Tubulin stimulates adenylyl cyclase activity in C6 glioma cells by bypassing the β‐adrenergic receptor: a potential mechanism of G protein activation

While the cytoskeleton is known to play several roles in the biology of the cell, one role, which has been revealed only recently, is that of a participant in the signal transduction process. Tubulin binds specifically to the α subunits of Gs (stimulatory GTP‐binding regulatory protein of adenylyl cyclase), Gi1 (inhibitory protein of adenylyl cyclase), and Gq and transactivates those molecules through direct transfer of GTP. The relevance of this transactivation process to G proteins which are normally activated by a neurotransmitter‐occupied receptor is the subject of this study. C6 glioma cells, made permeable with saponin, retained tight coupling between Gs and the β‐adrenergic receptor. Although 5‐guanylylimidodiphosphate (GppNHp) was incapable of activating Gs (and subsequently, adenylyl cyclase) in the absence of agonist, tubulin with GppNHp bound (tubulin‐GppNHp) activated adenylyl cyclase with an EC 50 of 30 n m . Desensitization of β‐adrenergic receptors by isoproterenol exposure had no effect on the ability of tubulin‐GppNHp to activate Gs and adenylyl cyclase. When the photoaffinity GTP analog, azidoanilido GTP (AAGTP; P3(4‐azidoanilido)‐P1‐5'‐GTP), was added to C6 membranes or permeable C6 cells, it was only weakly incorporated by Gαs in the absence of isoproterenol. When the same concentration of dimeric tubulin with AAGTP bound was introduced, AAGTP was transferred from tubulin to Gαs, activating the latter species. Similar ‘preferential’ activation of Gαs by tubulin‐AAGTP versus the free nucleotide was seen using purified components. Thus, membrane‐associated tubulin may serve to activate Gαs, independent of signals not normally coupled to that protein. Tubulin may act as an agent to link a variety of membrane‐associated signalling systems.