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 GTP to them, and such nucleotide transfer correlates well with the inhibition of adenylyl cyclase. A previous study showed that tubulin-G protein complex formation occurred at regions of tubulin which were likely to be involved in binding to other tubulin dimers during the process of microtubule polymerization [Wang, N., & Rasenick, M. M. (1991) Biochemistry 30, 10957-10965]. This study represents an attempt to investigate the regions of tubulin which orchestrate the transfer of GTP from tubulin to G protein. It is demonstrated that two monoclonal antibodies, with determinants in similar regions of alpha (DM1a) or beta (DM1b) tubulin, blocked (by 70-80%) the ability of tubulin dimers (with GppNHp bound) to promote a stable inhibition of adenylyl cyclase. Under the same conditions, a polyclonal antitubulin antibody caused only a slight reduction in adenylyl cyclase inhibition. None of the antibodies altered the inhibition of adenylyl cyclase induced by GppNHp in the absence of tubulin. Under conditions where tubulin-GppNHp inhibited synaptic membrane adenylyl cyclase, tubulin dimers with the photoaffinity GTP analog azidoanilido-GTP (32P-AAGTP) bound transferred that nucleotide to G alpha i1 on the membrane. DM1a and DM1b blocked this nucleotide 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 Gi1 by tubulin, neither antibody altered the ability of tubulin to bind to native Gi alpha affixed to nitrocellulose.(ABSTRACT TRUNCATED AT 250 WORDS)