scholarly journals A G-protein activation cascade from Arl13B to Arl3 and implications for ciliary targeting of lipidated proteins

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Katja Gotthardt ◽  
Mandy Lokaj ◽  
Carolin Koerner ◽  
Nathalie Falk ◽  
Andreas Gießl ◽  
...  
Keyword(s):  
G Protein ◽  
Joubert Syndrome ◽  
Nucleotide Exchange ◽  
Protein Activation ◽  
Guanine Nucleotide Exchange ◽  
Mammalian Cell Lines ◽  
Nucleotide Exchange Factor ◽  
Cargo Proteins ◽  
Switch Regions ◽  
Activation Cascade

Small G-proteins of the ADP-ribosylation-factor-like (Arl) subfamily have been shown to be crucial to ciliogenesis and cilia maintenance. Active Arl3 is involved in targeting and releasing lipidated cargo proteins from their carriers PDE6δ and UNC119a/b to the cilium. However, the guanine nucleotide exchange factor (GEF) which activates Arl3 is unknown. Here we show that the ciliary G-protein Arl13B mutated in Joubert syndrome is the GEF for Arl3, and its function is conserved in evolution. The GEF activity of Arl13B is mediated by the G-domain plus an additional C-terminal helix. The switch regions of Arl13B are involved in the interaction with Arl3. Overexpression of Arl13B in mammalian cell lines leads to an increased Arl3·GTP level, whereas Arl13B Joubert-Syndrome patient mutations impair GEF activity and thus Arl3 activation. We anticipate that through Arl13B’s exclusive ciliary localization, Arl3 activation is spatially restricted and thereby an Arl3·GTP compartment generated where ciliary cargo is specifically released.

scholarly journals The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

2015 ◽  
Vol 112 (5) ◽  
pp. 1404-1409 ◽  
Author(s):  
Ned Van Eps ◽  
Celestine J. Thomas ◽  
Wayne L. Hubbell ◽  
Stephen R. Sprang
Keyword(s):  
G Protein ◽  
Bound States ◽  
Guanine Nucleotide Exchange Factor ◽  
Nucleotide Binding ◽  
Conformational Ensemble ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF.

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 Ric-8B Is a GTP-dependent G Protein αs Guanine Nucleotide Exchange Factor

2011 ◽  
Vol 286 (22) ◽  
pp. 19932-19942 ◽  
Author(s):  
PuiYee Chan ◽  
Meital Gabay ◽  
Forrest A. Wright ◽  
Gregory G. Tall
Keyword(s):  
G Protein ◽  
Asymmetric Cell Division ◽  
Odorant Receptor ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Gtpγs Binding

ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including Gαq and Gαs regulation of neurotransmission, Gαi-dependent mitotic spindle positioning during (asymmetric) cell division, and Gαolf-dependent odorant receptor signaling. Mammalian Ric-8 activities are partitioned between two genes, ric-8A and ric-8B. Ric-8A is a guanine nucleotide exchange factor (GEF) for Gαi/αq/α12/13 subunits. Ric-8B potentiated Gs signaling presumably as a Gαs-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be Gα subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed Gαs and lesser amounts of Gαq and Gα13. Ric-8BFL stimulated guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding to these subunits and Gαolf, whereas the Ric-8BΔ9 isoform stimulated Gαs short GTPγS binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the Vmax of Gαs steady state GTP hydrolysis and the apparent Km values of GTP binding to Gαs from ∼385 nm to an estimated value of ∼42 μm. Directionality of the Ric-8BFL-catalyzed Gαs exchange reaction was GTP-dependent. At sub-Km GTP, Ric-BFL was inhibitory to exchange despite being a rapid GDP release accelerator. Ric-8BFL binds nucleotide-free Gαs tightly, and near-Km GTP levels were required to dissociate the Ric-8B·Gα nucleotide-free intermediate to release free Ric-8B and Gα-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce Gα-GTP in cells.

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