[Effects of tubulin on beta adrenergic receptor linked to adenylyl cyclase system]

We developed a newly devised method for receptor binding assays in permeable C6 cell suspensions, which permits investigation of beta-adrenergic receptor behavior under conditions where the receptor appears tightly coupled to Gs protein to activate adenylyl cyclase. Tubulin, the cytoskeletal element, is a G protein with similarities to other G proteins. Although dimeric tubulins have been implicated as modulators of the adenylyl cyclase system, the total mechanism of this regulation has not been clear. Guanine nucleotides have been known to modulate agonist binding affinity for G protein-linked receptors. We demonstrate that tubulin alters the coupling between receptors and G proteins involved in the stimulation or inhibition of adenylyl cyclase system via direct transfer of GTP from tubulin to Gs alpha and Gi 1 alpha. In this study, the effects of tubulin-GppNHp have been compared to those of GppNHp in changing beta-adrenergic agonist affinity in both permeable C6 cells and C6 membranes. Competitive binding studies, including time-course assays, were performed with isoproterenol and the beta-adrenergic antagonist, [125I]pindolol. These results show that tubulin-GppNHp is more effective than GppNHp in decreasing receptor affinity in both permeable C6 cells and C6 membranes. These results imply that tubulin plays a dynamic role in neuronal signal transduction system at multiple loci.