scholarly journals Structure of small G proteins and their regulators.

2001 ◽  
Vol 48 (4) ◽  
pp. 829-850 ◽  
Author(s):  
M Paduch ◽  
F Jeleń ◽  
J Otlewski
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Protein Interactions ◽  
Sequence Similarity ◽  
Nucleotide Exchange ◽  
Microtubule Organization ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Small G Protein ◽  
Small G Proteins

In recent years small G proteins have become an intensively studied group of regulatory GTP hydrolases involved in cell signaling. More than 100 small G proteins have been identified in eucaryotes from protozoan to human. The small G protein superfamily includes Ras, Rho Rab, Rac, Sarl/Arf and Ran homologs, which take part in numerous and diverse cellular processes, such as gene expression, cytoskeleton reorganization, microtubule organization, and vesicular and nuclear transport. These proteins share a common structural core, described as the G domain, and significant sequence similarity. In this paper we review the available data on G domain structure, together with a detailed analysis of the mechanism of action. We also present small G protein regulators: GTPase activating proteins that bind to a catalytic G domain and increase its low intrinsic hydrolase activity, GTPase dissociation inhibitors that stabilize the GDP-bound, inactive state of G proteins, and guanine nucleotide exchange factors that accelerate nucleotide exchange in response to cellular signals. Additionally, in this paper we describe some aspects of small G protein interactions with down-stream effectors.

scholarly journals The small G protein Arl1 directs the trans-Golgi–specific targeting of the Arf1 exchange factors BIG1 and BIG2

2012 ◽  
Vol 196 (3) ◽  
pp. 327-335 ◽  
Author(s):  
Chantal Christis ◽  
Sean Munro
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mammalian Cells ◽  
Affinity Purification ◽  
Nucleotide Exchange ◽  
Purification Method ◽  
Guanine Nucleotide Exchange ◽  
Small G Protein ◽  
Nucleotide Exchange Factor ◽  
Small G Proteins

The small G protein Arf1 regulates Golgi traffic and is activated by two related types of guanine nucleotide exchange factor (GEF). GBF1 acts at the cis-Golgi, whereas BIG1 and its close paralog BIG2 act at the trans-Golgi. Peripheral membrane proteins such as these GEFs are often recruited to membranes by small G proteins, but the basis for specific recruitment of Arf GEFs, and hence Arfs, to Golgi membranes is not understood. In this paper, we report a liposome-based affinity purification method to identify effectors for small G proteins of the Arf family. We validate this with the Drosophila melanogaster Arf1 orthologue (Arf79F) and the related class II Arf (Arf102F), which showed a similar pattern of effector binding. Applying the method to the Arf-like G protein Arl1, we found that it binds directly to Sec71, the Drosophila ortholog of BIG1 and BIG2, via an N-terminal region. We show that in mammalian cells, Arl1 is necessary for Golgi recruitment of BIG1 and BIG2 but not GBF1. Thus, Arl1 acts to direct a trans-Golgi–specific Arf1 GEF, and hence active Arf1, to the trans side of the Golgi.

scholarly journals Activation of G proteins by guanine nucleotide exchange factors relies on GTPase activity

2015 ◽  
Author(s):  
Rob J Stanley ◽  
Geraint MH Thomas
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Gtpase Activity ◽  
Nucleotide Exchange ◽  
Signalling Molecules ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Experimental Strategies

G proteins are an important family of signalling molecules controlled by guanine nucleotide exchange and GTPase activity in what is commonly called an 'activation/inactivation cycle'. The molecular mechanism by which guanine nucleotide exchange factors (GEFs) catalyse the activation of monomeric G proteins is well-established, however the complete reversibility of this mechanism is often overlooked. Here, we use a theoretical approach to prove that GEFs are unable to positively control G protein systems at steady-state in the absence of GTPase activity. Instead, positive regulation of G proteins must be seen as a product of the competition between guanine nucleotide exchange and GTPase activity -- emphasising a central role for GTPase activity beyond merely signal termination. We conclude that a more accurate description of the regulation of G proteins via these processes is as a 'balance/imbalance' mechanism. This result has implications for the understanding of many intracellular signalling processes, and for experimental strategies that rely on modulating G protein systems.

scholarly journals Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mikel Garcia-Marcos
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Protein Signaling ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Cytoplasmic Proteins ◽  
Nucleotide Exchange Factor ◽  
In Cells

It has become evident that activation of heterotrimeric G-proteins by cytoplasmic proteins that are not G-protein-coupled receptors (GPCRs) plays a role in physiology and disease. Despite sharing the same biochemical guanine nucleotide exchange factor (GEF) activity as GPCRs in vitro, the mechanisms by which these cytoplasmic proteins trigger G-protein-dependent signaling in cells have not been elucidated. Heterotrimeric G-proteins can give rise to two active signaling species, Gα-GTP and dissociated Gβγ, with different downstream effectors, but how non-receptor GEFs affect the levels of these two species in cells is not known. Here, a systematic comparison of GPCRs and three unrelated non-receptor proteins with GEF activity in vitro (GIV/Girdin, AGS1/Dexras1, and Ric-8A) revealed high divergence in their contribution to generating Gα-GTP and free Gβγ in cells directly measured with live-cell biosensors. These findings demonstrate fundamental differences in how receptor and non-receptor G-protein activators promote signaling in cells despite sharing similar biochemical activities in vitro.

Arf, Sec7 and Brefeldin A: a model towards the therapeutic inhibition of guanine nucleotide-exchange factors

2005 ◽  
Vol 33 (6) ◽  
pp. 1265-1268 ◽  
Author(s):  
M. Zeghouf ◽  
B. Guibert ◽  
J.-C. Zeeh ◽  
J. Cherfils
Keyword(s):  
Exchange Reaction ◽  
G Proteins ◽  
Brefeldin A ◽  
Membrane Traffic ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Small G Proteins

GEFs (guanine nucleotide-exchange factors), which stimulate GDP dissociation from small G-proteins, are pivotal regulators of signalling pathways activated by small G-proteins. In the case of Arf proteins, which are major regulators of membrane traffic in the cell and have recently been found to be involved in an increasing number of human diseases, GDP/GTP exchange is stimulated by GEFs that carry a catalytic Sec7 domain. Recent structural results captured snapshots of the exchange reaction, revealing that Sec7 domains secure Arf-GDP to membranes before nucleotide exchange takes place, taking advantage of a built-in structural device in Arf proteins that couples their affinity for membranes to the nature of the bound nucleotide. One of the Arf–Sec7 intermediates was trapped by BFA (Brefeldin A), an uncompetitive inhibitor of Arf activation that has been instrumental in deciphering the molecular principles of membrane traffic at the Golgi. BFA targets a low-affinity Arf–Sec7 intermediate of the exchange reaction. It binds at the Arf-GDP/Sec7 interface, thus freezing the complex in an abortive conformation that cannot proceed to nucleotide dissociation. In the cell, this results in the specific inhibition of Arf1 by a subset of its GEFs, and the efficient and reversible block of membrane traffic at the Golgi. The mechanism of BFA leads to the concept of ‘interfacial inhibition’, in which a protein–protein interaction of therapeutic interest is stabilized, rather than impaired, by a drug. Up-regulated activity of small G-proteins is involved in various human diseases, making their GEFs attractive candidates to interrupt specifically the corresponding signalling pathway. Interfacial inhibitors are proposed as an alternative to competitive inhibitors that may be explored for their inhibition.

Clues to the mechanism of action of eIF2B, the guanine-nucleotide-exchange factor for translation initiation

2008 ◽  
Vol 36 (4) ◽  
pp. 658-664 ◽  
Author(s):  
Sarah S. Mohammad-Qureshi ◽  
Martin D. Jennings ◽  
Graham D. Pavitt
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Structural Similarity ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Eukaryotic Translation ◽  
Guanine Nucleotide Exchange

A variety of cellular processes rely on G-proteins, which cycle through active GTP-bound and inactive GDP-bound forms. The switch between these states is commonly regulated by GEFs (guanine-nucleotide-exchange factors) and GAPs (GTPase-activating proteins). Although G-proteins have structural similarity, GEFs are very diverse proteins. A complex example of this system is seen in eukaryotic translation initiation between eIF (eukaryotic initiation factor) 2, a G-protein, its five-subunit GEF, eIF2B, and its GAP, eIF5. eIF2 delivers Met-tRNAi (initiator methionyl-tRNA) to the 40S ribosomal subunit before mRNA binding. Upon AUG recognition, eIF2 hydrolyses GTP, aided by eIF5. eIF2B then re-activates eIF2 by removing GDP, thereby promoting association of GTP. In the present article, we review data from studies of representative G-protein–GEF pairs and compare these with observations from our research on eIF2 and eIF2B to propose a model for how interactions between eIF2B and eIF2 promote guanine nucleotide exchange.

scholarly journals A biochemical and genetic discovery pipeline identifies PLCδ4b as a nonreceptor activator of heterotrimeric G-proteins

2018 ◽  
Vol 293 (44) ◽  
pp. 16964-16983 ◽  
Author(s):  
Marcin Maziarz ◽  
Stefan Broselid ◽  
Vincent DiGiacomo ◽  
Jong-Chan Park ◽  
Alex Luebbers ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Cell Function ◽  
Sequence Similarity ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Protein Activity ◽  
Conserved Sequence ◽  
Discovery Pipeline

Recent evidence has revealed that heterotrimeric G-proteins can be activated by cytoplasmic proteins that share an evolutionarily conserved sequence called the Gα-binding-and-activating (GBA) motif. This mechanism provides an alternative to canonical activation by G-protein–coupled receptors (GPCRs) and plays important roles in cell function, and its dysregulation is linked to diseases such as cancer. Here, we describe a discovery pipeline that uses biochemical and genetic approaches to validate GBA candidates identified by sequence similarity. First, putative GBA motifs discovered in bioinformatics searches were synthesized on peptide arrays and probed in batch for Gαi3 binding. Then, cDNAs encoding proteins with Gαi3-binding sequences were expressed in a genetically-modified yeast strain that reports mammalian G-protein activity in the absence of GPCRs. The resulting GBA motif candidates were characterized by comparison of their biochemical, structural, and signaling properties with those of all previously described GBA motifs in mammals (GIV/Girdin, DAPLE, Calnuc, and NUCB2). We found that the phospholipase Cδ4 (PLCδ4) GBA motif binds G-proteins with high affinity, has guanine nucleotide exchange factor activity in vitro, and activates G-protein signaling in cells, as indicated by bioluminescence resonance energy transfer (BRET)-based biosensors of G-protein activity. Interestingly, the PLCδ4 isoform b (PLCδ4b), which lacks the domains required for PLC activity, bound and activated G-proteins more efficiently than the full-length isoform a, suggesting that PLCδ4b functions as a G-protein regulator rather than as a PLC. In summary, we have identified PLCδ4 as a nonreceptor activator of G-proteins and established an experimental pipeline to discover and characterize GBA motif–containing proteins.

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