Previous studies have demonstrated that dimeric tubulin, associated with synaptic membrane, is capable of activating the G-proteins Gs and G alpha i1 via transfer of GTP. To clarify the mechanism of intracellular interaction between tubulin and G alpha s as it refers to adenylyl cyclase activation, wild type and chimeric G alpha s/G alpha i2 proteins were transiently overexpressed in COS 1 cells. Effects of tubulin dimers with guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p) bound (tubulin-Gpp(NH)p) or Gpp NH)p with/without isoproterenol on adenylyl cyclase were assessed in cells made permeable with saponin. In naive and wild type G alpha s-overexpressing COS 1 cells, the beta-adrenergic agonist isoproterenol potentiated significantly the stimulatory effects of Gpp(NH)p and, to an even greater extent, tubulin-Gpp(NH)p on adenylyl cyclase. In COS 1 cells expressing the chimera G alpha i(54)/s (G alpha i2 1-54, G alpha s 62-394 amino acids), tubulin-Gp-p (NH)p was more potent than Gpp(NH)p in the presence of isoproterenol, but the maximal activity was equal. In chimera G alpha s/i(38) (G alpha s 1-356, G alpha i2 357-392) tubulin-Gp-p(NH)p or Gpp(NH)p stimulated adenylyl cyclase activity 11-14 times above the control whether or not beta-adrenergic receptor was activated, suggesting that G alpha chimera and the beta-adrenergic receptor are uncoupled. The chimera G alpha i/s(Bam) (G alpha i2 1-212, G alpha s 213-292) was nearly identical to native COS 1 cells, but isoproterenol potentiated Gpp(NH)p but not the tubulin-Gpp(NH)p response. The construct G alpha i(Bam)/s/i(38) (G alpha i2 1-212, G alpha s 213-356, G alpha i2 357-392) was weakly responsive to Gpp(NH)p or tubulin-Gpp(NH)p and unresponsive to isoproterenol. In photoaffinity labeling studies with tubulin-[32P]azidoanilido-GTP (tubulin-[32P]AAGTP), isoproterenol increased the amount of tubulin associated with membranes and the transfer of [32P]AAGTP from tubulin to G alpha i(54)/s, G alpha s, and G alpha i/s(Bam), but not to G alpha i(Bam)/s/i(38) and very slightly to G alpha s/i(38). These results suggest that regions between the 54th and 212th amino acids of G alpha s are important for guanine nucleotide transfer from tubulin, while the 1st to 54th amino acids of G alpha s are required for the ability of tubulin to activate adenylyl cyclase. We speculate that the active G alpha s conformation provoked by nucleotide transfer from tubulin is stabilized by G alpha s-tubulin interaction leading to extended stimulation of adenylyl cyclase.