Abstract

Accumulating evidence suggests that heterotrimeric G protein activation may not require G protein subunit dissociation. Results presented here provide evidence for a subunit dissociation-independent mechanism for G protein activation by a receptor-independent activator of G protein signaling, AGS8. AGS8 is a member of the AGS group III family of AGS proteins thought to activate G protein signaling primarily through interactions with Gbetagamma subunits. Results are presented demonstrating that AGS8 binds to the effector and alpha subunit binding "hot spot" on Gbetagamma yet does not interfere with Galpha subunit binding to Gbetagamma or phospholipase C beta2 activation. AGS8 stimulates activation of phospholipase C beta2 by heterotrimeric Galphabetagamma and forms a quaternary complex with Galpha(i1), Gbeta(1)gamma(2), and phospholipase C beta2. AGS8 rescued phospholipase C beta binding and regulation by an inactive beta subunit with a mutation in the hot spot (beta(1)(W99A)gamma(2)) that normally prevents binding and activation of phospholipase C beta2. This demonstrates that, in the presence of AGS8, the hot spot is not used for Gbetagamma interactions with phospholipase C beta2. Mutation of an alternate binding site for phospholipase C beta2 in the amino-terminal coiled-coil region of Gbetagamma prevented AGS8-dependent phospholipase C binding and activation. These data implicate a mechanism for AGS8, and potentially other Gbetagamma binding proteins, for directing Gbetagamma signaling through alternative effector activation sites on Gbetagamma in the absence of subunit dissociation.

Highlights

  • G protein-coupled receptor signaling systems play key roles in a number of biological processes [1]

  • The peptides SIGK and SIRK bind to a hot spot on G␤1␥2 that interacts with multiple effectors, including phospholipase C (PLC)␤2, and corresponds to the G␣ subunit switch II binding site [2, 14, 16, 25]

  • CAGS8 May Unmask a New Binding Surface at the Amino Terminus of ␤␥—The data support a model where AGS binds to the heterotrimer with simultaneous interactions with G␤␥ at the hot spot and the G␣ subunit and that the hot spot is no longer used for signal transfer to PLC␤2 activation, because it is occupied with cAGS8

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Summary

Introduction

G protein-coupled receptor signaling systems play key roles in a number of biological processes [1]. AGS8 forms a complex interacting with G␣ and G␤␥ simultaneously, occupies the G␤␥“hot spot,” a critical effector binding and signal transfer region on G␤␥ (14 – 17), yet does not dissociate G␣ from G␤␥ subunits.

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