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 signal transduction process through its association with the GTP-binding (G) proteins Gs and Gi1. Using the photoaffinity GTP analog, azidoanilido-GTP, it has been shown that the transfer of nucleotide from tubulin to G alpha s and G alpha i1 is the key step of this activation. The binding sites between tubulin and Gs or G alpha i1 appear to involve microtubule polymerization domains, since G protein alpha subunits were demonstrated to inhibit microtubule assembly [Wang, N., & Rasenick, M. M. (1991) Biochemistry 30, 10957-10965]. In order to understand tubulin-G protein interaction and the nucleotide transfer process in detail, tubulin was labeled with [alpha-32P]GTP or [35S]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 bound nucleotide was lost from tubulin during the filtration assay. However, the addition of G alpha i1 to [alpha-32P]-GTP-tubulin protected the nucleotide binding in a dose-dependent manner, suggesting a stabilization of GTP binding in the tubulin-G alpha i1 complex. G beta gamma mitigated this effect, and this was not dependent upon the presence of G alpha, suggesting a direct interaction between beta gamma and tubulin. The retinal G protein, transducin, which displayed a much lower affinity for tubulin, did not elicit similar stabilization of GTP binding, and transducin beta gamma did not release GTP from tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)