Regulators and Effectors of Small Gtpases: Ras Family
Small-scale GTPases
Adam Shutes , Channing J. Der , in Encyclopedia of Biological Chemistry, 2004
Identification and Classification of Small GTPases
The first and prototypical small GTPases were identified as the proteins encoded by the retrovirus oncogenes of the Harvey and Kirsten rat sarcoma (Ras) viruses ( Effigy ane). Since its initial discovery, over 150 mammalian proteins have been identified which show varying degrees of both amino acrid sequence and structural similarity with Ras proteins. Small-scale GTPases were identified by a variety of approaches, some fortuitously (e.thousand., Rho), in yeast genetic studies (due east.g., Rabs), and others past nucleic acrid sequence homology (e.g., Ral, Rap). These 'Ras superfamily' proteins are further divided into families (e.g., the Rho and Rab families) by sequence identity and cellular office. The major branches are the Ras, Rho, and Rab families, with smaller branches including Arf, Sar, Rad/Gem, and Ran proteins (Figure 2) . Modest GTPases are well conserved in evolution, with structural and functional homologues of many of the mammalian proteins found in yeast, flies, worms, and plants.
Figure 1. Pocket-size GTPases role as GDP/GTP-regulated molecular switches. The intrinsic GDP/GTP exchange activity is accelerated past guanine nucleotide-exchange factors (GEFs) to promote germination of the active, GTP-bound protein, whereas GTPase-activating proteins (GAPs) stimulate the intrinsic GTP hydrolysis activity to stimulate germination of the inactive, Gross domestic product-bound protein and the release of free phosphate (Pi). Guanine nucleotide dissociation inhibitors (GDIs) forbid nucleotide exchange likewise as GAP stimulation. Small GTPase activation is most normally mediated by a signal input that activates GEF function. The GTP-leap GTPase displays college analogousness for downstream effector targets, leading to stimulation of diverse cellular responses.
Figure 2. Small GTPase family.
In add-on to the GTPase action, a characteristic of many modest GTPases is their modification by lipids. Members of the Ras, Rho, and Rab families are modified by either the C15 farnesyl or C20 geranylgeranyl isoprenoid lipid (Figure 3) . Protein prenylation is signaled by carboxyl-last sequences. For Ras and Rho small-scale GTPases, this lipid modification occurs at a particular motif, the CAAX tetrapeptide motif, consisting of a cysteine followed by whatsoever ii hydrophobic amino acids, and a terminal 10 rest that determines whether the poly peptide will exist modified by farnesyltransferase (X=Due south, M) or geranylgeranyl transferase I (X=50, F). Rab proteins possess cysteine-containing carboxyl-terminal motifs (CC, CXC, CCX, CCXXX) that signal geranylgeranyl isoprenoid mail-translational modification past a third prenyltransferase, geranylgeranyltransferase. Arf proteins are cotranslationally modified past the C14 myristate fat acrid at the amino-termini. Lipid modification is typically critical for protein function and facilitates the association of small-scale GTPases with specific membrane compartments. Ran is unusual amid the small GTPases and does not undergo any lipid modification.
Figure 3. Postal service-translational modification by prenylation is important for the function of some small GTPases. Members of the Ras and Rho family of small-scale GTPases terminate with CAAX tetrapeptide sequences that bespeak a series of post-translational modifications that promote association with membranes that is critical for function. For example, Ras proteins are synthesized initially as inactive, cytosolic proteins. Ras proteins are offset modified by farnesyltransferase (FTase) which catalyzes the covalent improver of a C15 farnesyl isoprenoid (F) lipid to the cysteine residue of the CAAX motif. This is followed by Rce1-mediated proteolytic removal of the AAX peptide and Icmt-catalyzed carboxylmethylation (O-Me) of the now terminal farnesylated cysteine residue. Rho GTPases undergo the same series of modifications, with the first step catalyzed by geranylgeranyltransferase I (GGTaseI) and addition of the geranylgeranyl isoprenoid (GG). Rho GDIs recognize the prenylated form of Rho GTPases and prevent its association with membranes, thus leading to Rho GTPase inactivation.
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Imaging and Spectroscopic Assay of Living Cells
Xin Zhou , ... Jin Zhang , in Methods in Enzymology, 2012
3.two.1 Introduction
Small GTPases are enzymes that catalyze the hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). As the most well-known members, Ras GTPases play essential roles in regulating jail cell growth, cell differentiation, cell migration, and lipid vesicle trafficking. Ras GTPases bicycle between the active GTP-spring state and the inactive GDP-bound state; they are inactivated by GTPase activating proteins (GAP) and activated by guanine nucleotide substitution factors (GEF). The quondam activate the intrinsic GTPase activity of Ras GTPases, which hydrolyze GTP to GDP, while the latter cause dissociation of Gross domestic product from Ras GTPases and association of GTP. Characterizing the spatial and temporal regulation of minor GTPases using traditional biochemical methods have proven hard ( Walker and Lockyer, 2004), but these complications have been overcome past the development of genetically encodable biosensors.
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Enzymes as Sensors
Satoshi Okada , ... Hay-Oak Park , in Methods in Enzymology, 2017
5 Terminal Remarks
Small GTPases are cellular switches that play vital roles in various processes such as cell polarization, cell migration, membrane trafficking, and cytokinesis ( Etienne-Manneville, 2004; Park & Bi, 2007). Their activation and inactivation are exquisitely controlled to ensure that specific cellular events occur only at specific times and places. This also explains why there are far more GEFs and GAPs (GTPase-activating proteins) than their target GTPases (e.thou., in that location are ~ 70 GEFs and lxx GAPs for 17 Rho GTPases in the homo genome) (Bernards, 2003; Bernards & Settleman, 2004). Small GTPases also share two key features in executing specific cellular functions—displaying a unique pattern of individual activation dynamics (e.g., Cdc42) and requiring spatiotemporal coordination with other GTPases (Rac, Rho, Rab, etc.). Every bit we take demonstrated here, the biosensor for Cdc42-GTP in budding yeast has led to the discoveries of new activation and feedback mechanisms during polarization and bud morphogenesis. Strikingly, Cdc42 is inhibited while Rho1 is active during cytokinesis in budding yeast (Atkins et al., 2013; Okada et al., 2013; Okada, Wloka, & Bi, 2017; Onishi, Ko, Nishihama, & Pringle, 2013; Tong et al., 2007). Thus, it will exist critically important to explore the potential mutual antagonism between Cdc42 and Rho1 during cytokinesis (Atkins et al., 2013; Onishi et al., 2013). A like relationship betwixt the GTPases has besides been suggested for the budding procedure (Gao, Caviston, Tcheperegine, & Bi, 2004). Such studies require the development of a rigorously tested biosensor for Rho1 in budding yeast, which has still to be done. In mammalian cells, the activities of minor GTPases such as Ras, Rho, Cdc42, and Rac are coordinately regulated, frequently displaying mutual antagonism in processes such as cell migration (Machacek et al., 2009; Nobes & Hall, 1999; Tsubouchi et al., 2002), cell–cell adhesion (Arthur, Noren, & Burridge, 2002; Bruewer, Hopkins, Hobert, Nusrat, & Madara, 2004), and chemotaxis (Xu et al., 2003). Biosensors for different small GTPases have played a primal role in defining their spatiotemporal relationships in a specific process. These biosensors and newly developed ones will continue to be instrumental for understanding the functions and mechanisms of individual GTPases, discovering their new roles, and understanding their coordination with other GTPases in a given process.
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Optogenetics: Tools for Decision-making and Monitoring Neuronal Activity
Mathew Tantama , ... Gary Yellen , in Progress in Brain Research, 2012
Small GTPases
Small GTPases regulate cytoskeletal dynamics at the neurite growth cone ( Dickson, 2001). Small GTPases are G proteins that function similar to the α-subunits of heterotrimeric G proteins. Small GTPases tin can be activated by exchange of GTP for Gross domestic product, promoted by guanine nucleotide exchange factors, and they are inactivated when GTP is hydrolyzed to Gross domestic product, promoted by GTPase activating proteins. To prototype the activated conformation of a small GTPase, one successful strategy uses the target GTPase and a binding partner labeled with the corresponding partners of a FRET pair. The binding partner selectively recognizes the GTP-bound, activated conformation of the GTPase, leading to a dynamic FRET point that provides a readout for activation (Kraynov et al., 2000). This strategy has been adjusted to engineer GEIs based on intramolecular FRET, and sensors take been developed to detect the activation of Ras and Rho family members—Ras, Rap1, RhoA, Rac, Cdc42—besides every bit for other small-scale GTPases (Aoki et al., 2008; Itoh et al., 2002; Mochizuki et al., 2001; Pertz et al., 2006; Sabouri-Ghomi et al., 2008; Yoshizaki et al., 2003).
Neurite outgrowth in the PC12 cell line has been examined extensively using GEIs for Ras and Rho small GTPases (Nakamura et al., 2008). The Raichu serial of GEIs allow neurite outgrowth to be monitored in parallel with G protein activeness, revealing signaling requirements for the successful activity of outgrowth signals such equally nerve growth gene or dibutyryl-camp (Goto et al., 2011). Quantitative live-jail cell imaging has also provided spatial and temporal parameters characterizing the Rac1 and cdc42 signaling pathway for neurite outgrowth (Aoki et al., 2004, 2005, 2007). These parameters were used to create a signaling model, predicting that a PI-5phophatase negatively regulates outgrowth (Aoki et al., 2007). Experimental verification of this prediction shows that this blazon of quantitative assay and modeling may provide valuable insights into the convergence and departure of signaling pathways governing neurite outgrowth and guidance.
These GEIs have also been used to show that Rac and RhoA regulate dendritic spine morphogenesis in cultured hippocampal neurons. Both overexpression of a constitutively active RhoA mutant and knockdown of the polarity protein PAR-half dozen inhibit dendritic spine formation. Imaging the Raichu GEI for RhoA demonstrated that PAR-half dozen knockdown acquired increased RhoA activity, suggesting that PAR-vi promotes spine morphogenesis by negative upstream regulation of RhoA (Zhang and Macara, 2008). Similarly, imaging Rac activity suggested a delineation of several steps of NMDAR regulation of spine morphogenesis. The guanine nucleotide exchange factor PIX activates Rac, and PIX interaction with Rac is promoted by CaMKK/CaMKI. Ras imaging showed that Ras activeness in dendritic spines can be increased by depolarization in a CaMK-dependent manner, and overexpression of a phosphorylation-deficient PIX dominant negative tin attenuate activation (Saneyoshi et al., 2008). These imaging data may suggest that NMDAR activation causes calcium influx and CaMK activation, and these signaling events could lead to local phosphorylation of PIX and after local activation of Rac to promote spine morphogenesis.
Imaging RhoA activity in cultured cortical neurons and in brain slices has also shown that the subcellular localization of RhoA activation is important for the regulation of cortical neuron migration during evolution (Pacary et al., 2011). The regulatory proteins Rnd2 and Rnd3 promote neuronal migration past inhibiting RhoA action, but knockdown has unlike pathological phenotypes. In this study, Raichu was genetically targeted to unlike subcellular compartments, and imaging RhoA action demonstrated that only Rnd3 knockdown increases RhoA activeness at the plasma membrane. The spatial resolution resulting from this strategy was instrumental for showing that spatial organization of RhoA activity existed and that compartmentation of signaling components may exist a primal regulatory feature in neuronal migration.
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Development of Neuronal Polarity In Vivo
F. Polleux , in Cellular Migration and Germination of Neuronal Connections, 2013
ane.5.3.3 Ras- and Rho-Family of Small-scale GTPases
Small GTPases are critical regulators of cytoskeletal and membrane dynamics underlying cell motility, cell polarity, and cell growth. Small GTPase proteins are molecular switches that more often than not human action on downstream effectors when bound to GTP and are inactive when this GTP is hydrolyzed to guanosine diphosphate (GDP). Rho-GTPases possess relatively ho-hum intrinsic GTP hydrolysis activity, and their catalytic activeness is regulated past GAPs (53 predicted in the human genome). GAPs therefore act as negative regulators of GTPase activity past promoting the Gdp bound (inactive) country. Activation of small GTPases past exchanging Gdp for GTP is controlled by guanine nucleotide exchange factors (GEFs; 69 predicted in the homo genome). Not surprisingly, both Ras- and Rho-family unit small GTPases accept been involved in axon specification and axon growth.
Several members of the Ras-family of small-scale GTPases take been shown to regulate neuronal polarity including H-Ras, R-Ras, K-Ras, and North-Ras. Overexpressing either wild-type or a constitutively active mutant (V12 or Q61L) of the H-Ras or the related protein R-RasQ87L leads to the production of multiple axons (Fivaz et al., 2008; Oinuma et al., 2007; Yoshimura et al., 2006). Ras proteins regulate both the MAP kinase and PI3K pathways, and pharmacologic inhibition of either pathway was sufficient to inhibit the production of additional axons, but surprisingly it did not bear upon axon formation in full general (Yoshimura et al., 2006). Ras activation is coupled to many cell surface receptors including growth factor receptors, and a EGFR tyrosine kinase inhibitor, AG1478, can inhibit axon formation (Shi et al., 2003). Elegant work using a fluorescent reporter of Ras activation demonstrates the restricted nature of Ras signaling and its recruitment during axon determination to contribute to a positive feedback loop with PI3K (Fivaz et al., 2008). Additional work remains to identify which upstream activators may regulate Ras during neuronal symmetry breaking to make up one's mind the nascent axon; we discuss some potential candidates afterward in this affiliate.
The best studied of all mammalian Rho-family unit minor GTPases (22 total) are Cdc42, RhoA, and Rac1. Expression of dominant-negative (locked in Gross domestic product-jump state) or constitutively active (locked in GTP-bound land) mutants of each of these small GTPases in polarizing neurons, or treatment with the Rho-GTPase inhibitor toxin B (Bradke and Dotti, 1999), indicates a critical office for both cdc42 and Rac1 both in vitro in rodent neurons (Nishimura et al., 2005; Schwamborn and Puschel, 2004) and in Drosophila in vivo (Luo et al., 1994). Specifically, the expression of Cdc42L28, a cdc42 mutation that constitutively cycles between a GDP- and GTP-spring state, leads to the formation of multiple axons in rodent neurons. The loss of cdc42 expression, either through siRNA knockdown (Schwamborn and Puschel, 2004) or genetic ablation (Garvalov et al., 2007), leads to a strong axon specification defect. In the case of cdc42 conditional knockout mice, the axon phenotype may be due to increased levels of phosphorylated (inactive) cofilin, a regulator of actin dynamics enriched in developing axons (Garvalov et al., 2007). This phosphorylation is achieved by LIMK, an activity stimulated by a cdc42 effector kinase, Pak1. Paradoxically, Pak1 activity is greatly reduced in cdc42-zippo mice, suggesting that the deregulation of some other pathway regulating cofilin occurs in the absence of cdc42, well-nigh probable the RhoA-regulated kinase Stone (Maekawa et al., 1999). The loss of Pak1 itself also inhibits neuronal polarization, and conversely, constitutively active Pak1 induces multiple tau1-positive processes (Jacobs et al., 2007). The latter effect can exist partially reduced by coexpression of either an unphosphorylatable class of cofilin or a GDP-locked Rac1, suggesting that Rac1 may act downstream of Pak1 activation. Taken together, these results demonstrate a office for activated cdc42 in neuronal polarization beyond its association with the PAR3/six complex described later on in this review.
RhoA is another small GTPase, and it is typically associated with destabilization of the actin cytoskeleton and myosin-based contractility. Experiments using a constitutively active form of RhoA show that it inhibits neuritogenesis, whereas a ascendant-negative form of RhoA enhances neurite outgrowth (Schwamborn and Puschel, 2004). This finding is consistent with the regulatory role proposed for p190RhoGAP and the effect of inhibiting the RhoA-activated kinase, ROCK, on axogenesis (Bito et al., 2000). Future experiments will test if RhoA activation is regulated past local degradation through Smurf1/2 activity specifically in the axon downstream of local TGFß receptor activation and recruitment of Par6 (Yi et al., 2010) as previously shown in EMT (Ozdamar et al., 2005).
The examination of Rac1's role in neuronal polarization has led to some confounding results. In Drosophila, the expression of either ascendant-negative (GDP-locked) Rac (Luo et al., 1994) or loss of Rac expression (Hakeda-Suzuki et al., 2002; Ng and Luo, 2004; Ng et al., 2002) affects outgrowth but not polarity. Similarly, siRNA knockdown of mammalian Rac1 typically does not impact axon identity (Gualdoni et al., 2007), although some reports detected unpolarized neurons following expression of the ascendant-negative form of Rac1 (Nishimura et al., 2005). In cultured neurons, a constitutively active version of Rac1 does not touch on axon specification (Schwamborn and Puschel, 2004). These results, while mixed, do hint at a more than complex regulation of Rac1 in neuronal polarization. This fact becomes clearer afterwards in this review because the just Gef proteins shown to be crucial for axon formation appear to control Rac1. This ascertainment's apparent disjunction with the lack of strong phenotype may reflect the importance of subcellular localization of activated pools of Rac1 and compensation by related small GTPases.
Small GTPases have a plethora of effectors within cells, and proper activation of these effectors, both spatially and temporally, requires exquisite control of both activation and inactivation by GEFs and GAPs, respectively. Autonomously from p190RhoGAP, most studies have and so far focused on the office of GEFs in neuronal polarity. This includes the two GEFs Tiam1 and STEF, described later, and DOCK7 GEF, recently reported to exist a regulator of axon specification by activating Rac1 triggering phosphorylation of Stathmin/Op18, a microtubule-destabilizing gene critical for axogenesis (Watabe-Uchida et al., 2006). Another axonally enriched, anarchistic Rac1 regulatory protein is the cytoplasmic dynein light chain TcTEX-1 (Chuang et al., 2005). Increased levels of TcTEX-i result in increases in GTP-loaded Rac1 and a drop in GTP-Rac1 levels following TcTEX-1 siRNA handling. Multiple axons outcome from overexpression, and this effect is preserved using a mutant form (T94E) that cannot bind dynein heavy chain. Consequent with a office in decision-making Rac1, the supranumerary axon phenotype is suppressed by constitutively active RhoA or dominant negative Rac1.
Rap1b, a member of the Ras superfamily of GTPases, is too required for proper neuronal polarization in vitro (Schwamborn and Puschel, 2004; Schwamborn et al., 2007b) and in vivo (Jossin and Cooper, 2011). It is found at the tip of the nascent axon, and its overexpression leads to hippocampal neurons bearing multiple axons. The loss of Rap1b post-obit siRNA knockdown abrogates axon germination, and expression of autocycling cdc42 can rescue the phenotype. Expression of a constitutively active Rap1b fails to contrary the loss of axons observed post-obit a loss of cdc42, indicating that Rap1b lies upstream of cdc42 in this pathway of neuron polarization. Similarly, suppressing axogenesis via pharmacological inhibition of PI3-kinase can exist reversed past autocycling either cdc42 or constitutively active Rap1b, placing both of these small GTPases downstream of PI3K signaling during axon specification. In addition to its place in 1 of the canonical polarity pathways, studies on Rap1b accept explored a novel mechanism for protein localization during neuronal polarity, namely, selective protein degradation (Schwamborn et al., 2007a,b). This ways of controlling protein activity appears to apply to several polarity-regulating proteins.
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International Review of Cell and Molecular Biology
Francisco Rivero , Huajiang Xiong , in International Review of Prison cell and Molecular Biology, 2016
Abstract
Small GTPases of the Rho family are ubiquitous molecular switches involved in the regulation of near actin cytoskeleton dependent processes and many other processes not direct linked to actin. D. discoideum is a well-established model organism for studies of the actin cytoskeleton and its regulation by signal transduction pathways. D. discoideum is equipped with a complex repertoire of Rho signaling components, with xx Rho GTPases, more than 100 regulators (including exchange factors, GTPase activating proteins and guanine nucleotide dissociation inhibitors), and nearly eighty effectors or components of effector complexes. In this review we examine the knowledge accumulated to engagement about proteins involved in Rho-regulated signaling pathways in D. discoideum, with an emphasis on functional studies. Nosotros integrate the information about individual components into defined signaling pathways, with a focus on three extensively investigated processes: chemotaxis, vesicle trafficking, and cytokinesis.
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The Molecular Biology of Arrestins
Audrey Claing , in Progress in Molecular Biology and Translational Science, 2013
1.1 The family unit of GTPases
Small GTPases are single-chain polypeptides of 20–xl kDa. These proteins are considered molecular switches that decide the temporal aspects of a broad diversity of signaling events involved in numerous cellular processes and responses. The One thousand domain responsible for guanine nucleotide binding is highly conserved between the different families of GTPases. When spring to guanosine diphosphate (Gross domestic product), small GTPases are inactive. Some families are maintained inactive through binding guanosine nucleotide dissociation inhibitors (GDIs). These accessory proteins not only prevent nucleotide exchange but also restrict relocalization of GTPases to membranes. Loading of guanosine triphosphate (GTP) results in the stabilization of a new 3D conformation that enables them to collaborate with and modulate the activity of different and specific effector proteins. The GTP-bound state is therefore regarded equally the active (ON) state. Typically, these proteins crave guanine nucleotide commutation factors (GEFs) to exchange GTP for Gross domestic product. Some GTPases possess limited intrinsic GTPase activity, which depends on a few conserved amino acids in critical positions. GTPase-activating proteins (GAPs) activate the intrinsic GTPase action, catalyzing the hydrolysis of GTP into GDP. It is the interplay between the GTPases, the GEFs, and the GAPs that coordinates signal transduction regulated by GTPases (Fig. 6.i).
Figure 6.1. Way of activation of small GTPases. Small GTPases are inactive (OFF) when leap to Gross domestic product and active when bound to GTP (ON). Cycling of these molecular switches is controlled by the combined activities of guanine nucleotide exchange factors (GEFs), which catalyze the substitution of GDP for GTP, and GTPase-activating proteins (GAPs), which increase the charge per unit of GTP hydrolysis. For some GTPases, another level of regulation is provided past guanosine dissociation inhibitors (GDIs) that assure stability of the inactive Gross domestic product-bound country. Upon their activation, GTPases command a broad diverseness of effectors to regulate the timing, localization, and specificity of the cellular response.
More than 150 modest Yard proteins take been identified in humans. These are traditionally classified into five families. 3 Ras proteins were the offset members of the unabridged superfamily and were initially discovered on the basis of their homology to rat sarcoma virus genes. iv,5 The best-characterized members are H-Ras, North-Ras, and K-Ras, which have been implicated in many types of cancers and considered protooncogenes. However, the Ral and Rap families of modest GTPases also vest to this prototypical grade of more thirty members. Members of the Ras homologous (Rho) family unit contain a second group of more than than twenty proteins. The three major members are Rho (A, B, C), Rac, 1–3 and Cdc42. This family of GTPase is well known for its role in remodeling of the actin cytoskeleton and gene expression. The third family, the Rabs, includes more than than threescore isoforms. Their main office in cells is to finely tune many steps of vesicle trafficking (vesicle formation, vesicle movement, and membrane fusion). The fourth family is the ADP-ribosylation factors (ARFs), which also regulate vesicle budding through regulation of coat polymerization and disassembly. Some ARF isoforms are likewise involved in actin rearrangement and lipid remodeling. The last family of GTPases consists of only 1 member, Ran (Ras-related Nuclear protein), which regulates nucleoplasmic send during interphase. This small G protein controls prison cell cycle progression. GTPases that have been linked directly or indirectly to β-arrestins are listed in Table 6.1.
Table vi.1. Cantankerous talk between β-arrestin and small GTPase family of proteins in mammals
| Ras family | Rho family unit | Rab family unit | ARF family | ||||
|---|---|---|---|---|---|---|---|
| GTPase | References | GTPase | References | GTPase | References | GTPase | References |
| Ras | 6,7 | Rho | eight–11 | Rab4 | 12 | ARF6 | 13–17 |
| Ral | ix,18,nineteen | Rac | 20–23 | Rab5 | 12,24 | ||
| Cdc42 | 25,26 | Rab7 | 12,27 | ||||
| Cellular part | Cellular function | Cellular function | Cellular function | ||||
|---|---|---|---|---|---|---|---|
| Proliferation Death/survival | Prison cell shape Migration Contraction | Intracellular trafficking | Vesicular trafficking Actin remodeling Lipid transformation | ||||
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Endosome Signaling Function B
Ahmed Zahraoui , in Methods in Enzymology, 2014
Abstract
Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates tight junction assembly and polarized membrane transport in epithelial cells. Using yeast 2-hybrid screen, we identified MICAL-like1 (MICAL-L1), a protein that interacts with GTP-bound Rab13 and shares a like domain organisation with MICAL poly peptide family. MICAL-L1 has a calponin homology, Lin11, Isl-1 & Mec-three (LIM), proline-rich, and coiled-coil domains. Information technology is associated with late and recycling endosomes. Time-lapse video microscopy shows that GFP–Rab7 and carmine–MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by brusque hairpin RNA does non modify the distribution of tight junction proteins, but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in late endosomal compartments. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its deposition. Our data evidence that MICAL-L1 inhibits EGFR degradation, suggesting that MICAL-L1 is involved in sorting/targeting the receptor to the recycling pathway. They provide novel insights into MICAL-L1/Rab protein circuitous that can regulate EGFR trafficking/signaling.
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Inhibitors of the Ras superfamily G-proteins, Office B
Fumi Shima , ... Tohru Kataoka , in The Enzymes, 2013
ane Introduction
Small GTPases H-Ras, Thou-Ras, and Due north-Ras, collectively called Ras, office equally a molecular switch by cycling between GTP-spring active and Gdp-bound inactive forms (Ras·GTP and Ras·GDP, respectively) in a variety of intracellular signaling pathways controlling cell growth, differentiation, and apoptosis [one]. Ras·GTP binds straight and activates downstream effectors such every bit Raf kinases (c-Raf-1, B-Raf, and A-Raf, collectively called Raf), phosphoinositide 3-kinases (PI3Ks), Ral guanine nucleotide dissociation stimulator (RalGDS) family proteins, and phospholipase Cɛ. Raf and PI3Ks induce activation of downstream kinase cascades MEK/ERK and PDK/Akt, respectively, while RalGDS activates small GTPase RalA. Not only Raf but also PI3Ks and RalGDS are implicated in malignant transformation. Interconversion between the two forms is reciprocally catalyzed by guanine nucleotide commutation factors (GEFs) and GTPase-activating proteins (GAPs) [ii]. In detail, GEFs such as Son-of-sevenless (Sos) mediate diverse upstream signals to induce germination of Ras·GTP. The GTP/Gdp exchange induces allosteric conformational changes in two flexible regions, termed switch I (residues 32–38) and switch Two (residues lx–75), both of which constitute a primary interface for effector recognition [2]. Oncogenic potential of Ras is enhanced by signal mutations at particular residues such every bit Gly12 and Gln61, which not only impair the intrinsic GTPase activity only also render Ras insensitive to the GAP activeness, resulting in the constitutive activation of the downstream effectors [1]. Such mutational activation of Ras is observed in a multifariousness of human cancers at an overall frequency of 15–20%, and this frequency goes upwardly to lx–ninety% and 30–fifty% in pancreatic and colorectal cancers, respectively [i,3,4]. Cancer cells with activated oncogenes such as ras are known to showroom a miracle called "oncogene addiction," where their survival becomes dependent on the activated oncogene functions [3]. In such a case, inhibition of the activated Ras function causes the reversal of transformed phenotypes of cancer cells, eventually leading to cell death and tumor regression [4,5]. Although these data characteristic Ras equally one of the most promising target for anticancer drug development, at that place is no effective molecular targeted therapy for Ras at nowadays now that in one case highly predictable farnesyl transferase inhibitors, which cake posttranslational farnesylation of Ras necessary for membrane targeting, accept failed in clinical trials [ane,vi]. Farnesylthiosalicylic acids, Due south-farnesyl cysteine mimetics which inhibit bounden of Ras to the Ras-escort proteins in the plasma membrane, have likewise been developed but their antitumor activeness remains unclear [vii]. Withal, in these 2 years, there take been meaning advances in developing new strategies for Ras inhibitor discovery, which volition be discussed in Section vii.
In this chapter, we review our strategy for Ras inhibitor development, where structural information on drug-accepting pockets found in a novel crystal structure of Ras·GTP is effectively utilized for construction-based drug design (SBDD) of a novel form of small-molecule Ras inhibitors, which cake binding of Ras·GTP to multiple effector molecules and exhibit antitumor activeness toward a xenograft of human colon carcinoma cells carrying the activated Chiliad-rasG12V gene.
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Regulators and Effectors of Small GTPases
Irit Huber , ... Dan Cassel , in Methods in Enzymology, 2001
Introduction
Small GTPases of the ADP-ribosylation gene (ARF) family act as regulators of vesicular trafficking of proteins in all eukaryotic cells. 1–iii In mammalian cells the family consists of six proteins classified into class I (ARF1, 2, and iii), course II (ARF4 and v), and grade 3 (ARF6). The most arable and best characterized ARF protein, ARF1, regulates the budding of COPI-coated vesicles from Golgi stacks 4, 5 and of clathrin-coated vesicles from the trans-Golgi network (TGN). vi, 7 The conversion of ARF to the GTP-jump course through the action of a guanine nucleotide exchange protein (GEP) 8, 9 triggers the recruitment of a cytosolic coat protein onto the membrane, whereas coat protein dissociation depends on the hydrolysis of GTP, a process that requires the action of a GTPase-activating protein (GAP). Thus, regulators of the GTPase cycle of ARF1 play a critical part in membrane traffic. ten
Our laboratory has reported the purification and cloning of a 45-kDa ARF1-directed GAP from rats (GAP1). xi, 12 The "catalytic" domain of GAP1 was plant to reside within the first 130–140 amino acids and to contain an essential Cys4 zinc finger structure. Several lines of show suggest that GAP1 functions in the regulation of Golgi traffic. GAP1 cycles between cytosol and Golgi, 12 its overexpression results in an expected phenotype of Golgi disassembly, thirteen and the protein interacts with a Golgilocalized receptor that mediates the retrieval of escaped endoplasmic reticulum (ER) glycoproteins bearing a carboxy- last KDEL tag. 14–16
ARF GAPs have been identified in yeast (encounter [34] in this book) 17, 18 and new mammalian ARF GAPs have been identified and characterized (run across [36] and [37] in this book). 19–21 All of these proteins show high similarity to GAP1 in their catalytic domains, but otherwise mammalian ARF GAPs vary in size, contain different protein motifs, and may take singled-out biological functions. In this chapter, we draw the grooming and backdrop of GAP1.
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