Muscarinic receptor activation promotes the membrane association of tubulin for the regulation of Gq-mediated phospholipase Cbeta(1) signaling.
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.
Popova
J S
JS
Department of Physiology, University of Illinois at Chicago, College of Medicine, Chicago, Illinois 60612-7342, USA.
Rasenick
M M
MM
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology; D018679:Cholinergic Agonists / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000187:drug effects / Q000502:physiology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000502:physiology*; D007527:Isoenzymes / Q000187:drug effects / Q000502:physiology*; D009447:Neuroblastoma; D054799:Phospholipase C beta; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014404:Tubulin / Q000187:drug effects / Q000502:physiology*; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000187:drug effects / Q000502:physiology*
D018679:Cholinergic Agonists; D003598:Cytoskeletal Proteins; D007527:Isoenzymes; D011976:Receptors, Muscarinic; D014404:Tubulin; D002217:Carbachol; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11