TUBULIN-G PROTEIN ASSOCIATION STABILIZES GTP-BINDING AND ACTIVATES GTPASE - CYTOSKELETAL PARTICIPATION IN NEURONAL SIGNAL-TRANSDUCTION

It has been suggested that dimeric tubulin can participate in the sign al transduction process through its association with the GTP-binding ( G) proteins G(s) and G(i1). Using the photoaffinity GTP analog, azidoa nilido-GTP, it has been shown that the transfer of nucleotide from tub ulin to G(alpha s) and G(alpha i1) is the key step of this activation. The binding sites between tubulin and G(s) or G(alpha i1) appear to i nvolve microtubule polymerization domains, since G protein alpha subun its were demonstrated to inhibit microtubule assembly [Wang, N., & Ras enick, M. M. (1991) Biochemistry 30, 10957-10965]. In order to underst and tubulin-G protein interaction and the nucleotide transfer process in detail, tubulin was labeled with [alpha-P-32]GTP or [S-35]GTP gamma S and was incubated with recombinant G(alpha i1) at increasing molar ratios. Rapid filtration through nitrocellulose was used to determine nucleotide binding in the protein complex. A substantial amount of bou nd nucleotide was lost from tubulin during the filtration assay. Howev er, the addition of G(alpha i1) to [alpha-P-32]GTP-tubulin protected t he nucleotide binding in a dose-dependent manner, suggesting a stabili zation of GTP binding in the tubulin-(alpha i1) complex. G(beta gamma) mitigated this effect, and this was not dependent upon the presence o f G(alpha), suggesting a direct interation between beta gamma and tubu lin. The retinal G protein, transducin, which displayed a much lower a ffinity for tubulin, did not elicit similar stabilization of GTP bindi ng, and transducin py did not release GTP from tubulin. The [alpha-P-3 2]GTP bound to tubulin was displaced completely by a 10-fold excess of free GTP but only by similar to 60% when GTP binding was stabilized i n presence of G(alpha i1). The residual nucleotide stayed bound to the tubulin-G(alpha i) complex even in the presence of 1 mM GTP, suggesti ng a partial nonexchangeability of nucleotide in the complex. Thin lay er chromatography was used to identify the nucleotide bound to the pro tein. When tubulin and G(i) were present, 60-80% of the GTP was hydrol yzed. Thus G(alpha i1) may evoke some change in tubulin similar to tha t seen during the formation of a microtubule. The transfer and hydroly sis of GTP from synaptic membrane tubulin to G(alpha s) and G(alpha i1 ) could provide a mechanism for cross-talk among signal transduction p athways in the brain and might represent a function of tubulin dimers which is independent of microtubule formation.