Phosphoinositide 3-kinase (PI3K) mediates insulin actions by relaying signals 
from insulin receptors (IRs) to downstream targets. The p110α catalytic subunit 
of class IA PI3K is the primary insulin-responsive PI3K implicated in insulin 
signaling. We demonstrate here a new mode of spatial regulation for the p110α 
subunit of PI3K by PAQR3 that is exclusively localized in the Golgi apparatus. 
PAQR3 interacts with p110α, and the intracellular targeting of p110α to the 
Golgi apparatus is reduced by PAQR3 downregulation and increased by PAQR3 
overexpression. Insulin-stimulated PI3K activity and phosphoinositide 
(3,4,5)-triphosphate production are enhanced by Paqr3 deletion and reduced by 
PAQR3 overexpression in hepatocytes. Deletion of Paqr3 enhances 
insulin-stimulated phosphorylation of AKT and glycogen synthase kinase 3β, but 
not phosphorylation of IR and IR substrate-1 (IRS-1), in hepatocytes, mouse 
liver, and skeletal muscle. Insulin-stimulated GLUT4 translocation to the plasma 
membrane and glucose uptake are enhanced by Paqr3 ablation. Furthermore, PAQR3 
interacts with the domain of p110α involved in its binding with p85, the 
regulatory subunit of PI3K. Overexpression of PAQR3 dose-dependently reduces the 
interaction of p85α with p110α. Thus, PAQR3 negatively regulates insulin 
signaling by shunting cytosolic p110α to the Golgi apparatus while competing 
with p85 subunit in forming a PI3K complex with p110α.