes was dependent on the presence of guanosine‐5′‐O‐(3‐thio)triphosphate (1 µM), thus suggesting that G protein(s) link melatonin receptor activation to phospholipase C stimulation. The competitive melatonin receptor antagonist luzindole (10–100 µM) inhibited in a concentration‐dependent manner the IP1 accumulation stimulated by 6‐chloromelatonin (10–100 µM); however, it did not affect the accumulation stimulated by 5‐hydroxytryptamine (10 µM). By contrast, methysergide (10 µM) completely inhibited 5‐hydroxytryptamine (10 µM)‐, but not 6‐chloromelatonin (10 µM)‐, induced IP1 accumulation. Melatonin receptor agonists increased IP1 accumulation in a concentration‐dependent manner reaching different maximal responses. N‐Acetyl‐5‐hydroxytryptamine was more potent than melatonin in increasing IP1 accumulation, suggesting activation of a melatonin receptor site other than the ML‐1 melatonin receptor (i.e., N‐acetyl‐5‐hydroxytryptamine ≥ melatonin). In conclusion, these results demonstrate that activation of a melatonin receptor with pharmacological characteristics different from those of the ML‐1 subtype leads to activation of the phospholipase C‐mediated signal transduction pathway.
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