CHIMERIC G(ALPHA-S) G(ALPHA-I2), PROTEINS DEFINE DOMAINS ON G(ALPHA-S) THAT INTERACT WITH TUBULIN FOR BETA-ADRENERGIC ACTIVATION OF ADENYLYL-CYCLASE/

Previous Studies have demonstrated that dimeric tubulin, associated wi th synaptic membrane, is capable of activating the G-proteins G(s) and G(alpha i1) via transfer of GTP. To clarify the mechanism of intracel lular interaction between tubulin and G(alpha s) as it refers to adeny lyl cyclase activation wild type and chimeric G(alpha s/alpha i2) prot eins were transiently overexpressed in COS 1 cells. Effects of tubulin dimers with guanosine 5'-(beta,gamma-imido)triphosphate (Gpp(NH)p) bo und (tubulin-Gpp(NH)p) or Gpp(NH)p with/without isoproterenol on adeny lyl cyclase were assessed in cells made permeable with saponin. In nai ve and wild type G(alpha s)-overexpressing COS 1 cells, the beta-adren ergic agonist isoproterenol potentiated significantly the stimulatory effects of Gpp(NH)p and, to an even greater extent, tubulin-Gpp(NH)p o n 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-Gpp(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) tublin-Gpp(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) chim era and the beta-adrenergic receptor are uncoupled. The chimera G(alph a i/s(Bam)) (G(alpha i2) 1-212, G(alpha s) 213-292) was nearly identic al to native COS 1 cells, but isoproterenol potentiated Gpp(NH)p but n ot 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 wea kly responsive to Gpp(NH)p or tubulin-Gpp(NH)p and unresponsive to iso proterenol. In photoaffinity labeling studies with tubulin-[P-32]azido anilido-GTP (tubulin-[P-32]AAGTP), isoproterenol increased the amount of tubulin associated with membranes and the transfer of [P-32]AAGTP f rom 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 a cids 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 transfe r from tubulin is stabilized by G(alpha s)-tubulin interaction leading to extended stimulation of adenylyl cyclase.