Tubulin stimulates adenylyl cyclase activity in C6 glioma cells by bypassing the beta-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 alpha 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 beta-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 nM. Desensitization of beta-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 G alpha s in the absence of isoproterenol. When the same concentration of dimeric tubulin with AAGTP bound was introduced, AAGTP was transferred from tubulin to G alpha s, activating the latter species. Similar 'preferential' activation of G alpha s by tubulin-AAGTP versus the free nucleotide was seen using purified components. Thus, membrane-associated tubulin may serve to activate G alpha s, independent of signals not normally coupled to that protein. Tubulin may act as an agent to link a variety of membrane-associated signalling systems.
Yan
K
K
Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, Illinois 60612-7342, USA.
Popova
J S
JS
Moss
A
A
Shah
B
B
Rasenick
M M
MM
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001386:Azides / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005910:Glioma / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D011233:Precipitin Tests; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D012503:Saponins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014404:Tubulin / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000318:Adrenergic beta-Agonists; D001386:Azides; D011943:Receptors, Adrenergic, beta; D012503:Saponins; D014404:Tubulin; D006165:Guanylyl Imidodiphosphate; C031907:GTP gamma-4-azidoanilide; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases