RESUMEN
G protein-coupled receptors (GPCRs) couple to heterotrimeric G proteins, comprised of α and ßγ subunits, to convert extracellular signals into activation of intracellular signaling pathways. Canonically, GPCR-mediated activation results in the exchange of GDP for GTP on Gα and the dissociation of Gα-GTP and Gßγ, both of which can regulate a variety of signaling pathways. Hydrolysis of bound GTP by Gα returns the protein to Gα-GDP and allows reassociation with Gßγ to re-form the inactive heterotrimer. Naturally occurring mutations in Gα have been found at conserved glutamine and arginine amino acids that disrupt the canonical G protein cycle by inhibiting GTP hydrolysis, rendering these mutants constitutively active. Interestingly, these dysregulated Gα mutants are found in many different cancers due to their ability to sustain aberrant signaling without a need for activation by GPCRs. This review will highlight an increased recognition of the prevalence of such constitutively activating Gα mutations in cancers and the signaling pathways activated. In addition, we will discuss new knowledge regarding how these constitutively active Gα are regulated, how different mutations are biochemically distinct, and how mutationally activated Gα are unique compared to GPCR-activated Gα. Lastly, we will discuss recent progress in developing inhibitors directly targeting constitutively active Gα mutants. Significance Statement Constitutively activating mutations in G protein α subunits (Gα) widely occur in and contribute to the development of many human cancers. To develop ways to inhibit dysregulated, oncogenic signaling by these mutant Gα, it is crucial to better understand mechanisms that lead to constitutive Gα activation and unique mechanisms that regulate mutationally activated Gα in cells. The prevalence of activating mutations in Gα in various cancers make Gα proteins compelling targets for the development of therapeutics.
RESUMEN
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.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas , Transducción de Señal , Humanos , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Mutación , Transducción de Señal/genéticaRESUMEN
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.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Péptidos Cíclicos , Receptores Acoplados a Proteínas G , Membrana Celular/metabolismo , Péptidos Cíclicos/química , Unión Proteica , Transducción de Señal , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
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.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Dobutamina/farmacología , Epinefrina/farmacología , Receptores Adrenérgicos beta 1/metabolismo , Antagonistas Adrenérgicos beta/química , Dobutamina/química , Epinefrina/química , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Ligandos , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Transducción de Señal/fisiología , Red trans-Golgi/metabolismo , Animales , Membrana Celular/genética , Perros , Subunidades beta de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades gamma de la Proteína de Unión al GTP/genética , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Red trans-Golgi/genéticaRESUMEN
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.
Asunto(s)
Senescencia Celular/genética , Quinasa 4 del Receptor Acoplado a Proteína-G/fisiología , Perfilación de la Expresión Génica , División Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Citometría de Flujo , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Células MCF-7 , Transcriptoma , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Humanos , Transporte de Proteínas , Transducción de SeñalRESUMEN
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.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Nocodazol/farmacología , Toxina del Pertussis , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Microtúbulos/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Aparato de Golgi/metabolismo , Subunidades de Proteína/metabolismo , Red trans-Golgi/metabolismo , Animales , Vesículas Citoplasmáticas/metabolismo , Inhibidores Enzimáticos/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Células HeLa , Humanos , Proteína Quinasa C/metabolismo , Subunidades de Proteína/genética , Transporte de Proteínas/fisiología , Fosfolipasas de Tipo C/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Xantenos/metabolismoRESUMEN
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.
Asunto(s)
Puntos de Control de la Fase G2 del Ciclo Celular/fisiología , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Aparato de Golgi/fisiología , Ciclo Celular/fisiología , Membrana Celular/metabolismo , Fase G2/fisiología , Aparato de Golgi/metabolismo , Proteínas de la Matriz de Golgi/metabolismo , Células HeLa , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Mitosis/fisiología , Fosforilación , Proteína Quinasa C/metabolismo , Transporte de Proteínas/fisiología , ARN Interferente Pequeño/metabolismo , Transducción de Señal/fisiologíaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Aminoácidos Básicos/fisiología , Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Nanoestructuras/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/fisiología , Transducción de Señal/fisiología , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Aminoácidos Básicos/genética , Animales , Línea Celular , Membrana Celular/química , Membrana Celular/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Spodoptera/citología , Spodoptera/genéticaRESUMEN
G protein-coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization.
Asunto(s)
Membrana Celular/enzimología , Núcleo Celular/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , Membrana Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Chlorocebus aethiops , Ácidos Grasos Insaturados/farmacología , Quinasas de Receptores Acoplados a Proteína-G , Humanos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Palmitatos/farmacología , Ácido Palmítico/metabolismo , Proteínas Serina-Treonina Quinasas/química , Estructura Secundaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimologíaRESUMEN
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.
Asunto(s)
Actinas/química , Actinas/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Secuencia Conservada , Dimerización , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factores de Intercambio de Guanina Nucleótido Rho , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Heterotrimeric G proteins typically localize at the cytoplasmic face of the plasma membrane where they interact with heptahelical receptors. For G protein alpha subunits, multiple membrane targeting signals, including myristoylation, palmitoylation, and interaction with betagamma subunits, facilitate membrane localization. Here we show that an additional membrane targeting signal, an N-terminal polybasic region, plays a key role in plasma membrane localization of non-myristoylated alpha subunits. Mutations of N-terminal basic residues in alpha(s) and alpha(q) caused defects in plasma membrane localization, as assessed through immunofluorescence microscopy and biochemical fractionations. In alpha(s), mutation of four basic residues to glutamine was sufficient to cause a defect, whereas in alpha(q) a defect in membrane localization was not observed unless nine basic residues were mutated to glutamine or if three basic residues were mutated to glutamic acid. betagamma co-expression only partially rescued the membrane localization defects; thus, the polybasic region is also important in the context of the heterotrimer. Introduction of a site for myristoylation into the polybasic mutants of alpha(s) and alpha(q) recovered strong plasma membrane localization, indicating that myristoylation and polybasic motifs may have complementary roles as membrane targeting signals. Loss of plasma membrane localization coincided with defects in palmitoylation. The polybasic mutants of alpha(s) and alpha(q) were still capable of assuming activated conformations and stimulating second messenger production, as demonstrated through GST-RGS4 interaction assays, cAMP assays, and inositol phosphate assays. Electrostatic interactions with membrane lipids have been found to be important in plasma membrane targeting of many proteins, and these results provide evidence that basic residues play a role in localization of G protein alpha subunits.
Asunto(s)
Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Mutación/genética , Ácido Mirístico/metabolismo , Conformación Proteica , Transporte de Proteínas , Transducción de Señal , Relación Estructura-ActividadRESUMEN
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.
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Depsipéptidos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Neoplasias de la Úvea/tratamiento farmacológico , Animales , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/aislamiento & purificación , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Insectos/citología , Sistema de Señalización de MAP Quinasas , Melanoma/metabolismo , Melanoma/patología , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Neoplasias de la Úvea/metabolismo , Neoplasias de la Úvea/patologíaRESUMEN
Signal transduction via G-protein-coupled receptors (GPCRs) is a fundamental pathway through which the functions of an individual cell can be integrated within the demands of a multicellular organism. Since this family of receptors first discovered, the proteins that constitute this signaling cascade and their interactions with one another have been studied intensely. In parallel, the pivotal role of lipids in the correct and efficient propagation of extracellular signals has attracted ever increasing attention. This is not surprising given that most of the signal transduction machinery is membrane-associated and therefore lipid-related. Hence, lipid-protein interactions exert a considerable influence on the activity of these proteins. This review focuses on the post-translational lipid modifications of GPCRs and G proteins (palmitoylation, myristoylation, and isoprenylation) and their significance for membrane binding, trafficking and signaling. Moreover, we address how the particular biophysical properties of different membrane structures may regulate the localization of these proteins and the potential functional consequences of this phenomenon in signal transduction. Finally, the interactions that occur between membrane lipids and GPCR effector enzymes such as PLC and PKC are also considered.
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Proteínas de Unión al GTP Heterotriméricas/metabolismo , Lípidos de la Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Unión al GTP Heterotriméricas/química , Humanos , Lípidos de la Membrana/química , Receptores Acoplados a Proteínas G/químicaRESUMEN
Small GTPases of the Rho family are crucial regulators of actin cytoskeleton rearrangements. Rho is activated by members of the Rho guanine-nucleotide exchange factor (GEF) family; however, mechanisms that regulate RhoGEFs are not well understood. This report demonstrates that PDZ-RhoGEF, a member of a subfamily of RhoGEFs that contain regulator of G protein signaling domains, is partially localized at or near the plasma membranes in 293T, COS-7, and Neuro2a cells, and this localization is coincident with cortical actin. Disruption of the cortical actin cytoskeleton in cells by using latrunculin B prevents the peri-plasma membrane localization of PDZ-RhoGEF. Coimmunoprecipitation and F-actin cosedimentation assays demonstrate that PDZ-RhoGEF binds to actin. Extensive deletion mutagenesis revealed the presence of a novel 25-amino acid sequence in PDZ-RhoGEF, located at amino acids 561-585, that is necessary and sufficient for localization to the actin cytoskeleton and interaction with actin. Last, PDZ-RhoGEF mutants that fail to interact with the actin cytoskeleton display enhanced Rho-dependent signaling compared with wild-type PDZ-RhoGEF. These results identify interaction with the actin cytoskeleton as a novel function for PDZ-RhoGEF, thus implicating actin interaction in organizing PDZ-RhoGEF signaling.
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Actinas/química , Citoesqueleto/metabolismo , Receptores de Glutamato/química , Actinas/metabolismo , Alanina/química , Secuencia de Aminoácidos , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células COS , Línea Celular , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Factores de Intercambio de Guanina Nucleótido , Humanos , Immunoblotting , Luciferasas/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Neuronas/metabolismo , Pruebas de Precipitina , Estructura Terciaria de Proteína , Receptores de Glutamato/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Tiazoles/farmacología , Tiazolidinas , TransfecciónRESUMEN
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.