Tubulin-G protein association stabilizes GTP binding and activates GTPase: cytoskeletal participation in neuronal signal transduction

Biochemistry. 1994 Aug 16;33(32):9800-5. doi: 10.1021/bi00198a052.

Abstract

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)

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Chromatography, Thin Layer
  • Cytoskeleton / metabolism
  • Enzyme Activation
  • Filtration / methods
  • GTP Phosphohydrolases / metabolism*
  • GTP-Binding Proteins / metabolism*
  • Guanosine Diphosphate / analysis
  • Guanosine Triphosphate / analysis
  • Guanosine Triphosphate / metabolism*
  • Neurons / metabolism
  • Protein Binding
  • Signal Transduction
  • Time Factors
  • Tubulin / metabolism*

Substances

  • Tubulin
  • Guanosine Diphosphate
  • Guanosine Triphosphate
  • GTP Phosphohydrolases
  • GTP-Binding Proteins