Recent cancer genome deep sequencing efforts have revealed an unanticipated high frequency of mutations in G proteins and G-protein-coupled receptors (GPCRs) in most tumour types. A striking 4.2% of all tumour sequences deposited to date show activating mutations in GNAS (a complex locus that encodes Gα_s). Transforming mutations in GNAS have been well documented in human thyroid and pituitary tumours, and recent sequencing efforts have shown these mutations to be present in a wide variety of additional tumour types, including colon cancer, hepatocellular carcinoma, and parathyroid, ovarian, endometrial, biliary tract and pancreatic tumours. Mutually exclusive activating mutations in GNAQ or GNA11 (encoding Gα_q family members) occur in 5.6% of tumours, and they are present in ∼66% and ∼6% of melanomas arising in the eye and skin, respectively, where they can act as driver oncogenes. Hotspot mutations in Gα_s (R201 and Q227) as well as Gα_q and Gα₁₁ (R183 and Q209) disrupt the GTPase activity, thereby leading to constitutive activity and persistent signalling. Nearly 20% of human cancers harbour mutations in GPCRs. The most frequently mutated GPCRs include thyroid-stimulating hormone receptor (TSHR), Smoothened (SMO), glutamate metabotropic receptors (GRMs), members of the adhesion family of GPCRs and receptors for bioactive lipid mediators such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) that accumulate in the tumour microenvironment. Many GPCR mutations are still uncharacterized with respect to their potential contribution to tumorigenesis and cancer progression. Aberrant expression, overexpression or signal reprogramming of GPCRs and G proteins in tumour cells can contribute to cancer development and progression. These alterations may arise from cancer-specific changes in gene copy number, as well as from other genetic, epigenetic and post-translational changes resulting in higher protein expression, thereby enhancing tumour progression and metastasis. Detailed three dimensional structures of GPCRs in various activation states can now help to explain the functional impact of cancer-associated GPCR mutations, and guide the rational design of signalling-selective GPCR agonists, antagonists and allosteric modulators. G proteins, GPCRs and their linked signalling circuitry represent novel therapeutic targets for cancer prevention and treatment.

Plexins represent a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. Emerging evidence implicates Rho GTPases in these biological events. However, Plexins lack any known catalytic activity in their conserved cytoplasmic tails, and how they transduce signals from semaphorins to Rho is still unknown. Here we show that Plexin B2 associates directly with two members of a recently identified family of Dbl homology/pleckstrin homology containing guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and Leukemia-associated Rho GEF (LARG). This physical interaction is mediated by their PDZ domains and a PDZ-binding motif found only in Plexins of the B family. In addition, we show that ligand-induced dimerization of Plexin B is sufficient to stimulate endogenous RhoA potently and to induce the reorganization of the cytoskeleton. Moreover, overexpression of the PDZ domain of PDZ-RhoGEF but not its regulator of G protein signaling domain prevents cell rounding and neurite retraction of differentiated PC12 cells induced by activation of endogenous Plexin B1 by semaphorin 4D. The association of Plexins with LARG and PDZ-RhoGEF thus provides a direct molecular mechanism by which semaphorins acting on Plexin B can control Rho, thereby regulating the actin-cytoskeleton during axonal guidance and cell migration.

Heterotrimeric G proteins of the G_i, G_s, and G_q family control a wide array of physiological functions primarily by regulating the activity of key intracellular second messenger-generating systems. α subunits of the G₁₂ family, Gα₁₂ and Gα₁₃, however, can promote cellular responses that are independent of conventional second messengers but that result from the activation of small GTP-binding proteins of the Rho family and their downstream targets. These findings led to the identification of a novel family of guanine-nucleotide exchange factors (GEFs) that provides a direct link between Gα_12/13 and Rho stimulation. Recent observations suggest that many cellular responses elicited by Gα_q and its coupled receptors also require the functional activity of Rho. However, available evidence suggests that Gα_q may act on pathways downstream from Rho rather than by promoting Rho activation. These seemingly conflicting observations and the recent development of sensitive assays to assess the in vivo levels of active Rho prompted us to ask whether Gα_q and its coupled receptors can stimulate endogenous Rho. Here we show that the expression of activated forms of Gα_q and the stimulation of G_q-coupled receptors or chimeric Gα_q molecules that respond to G_i-linked receptors can promote a robust activation of endogenous Rho in HEK-293T cells. Interestingly, this response was not prevented by molecules interfering with the ability of Gα₁₃ to stimulate its linked RhoGEFs, together suggesting the existence of a novel molecular mechanism by which Gα_q and the large family of G_q-coupled receptors can regulate the activity of Rho and its downstream signaling pathways.

Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gbetagamma and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration.

Somatic gain-of-function mutations of GNAQ and GNA11, which encode α subunits of heterotrimeric Gα_q/11 proteins, occur in about 85% of cases of uveal melanoma (UM), the most common cancer of the adult eye. Molecular therapies to directly target these oncoproteins are lacking, and current treatment options rely on radiation, surgery, or inhibition of effector molecules downstream of these G proteins. A hallmark feature of oncogenic Gα_q/11 proteins is their reduced intrinsic rate of hydrolysis of guanosine triphosphate (GTP), which results in their accumulation in the GTP-bound, active state. Here, we report that the cyclic depsipeptide FR900359 (FR) directly interacted with GTPase-deficient Gα_q/11 proteins and preferentially inhibited mitogenic ERK signaling rather than canonical phospholipase Cβ (PLCβ) signaling driven by these oncogenes. Thereby, FR suppressed the proliferation of melanoma cells in culture and inhibited the growth of Gα_q-driven UM mouse xenografts in vivo. In contrast, FR did not affect tumor growth when xenografts carried mutated B-Raf^V600E as the oncogenic driver. Because FR enabled suppression of malignant traits in cancer cells that are driven by activating mutations at codon 209 in Gα_q/11 proteins, we envision that similar approaches could be taken to blunt the signaling of non-Gα_q/11 G proteins.