Co-Targeting FASN and mTOR Suppresses Uveal Melanoma Growth.

Uveal melanoma (UM) displays a high frequency of metastasis; however, effective therapies for metastatic UM are limited. Identifying unique metabolic features of UM may provide a potential targeting strategy. A lipid metabolism protein expression signature was induced in a normal choroidal melanocyte (NCM) line transduced with GNAQ (Q209L), a driver in UM growth and development. Consistently, UM cells expressed elevated levels of fatty acid synthase (FASN) compared to NCMs. FASN upregulation was associated with increased mammalian target of rapamycin (mTOR) activation and sterol regulatory element-binding protein 1 (SREBP1) levels. FASN and mTOR inhibitors alone significantly reduced UM cell growth. Concurrent inhibition of FASN and mTOR further reduced UM cell growth by promoting cell cycle arrest and inhibiting glucose utilization, TCA cycle metabolism, and de novo fatty acid biosynthesis. Our findings indicate that FASN is important for UM cell growth and co-inhibition of FASN and mTOR signaling may be considered for treatment of UM.

Gßγ signaling regulates microtubule-dependent control of Golgi integrity.

Gßγ subunits regulate several non-canonical functions at distinct intracellular organelles. Previous studies have shown that Gßγ signaling at the Golgi is necessary to mediate vesicular protein transport function and to regulate mitotic Golgi fragmentation. Disruption of Golgi structure also occurs in response to microtubule depolymerizing agents, such as nocodazole. In this study, we use siRNA against Gß1/2 or specific Gγ subunits to deplete their expression, and show that their knockdown causes a significant reduction in nocodazole-induced Golgi fragmentation. We establish that knockdown of Gßγ or inhibition of Gßγ with gallein resulted in decreased activation of protein kinase D (PKD) in response to nocodazole treatment. We demonstrate that restricting the amount of free Gßγ available for signaling by either inhibiting Gαi activation using pertussis toxin or by knockdown of the non-GPCR GEF, Girdin/GIV protein, results in a substantial decrease in nocodazole-induced Golgi fragmentation and PKD phosphorylation. Our results also indicate that depletion of Gßγ or inhibition with gallein or pertussis toxin significantly reduces the microtubule disruption-dependent Golgi fragmentation phenotype observed in cells transfected with mutant SOD1, a major causative protein in familial amyotrophic lateral sclerosis (ALS). These results provide compelling evidence that Gßγ signaling is critical for the regulation of Golgi integrity.

Disruption of the interaction between mutationally activated Gαq and Gßγ attenuates aberrant signaling.

Heterotrimeric G protein stimulation via G protein-coupled receptors promotes downstream proliferative signaling. Mutations can occur in Gα proteins which prevent GTP hydrolysis; this allows the G proteins to signal independently of G protein-coupled receptors and can result in various cancers, such as uveal melanoma (UM). Most UM cases harbor Q209L, Q209P, or R183C mutations in Gαq/11 proteins, rendering the proteins constitutively active (CA). Although it is generally thought that active, GTP-bound Gα subunits are dissociated from and signal independently of Gßγ, accumulating evidence indicates that some CA Gα mutants, such as Gαq/11, retain binding to Gßγ, and this interaction is necessary for signaling. Here, we demonstrate that disrupting the interaction between Gßγ and Gαq is sufficient to inhibit aberrant signaling driven by CA Gαq. Introduction of the I25A point mutation in the N-terminal α helical domain of CA Gαq to inhibit Gßγ binding, overexpression of the G protein Gαo to sequester Gßγ, and siRNA depletion of Gß subunits inhibited or abolished CA Gαq signaling to the MAPK and YAP pathways. Moreover, in HEK 293 cells and in UM cell lines, we show that Gαq-Q209P and Gαq-R183C are more sensitive to the loss of Gßγ interaction than Gαq-Q209L. Our study challenges the idea that CA Gαq/11 signals independently of Gßγ and demonstrates differential sensitivity between the Gαq-Q209L, Gαq-Q209P, and Gαq-R183C mutants.

Enhanced membrane binding of oncogenic G protein αqQ209L confers resistance to inhibitor YM-254890.

Heterotrimeric G proteins couple activated G protein-coupled receptors (GPCRs) to intracellular signaling pathways. They can also function independently of GPCR activation upon acquiring mutations that prevent GTPase activity and result in constitutive signaling, as occurs with the αqQ209L mutation in uveal melanoma. YM-254890 (YM) can inhibit signaling by both GPCR-activated WT αq and GPCR-independent αqQ209L. Although YM inhibits WT αq by binding to αq-GDP and preventing GDP/GTP exchange, the mechanism of YM inhibition of cellular αqQ209L remains to be fully understood. Here, we show that YM promotes a subcellular redistribution of αqQ209L from the plasma membrane (PM) to the cytoplasm. To test if this loss of PM localization could contribute to the mechanism of inhibition of αqQ209L by YM, we developed and examined N-terminal mutants of αqQ209L, termed PM-restricted αqQ209L, in which the addition of membrane-binding motifs enhanced PM localization and prevented YM-promoted redistribution. Treatment of cells with YM failed to inhibit signaling by these PM-restricted αqQ209L. Additionally, pull-down experiments demonstrated that YM promotes similar conformational changes in both αqQ209L and PM-restricted αqQ209L, resulting in increased binding to ßγ and decreased binding to regulator RGS2, and effectors p63RhoGEF-DH/PH and phospholipase C-ß. GPCR-dependent signaling by PM-restricted WT αq is strongly inhibited by YM, demonstrating that resistance to YM inhibition by membrane-binding mutants is specific to constitutively active αqQ209L. Together, these results indicate that changes in membrane binding impact the ability of YM to inhibit αqQ209L and suggest that YM contributes to inhibition of αqQ209L by promoting its relocalization.

Prognostic Values of G-Protein Mutations in Metastatic Uveal Melanoma.

Uveal melanoma is the most common primary ocular malignancy in adults, characterized by gene mutations in G protein subunit alpha q (GNAQ) and G protein subunit alpha 11 (GNA11). Although they are considered to be driver mutations, their role in MUM remains elusive. We investigated key somatic mutations of MUM and their impact on patients' survival after development of systemic metastasis (Met-to-Death). Metastatic lesions from 87 MUM patients were analyzed by next generation sequencing (NGS). GNA11 (41/87) and GNAQ (39/87) mutations were most predominantly seen in MUM. Most GNA11 mutations were Q209L (36/41), whereas GNAQ mutations comprised Q209L (14/39) and Q209P (21/39). Epigenetic pathway mutations BAP1 (42/66), SF3B1 (11/66), FBXW7 (2/87), PBRM1 (1/66), and SETD2 (1/66) were found. No specimen had the EIF1AX mutation. Interestingly, Met-to-Death was longer in patients with GNAQ Q209P compared to GNAQ/GNA11 Q209L mutations, suggesting the difference in mutation type in GNAQ/GNA11 might determine the prognosis of MUM. Structural alterations of the GNAQ/GNA11 protein and their impact on survival of MUM patients should be further investigated.

Gßγ regulates mitotic Golgi fragmentation and G2/M cell cycle progression.

Heterotrimeric G proteins (αßγ) function at the cytoplasmic surface of a cell's plasma membrane to transduce extracellular signals into cellular responses. However, numerous studies indicate that G proteins also play noncanonical roles at unique intracellular locations. Previous work has established that G protein ßγ subunits (Gßγ) regulate a signaling pathway on the cytoplasmic surface of Golgi membranes that controls the exit of select protein cargo. Now, we demonstrate a novel role for Gßγ in regulating mitotic Golgi fragmentation, a key checkpoint of the cell cycle that occurs in the late G2 phase. We show that small interfering RNA-mediated depletion of Gß1 and Gß2 in synchronized cells causes a decrease in the number of cells with fragmented Golgi in late G2 and a delay of entry into mitosis and progression through G2/M. We also demonstrate that during G2/M Gßγ acts upstream of protein kinase D and regulates the phosphorylation of the Golgi structural protein GRASP55. Expression of Golgi-targeted GRK2ct, a Gßγ-sequestering protein used to inhibit Gßγ signaling, also causes a decrease in Golgi fragmentation and a delay in mitotic progression. These results highlight a novel role for Gßγ in regulation of Golgi structure.

Effects of Oncogenic Gαq and Gα11 Inhibition by FR900359 in Uveal Melanoma.

Uveal melanoma is the most common intraocular tumor in adults and often metastasizes to the liver, leaving patients with few options. Recurrent activating mutations in the G proteins, Gαq and Gα11, are observed in approximately 93% of all uveal melanomas. Although therapeutic intervention of downstream Gαq/11 targets has been unsuccessful in treating uveal melanoma, we have found that the Gαq/11 inhibitor, FR900359 (FR), effectively inhibits oncogenic Gαq/11 signaling in uveal melanoma cells expressing either mutant Gαq or Gα11. Inhibition of oncogenic Gαq/11 by FR results in cell-cycle arrest and induction of apoptosis. Furthermore, colony formation is prevented by FR treatment of uveal melanoma cells in 3D-cell culture, providing promise for future in vivo studies. This suggests direct inhibition of activating Gαq/11 mutants may be a potential means of treating uveal melanoma. IMPLICATIONS: Oncogenic Gαq/11 inhibition by FR900359 may be a potential treatment option for those with uveal melanoma.

G protein-coupled receptor kinase 4-induced cellular senescence and its senescence-associated gene expression profiling.

Senescent cells have lost their capacity for proliferation and manifest as irreversibly in cell cycle arrest. Many membrane receptors, including G protein-coupled receptors (GPCRs), initiate a variety of intracellular signaling cascades modulating cell division and potentially play roles in triggering cellular senescence response. GPCR kinases (GRKs) belong to a family of serine/threonine kinases. Although their role in homologous desensitization of activated GPCRs is well established, the involvement of the kinases in cell proliferation is still largely unknown. In this study, we isolated GRK4-GFP expressing HEK293 cells by fluorescence-activated cell sorting (FACS) and found that the ectopic expression of GRK4 halted cell proliferation. Cells expressing GRK4 (GRK4(+)) demonstrated cell cycle G1/G0 phase arrest, accompanied with significant increase of senescence-associated-ß-galactosidase (SA-ß-Gal) activity. Expression profiling analysis of 78 senescence-related genes by qRT-PCR showed a total of 17 genes significantly changed in GRK4(+) cells (≥ 2 fold, p < 0.05). Among these, 9 genes - AKT1, p16INK4, p27KIP1, p19INK4, IGFBP3, MAPK14, PLAU, THBS1, TP73 - were up-regulated, while 8 genes, Cyclin A2, Cyclin D1, CDK2, CDK6, ETS1, NBN, RB1, SIRT1, were down-regulated. The increase in cyclin-dependent kinase inhibitors (p16, p27) and p38 MAPK proteins (MAPK14) was validated by immunoblotting. Neither p53 nor p21Waf1/Cip1 protein was detectable, suggesting no p53 activation in the HEK293 cells. These results unveil a novel function of GRK4 on triggering a p53-independent cellular senescence, which involves an intricate signaling network.

Functional selectivity of GPCR-directed drug action through location bias.

G-protein-coupled receptors (GPCRs) are increasingly recognized to operate from intracellular membranes as well as the plasma membrane. The ß2-adrenergic GPCR can activate Gs-linked cyclic AMP (Gs-cAMP) signaling from endosomes. We show here that the homologous human ß1-adrenergic receptor initiates an internal Gs-cAMP signal from the Golgi apparatus. By developing a chemical method to acutely squelch G-protein coupling at defined membrane locations, we demonstrate that Golgi activation contributes significantly to the overall cellular cAMP response. Golgi signaling utilizes a preexisting receptor pool rather than receptors delivered from the cell surface, requiring separate access of extracellular ligands. Epinephrine, a hydrophilic endogenous ligand, accesses the Golgi-localized receptor pool by facilitated transport requiring the organic cation transporter 3 (OCT3), whereas drugs can access the Golgi pool by passive diffusion according to hydrophobicity. We demonstrate marked differences, among both agonist and antagonist drugs, in Golgi-localized receptor access and show that ß-blocker drugs currently used in the clinic differ markedly in ability to antagonize the Golgi signal. We propose 'location bias' as a new principle for achieving functional selectivity of GPCR-directed drug action.

Dysregulated GPCR Signaling and Therapeutic Options in Uveal Melanoma.

Uveal melanoma is the most common primary intraocular malignant tumor in adults and arises from the transformation of melanocytes in the uveal tract. Even after treatment of the primary tumor, up to 50% of patients succumb to metastatic disease. The liver is the predominant organ of metastasis. There is an important need to provide effective treatment options for advanced stage uveal melanoma. To provide the preclinical basis for new treatments, it is important to understand the molecular underpinnings of the disease. Recent genomic studies have shown that mutations within components of G protein-coupled receptor (GPCR) signaling are early events associated with approximately 98% of uveal melanomas.Implications: This review discusses the alterations in GPCR signaling components (GNAQ and GNA11), dysregulated GPCR signaling cascades, and viable targeted therapies with the intent to provide insight into new therapeutic strategies in uveal melanoma. Mol Cancer Res; 15(5); 501-6. ©2017 AACR.

Inducible Inhibition of Gßγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi.

Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on ßγ signaling at the Golgi, where ßγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous ßγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used ßγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit ßγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit ßγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by ßγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by ßγ inhibition, demonstrating that ßγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate ßγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for ßγ regulation of TGN to PM transport and a novel role for ßγ in mediating Golgi fragmentation.

Mitotic-dependent phosphorylation of leukemia-associated RhoGEF (LARG) by Cdk1.

Rho GTPases are integral to the regulation of actin cytoskeleton-dependent processes, including mitosis. Rho and leukemia-associated Rho guanine-nucleotide exchange factor (LARG), also known as ARHGEF12, are involved in mitosis as well as diseases such as cancer and heart disease. Since LARG has a role in mitosis and diverse signaling functions beyond mitosis, it is important to understand the regulation of the protein through modifications such as phosphorylation. Here we report that LARG undergoes a mitotic-dependent and cyclin-dependent kinase 1 (Cdk1) inhibitor-sensitive phosphorylation. Additionally, LARG is phosphorylated at the onset of mitosis and dephosphorylated as cells exit mitosis, concomitant with Cdk1 activity. Furthermore, using an in vitro kinase assay, we show that LARG can be directly phosphorylated by Cdk1. Through expression of phosphonull mutants that contain non-phosphorylatable alanine mutations at potential Cdk1 S/TP sites, we demonstrate that LARG phosphorylation occurs in both termini. Using phosphospecific antibodies, we confirm that two sites, serine 190 and serine 1176, are phosphorylated during mitosis in a Cdk1-dependent manner. In addition, these phosphospecific antibodies show phosphorylated LARG at specific mitotic locations, namely the mitotic organizing centers and flanking the midbody. Lastly, RhoA activity assays reveal that phosphonull LARG is more active in cells than phosphomimetic LARG. Our data thus identifies LARG as a phosphoregulated RhoGEF during mitosis.

PAQR3 regulates Golgi vesicle fission and transport via the Gßγ-PKD signaling pathway.

Heterotrimeric G proteins function at diverse subcellular locations, in addition to canonical signaling at the plasma membrane (PM). Gßγ signals at the Golgi, via protein kinase D (PKD), to regulate fission of PM-destined vesicles. However, the mechanism by which Gßγ is regulated at the Golgi in this process remains elusive. Recent studies have revealed that PAQR3 (Progestin and AdipoQ Receptor 3), also called RKTG (Raf Kinase Trapping to the Golgi), interacts with the Gß subunit and localizes Gß to the Golgi thereby inhibiting Gßγ signaling at the PM. Herein we show that, in contrast to this inhibition of canonical Gßγ signaling at the PM, PAQR3 promotes Gßγ signaling at the Golgi. Expression of PAQR3 causes fragmentation of the Golgi, while a Gß binding-deficient mutant of PAQR3 does not cause Golgi fragmentation. Also, a C-terminal fragment of GRK2 (GRK2ct), which interacts with Gßγ and inhibits Gßγ signaling, and gallein, a small molecule inhibitor of Gßγ, are both able to inhibit PAQR3-mediated Golgi fragmentation. Furthermore, a dominant negative form of PKD (PKD-DN) and a pharmacological inhibitor of PKD, Gö6976, also inhibit PAQR3-mediated fragmentation of the Golgi. Importantly, expression of the Gß binding-deficient mutant of PAQR3 inhibits the constitutive transport of the model cargo protein VSV-G from the Golgi to the PM, indicating the involvement of PAQR3 in Golgi-to PM vesicle transport and a dominant negative role for this mutant. Collectively, these results reveal a novel role for the newly characterized, Golgi-localized PAQR3 in regulating Gßγ at the non-canonical subcellular location of the Golgi and thus for controlling Golgi-to-PM protein transport via the Gßγ-PKD signaling pathway.

The regulator of G protein signaling (RGS) domain of G protein-coupled receptor kinase 5 (GRK5) regulates plasma membrane localization and function.

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs at the plasma membrane (PM). Here GRK5/GRK4 chimeras and point mutations in GRK5 identify a short sequence within the regulator of G protein signaling (RGS) domain in GRK5 that is critical for GRK5 PM localization. This region of the RGS domain of GRK5 coincides with a region of GRK6 and GRK1 shown to form a hydrophobic dimeric interface (HDI) in crystal structures. Coimmunoprecipitation (coIP) and acceptor photobleaching fluorescence resonance energy transfer assays show that expressed GRK5 self-associates in cells, whereas GRK5-M165E/F166E (GRK5-EE), containing hydrophilic mutations in the HDI region of the RGS domain, displays greatly decreased coIP interactions. Both forcing dimerization of GRK5-EE, via fusion to leucine zipper motifs, and appending an extra C-terminal membrane-binding region to GRK5-EE (GRK5-EE-CT) recover PM localization. In addition, GRK5-EE displays a decreased ability to inhibit PAR1-induced calcium release compared with GRK5 wild type (wt). In contrast, PM-localized GRK5-EE-CaaX (appending a C-terminal prenylation and polybasic motif from K-ras) or GRK5-EE-CT shows comparable ability to GRK5 wt to inhibit PAR1-induced calcium release. The results suggest a novel model in which GRK5 dimerization is important for its plasma membrane localization and function.

Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission.

Proper completion of mitosis requires the concerted effort of multiple RhoGEFs. Here we show that leukemia-associated RhoGEF (LARG), a RhoA-specific RGS-RhoGEF, is required for abscission, the final stage of cytokinesis, in which the intercellular membrane is cleaved between daughter cells. LARG colocalizes with α-tubulin at the spindle poles before localizing to the central spindle. During cytokinesis, LARG is condensed in the midbody, where it colocalizes with RhoA. HeLa cells depleted of LARG display apoptosis during cytokinesis with unresolved intercellular bridges, and rescue experiments show that expression of small interfering RNA-resistant LARG prevents this apoptosis. Moreover, live cell imaging of LARG-depleted cells reveals greatly delayed fission kinetics in abscission in which a population of cells with persistent bridges undergoes apoptosis; however, the delayed fission kinetics is rescued by Aurora-B inhibition. The formation of a Flemming body and thinning of microtubules in the intercellular bridge of cells depleted of LARG is consistent with a defect in late cytokinesis, just before the abscission event. In contrast to studies of other RhoGEFs, particularly Ect2 and GEF-H1, LARG depletion does not result in cytokinetic furrow regression nor does it affect internal mitotic timing. These results show that LARG is a novel and temporally distinct RhoGEF required for completion of abscission.

G protein trafficking.

The classical view of heterotrimeric G protein signaling places G -proteins at the cytoplasmic surface of the cell's plasma membrane where they are activated by an appropriate G protein-coupled receptor. Once activated, the GTP-bound Gα and the free Gßγ are able to regulate plasma membrane-localized effectors, such as adenylyl cyclase, phospholipase C-ß, RhoGEFs and ion channels. Hydrolysis of GTP by the Gα subunit returns the G protein to the inactive Gαßγ heterotrimer. Although all of these events in the G protein cycle can be restricted to the cytoplasmic surface of the plasma membrane, G protein localization is dynamic. Thus, it has become increasingly clear that G proteins are able to move to diverse subcellular locations where they perform non-canonical signaling functions. This chapter will highlight our current understanding of trafficking pathways that target newly synthesized G proteins to the plasma membrane, activation-induced and reversible translocation of G proteins from the plasma membrane to intracellular locations, and constitutive trafficking of G proteins.

Non-canonical signaling and localizations of heterotrimeric G proteins.

Heterotrimeric G proteins typically transduce signals from G protein-coupled receptors (GPCRs) to effector proteins. In the conventional G protein signaling paradigm, the G protein is located at the cytoplasmic surface of the plasma membrane, where, after activation by an agonist-bound GPCR, the GTP-bound Gα and free Gßγ bind to and regulate a number of well-studied effectors, including adenylyl cyclase, phospholipase Cß, RhoGEFs and ion channels. However, research over the past decade or more has established that G proteins serve non-canonical roles in the cell, whereby they regulate novel effectors, undergo activation independently of a GPCR, and/or function at subcellular locations other than the plasma membrane. This review will highlight some of these non-canonical aspects of G protein signaling, focusing on direct interactions of G protein subunits with cytoskeletal and cell adhesion proteins, the role of G proteins in cell division, and G protein signaling at diverse organelles.

Regulation of constitutive cargo transport from the trans-Golgi network to plasma membrane by Golgi-localized G protein betagamma subunits.

Observations of Golgi fragmentation upon introduction of G protein ßγ (Gßγ) subunits into cells have implicated Gßγ in a pathway controlling the fission at the trans-Golgi network (TGN) of plasma membrane (PM)-destined transport carriers. However, the subcellular location where Gßγ acts to provoke Golgi fragmentation is not known. Additionally, a role for Gßγ in regulating TGN-to-PM transport has not been demonstrated. Here we report that constitutive or inducible targeting of Gßγ to the Golgi, but not other subcellular locations, causes phospholipase C- and protein kinase D-dependent vesiculation of the Golgi in HeLa cells; Golgi-targeted ß(1)γ(2) also activates protein kinase D. Moreover, the novel Gßγ inhibitor, gallein, and the Gßγ-sequestering protein, GRK2ct, reveal that Gßγ is required for the constitutive PM transport of two model cargo proteins, VSV-G and ss-HRP. Importantly, Golgi-targeted GRK2ct, but not a PM-targeted GRK2ct, also blocks protein transport to the PM. To further support a role for Golgi-localized Gßγ, endogenous Gß was detected at the Golgi in HeLa cells. These results are the first to establish a role for Golgi-localized Gßγ in regulating protein transport from the TGN to the cell surface.

An N-terminal polybasic motif of Gαq is required for signaling and influences membrane nanodomain distribution.

Regions of basic amino acids in proteins can promote membrane localization through electrostatic interactions with negatively charged membrane lipid head groups. Previous work showed that the heterotrimeric G protein subunit α(q) contains a polybasic region in its N terminus that contributes to plasma membrane localization. Here, the role of the N-terminal polybasic region of α(q) in signaling was addressed. For α(q) mutants, loss of plasma membrane localization correlated with loss of signaling function, as measured by the ability to couple activated G protein-coupled receptors (GPCRs) to stimulation of inositol phosphate production. However, recovery of plasma membrane localization of α(q) polybasic mutants by introduction of a site for myristoylation or by coexpression of ßγ failed to recover signaling, suggesting a role for N-terminal basic amino acids of α(q) beyond simple plasma membrane localization. It is noteworthy that an α(q)4Q mutant, containing glutamine substitutions at arginines 27, 30, 31, and 34, was identified that failed to mediate signaling yet retained plasma membrane localization. Although α(q)4Q failed to couple activated receptors to inositol phosphate production, it was able to bind ßγ, bind RGS4 in an activation-dependent manner, stimulate inositol phosphate production in a receptor-independent manner, and productively interact with a GPCR in isolated membranes. It is noteworthy that α(q)4Q showed a differing localization to plasma membrane nanodomains compared with wild-type α(q). Thus, basic amino acids in the N terminus of α(q) can affect its lateral segregation on plasma membranes, and changes in such lateral segregation may be responsible for the observed signaling defects of α(q)4Q.

The amino acid motif L/IIxxFE defines a novel actin-binding sequence in PDZ-RhoGEF.

PDZ-RhoGEF is a member of the regulator family of G protein signaling (RGS) domain-containing RhoGEFs (RGS-RhoGEFs) that link activated heterotrimeric G protein alpha subunits of the G12 family to activation of the small GTPase RhoA. Unique among the RGS-RhoGEFs, PDZ-RhoGEF contains a short sequence that localizes the protein to the actin cytoskeleton. In this report, we demonstrate that the actin-binding domain, located between amino acids 561 and 585, directly binds to F-actin in vitro. Extensive mutagenesis identifies isoleucine 568, isoleucine 569, phenylalanine 572, and glutamic acid 573 as being necessary for binding to actin and for colocalization with the actin cytoskeleton in cells. These results define a novel actin-binding sequence in PDZ-RhoGEF with a critical amino acid motif of IIxxFE. Moreover, sequence analysis identifies a similar actin-binding motif in the N-terminus of the RhoGEF frabin, and as with PDZ-RhoGEF, mutagenesis and actin interaction experiments demonstrate an LIxxFE motif, consisting of the key amino acids leucine 23, isoleucine 24, phenylalanine 27, and glutamic acid 28. Taken together, results with PDZ-RhoGEF and frabin identify a novel actin-binding sequence. Lastly, inducible dimerization of the actin-binding region of PDZ-RhoGEF revealed a dimerization-dependent actin bundling activity in vitro. PDZ-RhoGEF exists in cells as a dimer, raising the possibility that PDZ-RhoGEF could influence actin structure in a manner independent of its ability to activate RhoA.