Muscarinic receptor activation promotes the membrane association of tubulin for the regulation of Gq-mediated phospholipase Cβ1 signaling

Abstract

The microtubule protein tubulin regulates adenylyl cyclase and phospholipase Cβ1 (PLCβ1) signaling via transactivation of the G-protein subunits Gαs, Gαi1, and Gαq. 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 PLCβ1-signaling pathway. Tubulin, at nanomolar concentrations, transactivated Gαq by the direct transfer of a GTP analog and potentiated carbachol-activated PLCβ1. 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 PLCβ1 cascade. The time course of this event was concordant both with transactivation of Gαq by the direct transfer of [32P]P3(4-azidoanilido)-P1-5'-GTP from tubulin as well as with the activation of PLCβ1. 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.