G-PROTEIN BINDING AND G-PROTEIN ACTIVATION BY NUCLEOTIDE TRANSFER INVOLVE DISTINCT DOMAINS ON TUBULIN - REGULATION OF SIGNAL TRANSDUCTION BY CYTOSKELETAL ELEMENTS

Cytoskeletal proteins have been demonstrated to interact with elements which mediate neuronal signal transduction. This laboratory has shown that tubulin forms complexes with certain G proteins and transfers GT P to them, and such nucleotide transfer correlates well with the inhib ition of adenylyl cyclase. A previous study showed that tubulin--G pro tein complex formation occurred at regions of tubulin which were likel y to be involved in binding to other tubulin dimers during the process of microtubule polymerization [Wang, N., & Rasenick, M.M. (1991) Bioc hemistry 30, 10957-10965]. This study represents an attempt to investi gate the regions of tubulin which orchestrate the transfer of GTP from tubulin to G protein. It is demonstrated that two monoclonal antibodi es, with determinants in similar regions of alpha (DM1a) or beta (DM1b ) tubulin, blocked (by 70-80%) the ability of tubulin dimers (with Gpp NHp bound) to promote a stable inhibition of adenylyl cyclase. Under t he same conditions, a polyclonal antitubulin antibody caused only a sl ight reduction in adenylyl cyclase inhibition. None of the antibodies altered the inhibition of adenylyl cyclase induced by GppNHp in the ab sence of tubulin. Under conditions where tubulin-GppNHp inhibited syna ptic membrane adenylyl cyclase, tubulin dimers with the photoaffinity GTP analog azidoanilido-GTP (P-32-AAGTP) bound transferred that nucleo tide to G(alphail) on the membrane. DM1a and DM1b blocked this nucleot ide transfer. Similarly, tubulin-AAGTP transferred AAGTP to purified G proteins in solution and DM1a and DM1b blocked this process as well. Despite their ability to block the activation of G(il) by tubulin, nei ther antibody altered the ability of tubulin to bind to native G(ialph a) affixed to nitrocellulose. These results suggest that multiple doma ins on tubulin are required for interactions with G protein, and the d omains which are required for binding are distinct from those required for the transfer of nucleotide. Dimeric tubulin may activate specific synaptic membrane G proteins and, in doing so, might link elements of the cytoskeleton to the signal transduction process.