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.

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.

scholarly journals DAPLE protein inhibits nucleotide exchange on Gαs and Gαq via the same motif that activates Gαi

2020 ◽  
Vol 295 (8) ◽  
pp. 2270-2284 ◽  
Author(s):  
Arthur Marivin ◽  
Marcin Maziarz ◽  
Jingyi Zhao ◽  
Vincent DiGiacomo ◽  
Isabel Olmos Calvo ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Birth Defects ◽  
High Frequency ◽  
G Protein Coupled Receptors ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Cytoplasmic Proteins ◽  
G Protein Coupled

Besides being regulated by G-protein–coupled receptors, the activity of heterotrimeric G proteins is modulated by many cytoplasmic proteins. GIV/Girdin and DAPLE (Dvl-associating protein with a high frequency of leucine) are the best-characterized members of a group of cytoplasmic regulators that contain a Gα-binding and -activating (GBA) motif and whose dysregulation underlies human diseases, including cancer and birth defects. GBA motif–containing proteins were originally reported to modulate G proteins by binding Gα subunits of the Gi/o family (Gαi) over other families (such as Gs, Gq/11, or G12/13), and promoting nucleotide exchange in vitro. However, some evidence suggests that this is not always the case, as phosphorylation of the GBA motif of GIV promotes its binding to Gαs and inhibits nucleotide exchange. The G-protein specificity of DAPLE and how it might affect nucleotide exchange on G proteins besides Gαi remain to be investigated. Here, we show that DAPLE's GBA motif, in addition to Gαi, binds efficiently to members of the Gs and Gq/11 families (Gαs and Gαq, respectively), but not of the G12/13 family (Gα12) in the absence of post-translational phosphorylation. We pinpointed Met-1669 as the residue in the GBA motif of DAPLE that diverges from that in GIV and enables better binding to Gαs and Gαq. Unlike the nucleotide-exchange acceleration observed for Gαi, DAPLE inhibited nucleotide exchange on Gαs and Gαq. These findings indicate that GBA motifs have versatility in their G-protein–modulating effect, i.e. they can bind to Gα subunits of different classes and either stimulate or inhibit nucleotide exchange depending on the G-protein subtype.

Anesthetics Inhibit Acetylcholine-promoted Guanine Nucleotide Exchange of Heterotrimeric G Proteins of Airway Smooth Muscle

2004 ◽  
Vol 101 (1) ◽  
pp. 120-126 ◽  
Author(s):  
Chie Sakihara ◽  
William J. Perkins ◽  
David O. Warner ◽  
Keith A. Jones
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
G Protein ◽  
Muscarinic Receptor ◽  
G Proteins ◽  
Airway Smooth Muscle ◽  
Smooth Muscle Contraction ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Heterotrimeric G Protein

Background Anesthetics inhibit airway smooth muscle contraction in part by a direct effect on the smooth muscle cell. This study tested the hypothesis that the anesthetics halothane and hexanol, which both relax airway smooth muscle in vitro, inhibit acetylcholine-promoted nucleotide exchange at the alpha subunit of the Gq/11 heterotrimeric G protein (Galphaq/11; i.e., they inhibit muscarinic receptor-Galphaq/11 coupling). Methods The effect of halothane (0.38 +/- 0.02 mm) and hexanol (10 mm) on basal and acetylcholine-stimulated Galphaq/11 guanosine nucleotide exchange was determined in membranes prepared from porcine tracheal smooth muscle. The nonhydrolyzable, radioactive form of guanosine-5'-triphosphate, [S]GTPgammaS, was used as the reporter for Galphaq/11 subunit dissociation from the membrane to soluble fraction, which was immunoprecipitated with rabbit polyclonal anti-Galphaq/11 antiserum. Results Acetylcholine caused a significant time- and concentration-dependent increase in the magnitude of Galphaq/11 nucleotide exchange compared with basal values (i.e., without acetylcholine), reaching a maximal difference at 100 microm (35.9 +/-2.9 vs. 9.8 +/-1.2 fmol/mg protein, respectively). Whereas neither anesthetic had an effect on basal Galphaq/11 nucleotide exchange, both halothane and hexanol significantly inhibited the increase in Galphaq/11 nucleotide exchange produced by 30 microm acetylcholine (by 59% and 68%, respectively). Conclusions Halothane and hexanol interact with the receptor-heterotrimeric G-protein complex in a manner that prevents acetylcholine-promoted exchange of guanosine-5(')-triphosphate for guanosine-5'-diphosphate at Galphaq/11. These data are consistent with the ability of anesthetics to interfere with cellular processes mediated by heterotrimeric G proteins in many cells, including effects on muscarinic receptor-G-protein regulation of airway smooth muscle contraction.

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 Optogenetic activation of heterotrimeric G-proteins by LOV2GIVe, a rationally engineered modular protein

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Mikel Garcia-Marcos ◽  
Kshitij Parag-Sharma ◽  
Arthur Marivin ◽  
Marcin Maziarz ◽  
Alex Luebbers ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Protein Domain ◽  
Heterotrimeric G Proteins ◽  
Protein Activity ◽  
Signal Transducers ◽  
Non Invasive ◽  
Experimental Systems ◽  
Extracellular Stimuli ◽  
G Protein Coupled

Heterotrimeric G-proteins are signal transducers involved in mediating the action of many natural extracellular stimuli and many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we developed LOV2GIVe, an engineered modular protein that allows the activation of heterotrimeric G-proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, that is metazoan opsins, which are light-activated G-protein-coupled receptors (GPCRs). Instead, LOV2GIVe consists of the fusion of a G-protein activating peptide derived from a non-GPCR regulator of G-proteins to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity.

scholarly journals Receptor tyrosine kinases activate heterotrimeric G proteins via phosphorylation within the interdomain cleft of Gαi

2020 ◽  
Author(s):  
Nicholas A. Kalogriopoulos ◽  
Inmaculada Lopez-Sanchez ◽  
Changsheng Lin ◽  
Tony Ngo ◽  
Krishna Midde ◽  
...  
Keyword(s):  
Growth Factor ◽  
G Protein ◽  
G Proteins ◽  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Receptor Tyrosine Kinases ◽  
Second Messengers ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Key Steps

AbstractThe molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs crosstalk has been a long-standing question, but answers remain elusive. Using linear-ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator, GIV, dissociates Gαi•βγ trimers, scaffolds monomeric Gαi with RTKs, and facilitates the phosphorylation on two tyrosines located within the inter-domain cleft of Gαi. Phosphorylation triggers the activation of Gαi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cell’s decision to ‘go’ vs. ‘grow’. These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes.Significance StatementGrowth factors and heterotrimeric G proteins are two of the most widely studied signaling pathways in eukaryotes; their crosstalk shapes some of the most fundamental cellular responses in both health and disease. Although mechanisms by which G protein pathways transactivate growth factor RTKs has been well-defined, how the reverse may happen is less understood. This study defines the key steps and cellular consequences of a fundamental mechanism of signal crosstalk that enables RTKs to transactivate heterotrimeric G protein, Gαi. Mutations found in tumors shed light on how derailing this mechanism impacts tumor cell behavior. Thus, findings not only show how cells integrate extracellular signals via pathway crosstalk, but also demonstrate the relevance of this pathway in cancers.

scholarly journals RGS3 interacts with 14-3-3 via the N-terminal region distinct from the RGS (regulator of G-protein signalling) domain

2002 ◽  
Vol 365 (3) ◽  
pp. 677-684 ◽  
Author(s):  
Jiaxin NIU ◽  
Astrid SCHESCHONKA ◽  
Kirk M. DRUEY ◽  
Amanda DAVIS ◽  
Eleanor REED ◽  
...  
Keyword(s):  
Binding Site ◽  
G Protein ◽  
G Proteins ◽  
Rgs Proteins ◽  
Heterotrimeric G Proteins ◽  
G Protein Signalling ◽  
Vitro Binding ◽  
Rgs Domain ◽  
Hybrid Screening

RGS3 belongs to a family of the regulators of G-protein signalling (RGS), which bind and inhibit the Gα subunits of heterotrimeric G-proteins via a homologous RGS domain. Increasing evidence suggests that RGS proteins can also interact with targets other than G-proteins. Employing yeast two-hybrid screening of a cDNA library, we identified an interaction between RGS3 and the phosphoserine-binding protein 14-3-3. This interaction was confirmed by in vitro binding and co-immunoprecipitation experiments. RGS3-deletion analysis revealed the presence of a single 14-3-3-binding site located outside of the RGS domain. Ser264 was then identified as the 14-3-3-binding site of RGS3. The S264A mutation resulted in the loss of RGS3 binding to 14-3-3, without affecting its ability to bind Gαq. Signalling studies showed that the S264A mutant was more potent than the wild-type RGS3 in inhibition of G-protein-mediated signalling. Binding experiments revealed that RGS3 exists in two separate pools, either 14-3-3-bound or G-protein-bound, and that the 14-3-3-bound RGS3 is unable to interact with G-proteins. These data are consistent with the model wherein 14-3-3 serves as a scavenger of RGS3, regulating the amounts of RGS3 available for binding G-proteins. This study describes a new level in the regulation of G-protein signalling, in which the inhibitors of G-proteins, RGS proteins, can themselves be regulated by phosphorylation and binding 14-3-3.

scholarly journals Specific inhibition of GPCR-independent G protein signaling by a rationally engineered protein

2017 ◽  
Vol 114 (48) ◽  
pp. E10319-E10328 ◽  
Author(s):  
Anthony Leyme ◽  
Arthur Marivin ◽  
Marcin Maziarz ◽  
Vincent DiGiacomo ◽  
Maria P. Papakonstantinou ◽  
...  
Keyword(s):  
G Protein ◽  
Rational Design ◽  
G Protein Signaling ◽  
Guanine Nucleotide ◽  
Protein Signaling ◽  
Nucleotide Exchange ◽  
Protein Activation ◽  
Conserved Sequence ◽  
G Protein Activation

Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gβγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.

scholarly journals Biochemical characterization of RGS14: RGS14 activity towards G-protein α subunits is independent of its binding to Rap2A

2006 ◽  
Vol 394 (1) ◽  
pp. 309-315 ◽  
Author(s):  
Vivek Mittal ◽  
Maurine E. Linder
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Biochemical Characterization ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Subunit Dissociation ◽  
Α Subunits

RGS (regulators of G-protein signalling) modulate signalling by acting as GAPs (GTPase-activating proteins) for α subunits of heterotrimeric G-proteins. RGS14 accelerates GTP hydrolysis by Giα family members through its RGS domain and suppresses guanine nucleotide dissociation from Giα1 and Giα3 subunits through its C-terminal GoLoco domain. Additionally, RGS14 binds the activated forms of the small GTPases Rap1 and Rap2 by virtue of tandem RBDs (Raf-like Ras/Rap binding domains). RGS14 was identified in a screen for Rap2 effectors [Traver, Splingard, Gaudriault and De Gunzburg (2004) Biochem. J. 379, 627–632]. In the present study, we tested whether Rap binding regulates RGS14's biochemical activities. We found that RGS14 activity towards heterotrimeric G-proteins, as either a GAP or a GDI (guanine nucleotide dissociation inhibitor), was unaffected by Rap binding. Extending our biochemical characterization of RGS14, we also examined whether RGS14 can suppress guanine nucleotide exchange on Giα1 in the context of the heterotrimer. We found that a heterotrimer composed of N-myristoylated Giα1 and prenylated Gβγ is resistant to the GDI activity of the GoLoco domain of RGS14. This is consistent with models of GoLoco domain action on free Gα and suggests that RGS14 alone cannot induce subunit dissociation to promote receptor-independent activation of Gβγ-mediated signalling pathways.

Small GTP-binding protein-coupled receptors

2004 ◽  
Vol 32 (6) ◽  
pp. 1040-1044 ◽  
Author(s):  
M. Bhattacharya ◽  
A.V. Babwah ◽  
S.S.G. Ferguson
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Rho Gtpases ◽  
Eukaryotic Cell ◽  
Small Gtpases ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Cell Functions ◽  
Effector Pathways ◽  
Small G Proteins

Heterotrimeric GPCRs (G-protein-coupled receptors) form the largest group of integral membrane receptor proteins and mediate diverse physiological processes. In addition to signalling via heterotrimeric G-proteins, GPCRs can also signal by interacting with various small G-proteins to regulate downstream effector pathways. The small G-protein superfamily is structurally classified into at least five families: the Ras, Rho/Rac/cdc42, Rab, Sar1/Arf and Ran families. They are monomeric G-proteins with molecular masses over the range 20–30 kDa, which function as molecular switches to control many eukaryotic cell functions. Several studies have provided evidence of crosstalk between GPCRs and small G-proteins. It is well documented that GPCR signalling through heterotrimeric G-proteins can lead to the activation of Ras and Rho GTPases. In addition, RhoA, Rabs, ARFs and ARF GEFs (guanine nucleotide-exchange factors) can associate directly with GPCRs, and GPCRs may also function as GEFs for small GTPases. In this review, we summarize the recent progress made in understanding the interaction between GPCRs and small GTPases, focusing on understanding how the association of small G-proteins with GPCRs and GPCR-regulatory proteins may influence GPCR signalling and intracellular trafficking.

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