Abstract
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.
Highlights
Previous studies have demonstrated that dimeric tu- interaction leading to extended stimulation of adenylyl bulin, associated with synaptic membrane, iscapable of cyclase. activating the G-proteins G, and Gail via transfer of GTP
To clarify the mechanism of intracellular interaction between tubulin and G, as it refers to adenylyl cyclase activation, wild type and chimeric GJG, pro- Heterotrimeric GTP binding proteins (G-proteins)l couple a teins were transiently overexpressed in COS 1 cells. wide range of cell surface receptors to membrane-boundeffecEffects of tubulin dimers with guanosine5’-(P,y-imi- tor molecules including adenylyl cyclase, phospholipase C, and do)triphosphate (Gpp(NH)p) bound (tubulin-Gpp(NH)p)ion channels [1,2,3,4,5,6,7]
When a G-protein is in its basal inactive or Gpp(NH)pwithlwithout isoproterenol onadenylyl state, thea subunit contains tightlybound GDP andis associcyclasewereassessed in cellsmadepermeable with ated with the P-y subunit complex
Summary
Construction of Plasmids-All methods used to construct plasmids for expression of rat G-protein Western Blotting-Immunoblot analysis was carried out by a modi- Schematic diagrams of the G,JGai chimeras used in thepresfication of the procedure described by Wang et al [24].Membrane proteins, resolved by SDS-polyacrylamide gel electrophoresis, were transferred to nitrocellulose using a semi-dry transfer apparatus(Bio-Rad) They were probed with antibodiesspecific for the carboxyl terminus of either G,, or Gmi,at a dilution of 1500 [36] or tubulin at a dilution of ent study areshown in the upper left corners ofpanels C-F in Fig. 1.The G,, polypeptide is 394 amino acid residues, andG, polypeptide is 354 amino acid residues. Adenylyl cyclase activity, depending on the nature of the chimera expressed [16, 17, 19, 28]
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