Activation of the regulator of G protein signaling 14-Gαi1-GDP signaling complex is regulated by resistance to inhibitors of cholinesterase-8A.

Conventional models of G protein signaling (1, 2) indicate that activated G protein-coupled receptors (GPCRs) serve as guanine nucleotide exchange factors (GEFs) towards coupled heterotrimeric (Gαβγ) G proteins. GPCR activation facilitates GDP release and subsequent GTP binding to the Gα subunit, which is followed by Gβγ dissociation from Gα-GTP. This allows free Gβγ and Gα-GTP to engage downstream effectors and linked signaling pathways. The lifetime of this signaling event is terminated by the regulators of G protein signaling (RGS) proteins, a large family of multifunctional signaling proteins that regulate the intrinsic GTPase activity of the Gα subunit and promote heterotrimer reassociation (3-5). RGS14 is a highly unusual RGS protein that is enriched in brain (6, 7) and binds to Gαi/o and H-Ras/Raf to integrate G protein and MAP kinase signaling pathways (8). RGS14 contains a conserved RGS domain, two adjacent Ras/Rap binding domains (RBDs), and a G protein regulatory (also known as a GoLoco [GL]) domain (7, 9). Like all RGS proteins, the RGS domain of RGS14 binds directly to active Gα (specifically Gαi and Gαo) to serve as a non-selective GTPase Activating Protein (GAP) towards both of these Gα subunits (6, 7, 10). Unlike other RGS proteins, the GL domain of RGS14 binds directly to inactive Gαi1-GDP and Gαi3-GDP to inhibit guanine nucleotide binding and exchange (11-13). Furthermore, the GL domain of RGS14 forms a tight complex at the plasma membrane with inactive Gαi1 and Gαi3 independent of Gβγ(13), suggesting RGS14 serves a different role in G protein signaling compared to other RGS proteins. Independent of conventional GPCR/G protein signaling, several unconventional G protein signaling pathways have been described recently that are involved in cell division and synaptic signaling (14-21). Ric-8A (Synembryn) is a cytosolic protein reported to bind to and act as a non-receptor GEF for Gαi1, Gαq, and Gαo proteins (22-24). Ric-8A recognizes inactive Gα-GDP proteins when they are in complex with several GL-domain containing proteins, including LGN/mPins and Activator of G protein Signaling 3 (AGS3). Like RGS14, LGN/mPins and AGS3 bind directly to inactive Gαi (22, 24), with LGN also being recruited to the plasma membrane by Gαi1 (25). However, unlike RGS14, these proteins lack an RGS domain. Given these similarities between RGS14, LGN/mPins, and AGS3, we sought to investigate if RGS14 functionally interacts with Ric-8A to regulate unconventional G protein signaling. Here we report that RGS14 is the first example of an RGS protein that also serves as a GL protein, forming a complex with Gαi1-GDP that is regulated by Ric-8A. We show that Ric-8A interacts with RGS14 in cells and acts on the RGS14:Gαi1-GDP protein complex in vitro, thereby promoting complex dissociation to affect the activation state of Gαi1. Moreover, we demonstrate that native RGS14 and Ric-8A co-exist within the same hippocampal neurons, further supporting a functional link between these two proteins. Taken together, these findings demonstrate that RGS14 serves as a multifunctional GL protein in addition to an RGS protein. We therefore propose a working molecular model to describe how Ric-8A could regulate RGS14:Gαi1 signaling functions in cells.