Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110α and β by protease-activated receptor 2 and β-arrestins

PAR-2 (protease-activated receptor 2) is a GPCR (G-protein-coupled receptor) that can elicit both G-protein-dependent and -independent signals. We have shown previously that PAR-2 simultaneously promotes G alpha q/Ca2+-dependent activation and arrestin-1-dependent inhibition of class IA PI3K (phosphoinositide 3-kinase), and we sought to characterize further the role of -arrestins in the regulation of PI3K activity. Whereas the ability of beta-arrestin-1 to inhibit p110 alpha (PI3K catalytic subunit alpha) has been demonstrated, the role of beta-arrestin-2 in PI3K regulation and possible differences in the regulation of the two catalytic subunits (p110 alpha and p110 beta) associated with p85 alpha (PI3K regulatory subunit) have not been examined. In the present study we have demonstrated that: (i) PAR-2 increases p110 alpha- and p110 beta associated lipid kinase activities, and both p110 alpha and p110 beta are inhibited by over-expression of either beta-arrestin-1 or -2; (ii) both beta-arrestin-1 and -2 directly inhibit the p110 alpha catalytic subunit in vitro, whereas only beta-arrestin-2 directly inhibited p110 beta; (iii) examination of upstream pathways revealed that PAR-2-induced PI3K activity required the small GTPase Cdc (celldivision cycle)42, but not tyrosine phosphorylation of p85; and (iv) beta-arrestins inhibit PAR-2-induced Cdc42 activation. Taken together, these results indicated that -arrestins could inhibit PAR-2-stimulated PI3K activity, both directly and through interference with upstream pathways, and that the two beta-arrestins differ in their ability to inhibit the p110 alpha and p110 beta catalytic subunits. These results are particularly important in light of the growing interest in PAR-2 as a pharmacological target, as commonly used biochemical assays that monitor G-protein coupling would not screen for beta-arrestin-dependent signalling events.