Exchange of Guanine Nucleotides Between Tubulin and GTP‐Binding Proteins That Regulate Adenylate Cyclase: Cytoskeletal Modification of Neuronal Signal Transduction

Abstract

Tubulin, the primary constituent of microtubules, is a GTP-binding proteins with structural similarities to other GTP-binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis-resistant GTP analog, 5'-guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP-binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis-resistant photoaffinity GTP analog, [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP-binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin-AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP-binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin-Gpp(NH)p could be overridden by the inclusion of 100 microM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23 degrees C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.

Lateral Diffusion of Gαs in the Plasma Membrane Is Decreased after Chronic but not Acute Antidepressant Treatment: Role of Lipid Raft and Non-Raft Membrane Microdomains

“…Lipid raft disruption by methyl-b-cyclodextrin has been previously shown to increase Ga s -adenylyl cyclase coupling (Donati and Rasenick, 2005a) and also induces a slower and less mobile recovery of GFPGa s after photobleaching as demonstrated here. The same is true for colchicine treatment, which disrupts Ga s anchoring to tubulin, releasing Ga s from rafts (Donati and Rasenick, 2005b;Rasenick, 1986;Rasenick and Wang, 1988;Rasenick et al, 2004).…”

Phosphatidylinositol 4,5-Bisphosphate Modifies Tubulin Participation in Phospholipase Cβ1Signaling

“…P 3 (4-azidoanilido)-P1-5Ј-GTP (AAGTP) and [ 32 P]AAGTP were synthesized as described previously . T ubulin -[ 32 P]AAGTP was made from PC -tubulin as indicated (Rasenick and Wang, 1988). The final preparations contained 0.4 -0.6 mol of nucleotide bound /mol of tubulin.…”

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

“…A role for membrane-associated tubulin in signal transduction has been proposed for a number of systems including lymphocyte activation (Offringa and Bierer, 1993) and regulation of G protein activities (Rasenick and Wang, 1988;Popova et al, 1994). The fact that palmitoylated proteins are often involved in signal transduction (James and Olson, 1990;Casey, 1995;Milligan et al, 1995;Ross, 1995) makes these studies even more compelling.…”