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)
Roychowdhury
S
S
Department of Physiology and Biophysics, University of Illinois, College of Medicine, Chicago 60612-7340.
Rasenick
M M
MM
D000818:Animals; D002855:Chromatography, Thin Layer; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation; D005374:Filtration / Q000379:methods; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000032:analysis; D006160:Guanosine Triphosphate / Q000032:analysis / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D015398:Signal Transduction; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism*
D014404:Tubulin; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins