GIV/Girdin activates Gαi and inhibits Gαs via the same motif

Abstract

We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK–ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV–Gαi complexes and activates GIV’s GEF function; then a second phosphomodification terminates GIV’s GEF function, triggers the assembly of GIV–Gαs complexes, and activates GIV’s GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK–ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses.

AMP-activated protein kinase fortifies epithelial tight junctions during energetic stress via its effector GIV/Girdin

“…Findings also reveal how deregulation of this pathway fuels the growth of tumor cells under energetic stress.

10.7554/eLife.20795.002Figure 1.AMPK binds and phosphorylates GIV at Ser ( S ) 245.( A ) Schematic showing the functional modules of the multimodular signal transducer GIV. From the N- to the C-terminus the domains are-- a Hook-domain (grey) which binds microtubules (Simpson et al, 2005); a long coiled-coil domain (green) assists in homo/oligomerization (Enomoto et al, 2005); a Gα-binding domain (GBD; yellow) which constitutively binds Gαi/s proteins (Le-Niculescu et al, 2005); a PI(4)P-binding motif (pink) which enables GIV to bind PI4P-enriched membranes at the Golgi and the PM (Enomoto et al, 2005); an evolutionarily conserved GEF motif (red) which binds and activates Gαi (Garcia-Marcos et al, 2009) and inactivates Gαs (Gupta et al, 2016), and releases ‘free’ Gβγ from both. The C-terminal ~200 aa of GIV (purple) also has key domains that enable GIV to bind and remodel actin (Enomoto et al, 2005), bind and enhance phosphorylation of Akt (Anai et al, 2005; Enomoto et al, 2005), bind ligand-activated RTKs (Ghosh et al, 2010; Lin et al, 2014), and bind and activate Class 1 PI3-Kinases (Lin et al, 2011).

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The GAPs, GEFs, GDIs and…now, GEMs: New kids on the heterotrimeric G protein signaling block

“…Gupta V., et al, 15 also revealed how the pleiotropic GEF/ GDI functions of GEMs may be controlled by post-translational modifications. They showed that in the case of the prototypical GEM GIV, sequential phosphorylation of 2 Ser residues that flank the bifunctional GEF/GDI motif on GIV by 2 kinases, CDK5 35 and PKCu, 36 ensures that GIV exerts its GEF and GDI activities on Gai and Gas, respectively, in a temporally and spatially segregated manner (Fig.…”

“…15 The members of this family share very little sequence homology and act within diverse signaling cascades; what unites them is the ability to couple activation of these cascades to G protein signaling (Fig. 1) via an evolutionarily conserved motif of »30 aa that directly binds to G proteins.…”

“…Consequently, they all serve as nonreceptor GEFs for Gai. In the case of GIV, Gupta V., et al 15 recently demonstrated that as KB-752, GIV is a GDI for Gas 15 : it inhibits the Gas!cAMP!PKA!pCREB signaling pathway in cells using the same KB-752-like motif. These findings exposed the need for a new nomenclature, i.e., "GEM," primarily to distinguish the KB-752-like family of proteins from the other non-receptor GEFs (like Ric8A/B or AGS1) that have no propensity for pleiotropy or dual (Gai activating/Gas inhibitory) function.…”

“…15,26 Using Gallein, 26 a compound that blocks Gbg-interactions with PI3Kg by binding to a protein-protein interaction "hot spot" on the Gb subunit, 39 it was demonstrated that the mechanism of PI3K-Akt enhancement brought about by GIV's GEM motif involves the release of 'free' Gbg-heterodimers from both Gi and Gs trimers. Because Gbg subunits interact with other components of canonical G protein signaling complexes (summarized in 40 ), e.g., receptors, Ga subunits, effectors (including GAPs and GEFs for small GTPases), and regulatory enzymes Figure 2.…”