Gem associates with Ezrin and acts via the Rho-GAP protein Gmip to down-regulate the Rho pathway.

Gem is a protein of the Ras superfamily that plays a role in regulating voltage-gated Ca2+ channels and cytoskeletal reorganization. We now report that GTP-bound Gem interacts with the membrane-cytoskeleton linker protein Ezrin in its active state, and that Gem binds to active Ezrin in cells. The coexpression of Gem and Ezrin induces cell elongation accompanied by the disappearance of actin stress fibers and collapse of most focal adhesions. The same morphological effect is elicited when cells expressing Gem alone are stimulated with serum and requires the expression of ERM proteins. We show that endogenous Gem down-regulates the level of active RhoA and actin stress fibers. The effects of Gem downstream of Rho, i.e., ERM phosphorylation as well as disappearance of actin stress fibers and most focal adhesions, require the Rho-GAP partner of Gem, Gmip, a protein that is enriched in membranes under conditions in which Gem induced cell elongation. Our results suggest that Gem binds active Ezrin at the plasma membrane-cytoskeleton interface and acts via the Rho-GAP protein Gmip to down-regulate the processes dependent on the Rho pathway.

The RGS (regulator of G-protein signalling) and GoLoco domains of RGS14 co-operate to regulate Gi-mediated signalling.

RGS (regulator of G-protein signalling) proteins stimulate the intrinsic GTPase activity of the a subunits of heterotrimeric G-proteins, and thereby negatively regulate G-protein-coupled receptor signalling. RGS14 has been shown previously to stimulate the GTPase activities of Ga(o) and Ga(i) subunits through its N-terminal RGS domain, and to down-modulate signalling from receptors coupled to G(i). It also contains a central domain that binds active Rap proteins, as well as a C-terminal GoLoco/G-protein regulatory motif that has been shown to act in vitro as a GDP-dissociation inhibitor for Ga(i). In order to elucidate the respective contributions of the three functional domains of RGS14 to its ability to regulate G(i) signalling, we generated RGS14 mutants invalidated in each of its domains, as well as truncated molecules, and assessed their effects on G(i) signalling via the bg pathway in a stable cell line ectopically expressing the G(i)-coupled M2 muscarinic acetylcholine receptor (HEK-m2). We show that the RGS and GoLoco domains of RGS14 are independently able to inhibit signalling downstream of G(i). Targeting of the isolated GoLoco domain to membranes, by myristoylation/palmitoylation or Rap binding, enhances its inhibitory activity on G(i) signalling. Finally, in the context of the full RGS14 molecule, the RGS and GoLoco domains co-operate to confer maximal activity on RGS14. We therefore propose that RGS14 combines the inhibition of G(i) activation or coupling to receptors via its GoLoco domain with stimulation of the GTPase activity of Ga(i)-GTP via its RGS domain to negatively regulate signalling downstream of G(i).

Biochemical and structural characterization of the gem GTPase.

RGK proteins, encompassing Rad, Gem, Rem1, and Rem2, constitute an intriguing branch of the Ras superfamily; their expression is regulated at the transcription level, they exhibit atypical nucleotide binding motifs, and they carry both large N- and C-terminal extensions. Biochemical and structural studies are required to better understand how such proteins function. Here, we report the first structure for a RGK protein: the crystal structure of a truncated form of the human Gem protein (G domain plus the first part of the C-terminal extension) in complex with Mg.GDP at 2.1 A resolution. It reveals that the G-domain fold and Mg.GDP binding site of Gem are similar to those found for other Ras family GTPases. The first part of the C-terminal extension adopts an alpha-helical conformation that extends along the alpha5 helix and interacts with the tip of the interswitch. Biochemical studies show that the affinities of Gem for GDP and GTP are considerably lower (micromolar range) compared with H-Ras, independent of the presence or absence of N- and C-terminal extensions, whereas its GTPase activity is higher than that of H-Ras and regulated by both extensions. We show how the bulky DXWEX motif, characteristic of the switch II of RGK proteins, affects the conformation of switch I and the phosphate-binding site. Altogether, our data reveal that Gem is a bona fide GTPase that exhibits striking structural and biochemical features that should impact its regulation and cellular activities.