Abstract
Some Gq-coupled receptors have been shown to antagonize growth factor activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. We used a constitutively active Galphaq(Q209L) mutant to explore the effects of Galphaq activation on signaling through the PI3K/Akt pathway. Transient expression of Galphaq(Q209L) in Rat-1 fibroblasts inhibited Akt activation induced by platelet-derived growth factor or insulin treatment. Expression of Galphaq(Q209L) also attenuated Akt activation promoted by coexpression of constitutively active PI3K in human embryonic kidney 293 cells. Galphaq(Q209L) had no effect on the activity of an Akt mutant in which the two regulatory phosphorylation sites were changed to acidic amino acids. Inducible expression of Galphaq(Q209L) in a stably transfected 293 cell line caused a decrease in PI3K activity in p110alpha (but not p110beta) immunoprecipitates. Receptor activation of Galphaq also selectively inhibited PI3K activity in p110alpha immunoprecipitates. Active Galphaq still inhibited PI3K/Akt in cells pretreated with the phospholipase C inhibitor U73122. Finally, Galphaq(Q209L) co-immunoprecipitated with the p110alpha-p85alpha PI3K heterodimer from lysates of COS-7 cells expressing these proteins, and incubation of immunoprecipitated Galphaq(Q209L) with purified recombinant p110alpha-p85alpha in vitro led to a decrease in PI3K activity. These results suggest that agonist binding to Gq-coupled receptors blocks Akt activation via the release of active Galphaq subunits that inhibit PI3K. The inhibitory mechanism seems to be independent of phospholipase C activation and might involve an inhibitory interaction between Galphaq and p110alpha PI3K.
Highlights
From ¶Research Service, Department of Veterans Affairs Medical Center, Northport, New York 11768 and the ‡Department of Medicine, Stony Brook University, Stony Brook, New York 11794
In cells cotreated with PE, the platelet-derived growth factor (PDGF)-induced increase in PI[3,4,5]P3 was reduced by 64% (Fig. 4B). These results suggest that activated G␣q inhibits p110␣ phosphatidylinositol 3-kinase (PI3K) activity, resulting in decreased growth factor-mediated PI[3,4,5]P3 production and Akt activation
We investigated whether the inhibitory effect of Gq-coupled receptors on the PI3K/Akt signaling pathway might be mediated by the activated G␣q subunit inhibiting PI3K
Summary
G␣q(Q209L) co-immunoprecipitated with the p110␣-p85␣ PI3K heterodimer from lysates of COS-7 cells expressing these proteins, and incubation of immunoprecipitated G␣q(Q209L) with purified recombinant p110␣-p85␣ in vitro led to a decrease in PI3K activity These results suggest that agonist binding to Gqcoupled receptors blocks Akt activation via the release of active G␣q subunits that inhibit PI3K. We reported that norepinephrine stimulation of ␣1adrenergic receptors in human aortic smooth muscle cells increases PI3K activity [20] This effect is completely blocked by pretreatment with pertussis toxin, indicating that it is not mediated by G␣q [20]. A second body of evidence suggests that Gq-coupled receptors do not activate PI3K or Akt and antagonize activation of these enzymes by growth factors that act through tyrosine kinase receptors [11, 21,22,23,24,25]. To further explore the mechanism by which the ␣1A-adrenergic receptor and other Gq-coupled receptors inhibit PI3K signaling, we used G␣q(Q209L) to test whether activated G␣q inhibits Akt activation by inhibiting PI3K
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