An inactive receptor-G protein complex maintains the dynamic range of agonist-induced signaling

Agonist binding promotes activation of G protein-coupled receptors (GPCRs) and association of active receptors with G protein heterotrimers. The resulting active-state ternary complex is the basis for conventional stimulus-response coupling. Although GPCRs can also associate with G proteins before agonist binding, the impact of such preassociated complexes on agonist-induced signaling is poorly understood. Here we show that preassociation of 5-HT7serotonin receptors with Gsheterotrimers is necessary for agonist-induced signaling. 5-HT7receptors in their inactive state associate with Gs, as these complexes are stabilized by inverse agonists and receptor mutations that favor the inactive state. Inactive-state 5-HT7–Gscomplexes dissociate in response to agonists, allowing the formation of conventional agonist–5-HT7–Gsternary complexes and subsequent Gsactivation. Inactive-state 5-HT7–Gscomplexes are required for the full dynamic range of agonist-induced signaling, as 5-HT7receptors spontaneously activate Gsvariants that cannot form inactive-state complexes. Therefore, agonist-induced signaling in this system involves two distinct receptor-G protein complexes, a conventional ternary complex that activates G proteins and an inverse-coupled binary complex that maintains the inactive state when agonist is not present.

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Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1417573112 ◽

2015 ◽

Vol 112 (11) ◽

pp. E1181-E1190 ◽

Cited By ~ 23

Author(s):

Matthias Hillenbrand ◽

Christian Schori ◽

Jendrik Schöppe ◽

Andreas Plückthun

Keyword(s):

G Protein ◽

G Proteins ◽

Conformational Changes ◽

Protein Complex ◽

Protein Complexes ◽

Heterotrimeric G Proteins ◽

Agonist Binding ◽

Transient Nature ◽

The Stability ◽

Neurotensin Receptor 1

Agonist binding to G-protein–coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1or αsLand all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1βγ combinations containing γ1and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.

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Heterotrimeric G-protein shuttling via Gip1 extends the dynamic range of eukaryotic chemotaxis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1516767113 ◽

2016 ◽

Vol 113 (16) ◽

pp. 4356-4361 ◽

Cited By ~ 13

Author(s):

Yoichiro Kamimura ◽

Yukihiro Miyanaga ◽

Masahiro Ueda

Keyword(s):

G Protein ◽

G Proteins ◽

Dynamic Range ◽

Protein Translocation ◽

Receptor Activation ◽

Dependent Manner ◽

Heterotrimeric G Protein ◽

Range Sensing ◽

Interacting Protein ◽

Wide Range

Chemotactic eukaryote cells can sense chemical gradients over a wide range of concentrations via heterotrimeric G-protein signaling; however, the underlying wide-range sensing mechanisms are only partially understood. Here we report that a novel regulator of G proteins, G protein-interacting protein 1 (Gip1), is essential for extending the chemotactic range of Dictyostelium cells. Genetic disruption of Gip1 caused severe defects in gradient sensing and directed cell migration at high but not low concentrations of chemoattractant. Also, Gip1 was found to bind and sequester G proteins in cytosolic pools. Receptor activation induced G-protein translocation to the plasma membrane from the cytosol in a Gip1-dependent manner, causing a biased redistribution of G protein on the membrane along a chemoattractant gradient. These findings suggest that Gip1 regulates G-protein shuttling between the cytosol and the membrane to ensure the availability and biased redistribution of G protein on the membrane for receptor-mediated chemotactic signaling. This mechanism offers an explanation for the wide-range sensing seen in eukaryotic chemotaxis.

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Lifetime of muscarinic receptor–G-protein complexes determines coupling efficiency and G-protein subtype selectivity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715751115 ◽

2018 ◽

Vol 115 (19) ◽

pp. 5016-5021 ◽

Cited By ~ 10

Author(s):

Olga S. Ilyaskina ◽

Horst Lemoine ◽

Moritz Bünemann

Keyword(s):

G Protein ◽

G Proteins ◽

Mammalian Cells ◽

Protein Complexes ◽

Coupling Efficiency ◽

Dissociation Kinetics ◽

Intact Cells ◽

Permeabilized Cells ◽

Subtype Selectivity ◽

Intracellular Signals

G-protein–coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR–G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of Gi/o, Gq/11, Gs, and G12/13 proteins to muscarinic M1, M2, and M3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein–M3–R interactions but rather the affinities of Gq and Go proteins to M3–Rs, their GPCR–G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

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A Novel Nuclear Signaling Pathway for Thromboxane A2 Receptors in Oligodendrocytes: Evidence for Signaling Compartmentalization during Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.00482-08 ◽

2008 ◽

Vol 28 (20) ◽

pp. 6329-6341 ◽

Cited By ~ 12

Author(s):

Fozia Mir ◽

Guy C. Le Breton

Keyword(s):

Signaling Pathway ◽

G Protein ◽

G Proteins ◽

Protein Complexes ◽

Functional Coupling ◽

Protein Signaling ◽

Creb Phosphorylation ◽

Expression Levels ◽

Nuclear Signaling ◽

Subcellular Compartmentalization

ABSTRACT The present study investigated G protein expression, localization, and functional coupling to thromboxane A2 receptors (TPRs) during oligodendrocyte (OLG) development. It was found that as OLGs mature, the expression levels of Gq increase while those of G13 decrease. In contrast, the expression levels of Gs, Go, and Gi do not change significantly. Localization studies revealed that Gq, G13, and Gi are present only in the extranuclear compartment, whereas Gs and Go are found in both the extranuclear and the nuclear compartments. Purification of TPR-G protein complexes demonstrated that TPRs couple to both Gq and G13 in the extranuclear compartment but only to Gs in the nuclear compartment. Furthermore, functional analysis revealed that stimulation of nuclear TPR in OLGs stimulates CREB phosphorylation and myelin basic protein transcription and increases survival. Collectively, these results demonstrate that (i) OLGs selectively modulate the expression of certain G proteins during development, (ii) G proteins are differentially localized in OLGs leading to subcellular compartmentalization, (iii) TPRs couple to Gq and G13 in the extranuclear compartment and to Gs only in the nucleus, (iv) mature OLGs have a functional nuclear TPR-Gs signaling pathway, and (v) nuclear TPR signaling can stimulate CREB phosphorylation and myelin gene transcription and increase cell survival. These findings represent a novel paradigm for selective modulation of G protein-coupled receptor-G protein signaling during cell development.

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Conformational plasticity of the intracellular cavity of GPCR−G-protein complexes leads to G-protein promiscuity and selectivity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1820944116 ◽

2019 ◽

pp. 201820944 ◽

Cited By ~ 10

Author(s):

Manbir Sandhu ◽

Anja M. Touma ◽

Matthew Dysthe ◽

Fredrik Sadler ◽

Sivaraj Sivaramakrishnan ◽

...

Keyword(s):

G Protein ◽

Protein Complexes ◽

Dependent Manner ◽

Secondary Messenger ◽

C Terminus ◽

Conformational Plasticity ◽

Small Ensemble ◽

Dynamics Simulations ◽

The Impact ◽

Dose Dependent

While the dynamics of the intracellular surface in agonist-stimulated GPCRs is well studied, the impact of GPCR dynamics on G-protein selectivity remains unclear. Here, we combine molecular dynamics simulations with live-cell FRET and secondary messenger measurements, for 21 GPCR−G-protein combinations, to advance a dynamic model of the GPCR−G-protein interface. Our data show C terminus peptides of Gαs, Gαi, and Gαq proteins assume a small ensemble of unique orientations when coupled to their cognate GPCRs, similar to the variations observed in 3D structures of GPCR−G-protein complexes. The noncognate G proteins interface with latent intracellular GPCR cavities but dissociate due to weak and unstable interactions. Three predicted mutations in β2-adrenergic receptor stabilize binding of noncognate Gαq protein in its latent cavity, allowing promiscuous signaling through both Gαs and Gαq in a dose-dependent manner. This demonstrates that latent GPCR cavities can be evolved, by design or nature, to tune G-protein selectivity, giving insights to pluridimensional GPCR signaling.

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Molecular basis for high-affinity agonist binding in GPCRs

Science ◽

10.1126/science.aau5595 ◽

2019 ◽

Vol 364 (6442) ◽

pp. 775-778 ◽

Cited By ~ 25

Author(s):

Tony Warne ◽

Patricia C. Edwards ◽

Andrew S. Doré ◽

Andrew G. W. Leslie ◽

Christopher G. Tate

Keyword(s):

Hydrogen Bonds ◽

G Protein ◽

Molecular Basis ◽

G Protein Coupled Receptors ◽

Active State ◽

Agonist Binding ◽

Inactive State ◽

High Affinity ◽

Wide Range ◽

G Protein Coupled

G protein–coupled receptors (GPCRs) in the G protein–coupled active state have higher affinity for agonists as compared with when they are in the inactive state, but the molecular basis for this is unclear. We have determined four active-state structures of the β1-adrenoceptor (β1AR) bound to conformation-specific nanobodies in the presence of agonists of varying efficacy. Comparison with inactive-state structures of β1AR bound to the identical ligands showed a 24 to 42% reduction in the volume of the orthosteric binding site. Potential hydrogen bonds were also shorter, and there was up to a 30% increase in the number of atomic contacts between the receptor and ligand. This explains the increase in agonist affinity of GPCRs in the active state for a wide range of structurally distinct agonists.

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Structures in G proteins important for subtype selective receptor binding and subsequent activation

Communications Biology ◽

10.1038/s42003-021-02143-9 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Volker Jelinek ◽

Nadja Mösslein ◽

Moritz Bünemann

Keyword(s):

G Protein ◽

G Proteins ◽

Protein Complexes ◽

Heterotrimeric G Proteins ◽

Structural Determinants ◽

C Terminus ◽

Subsequent Activation ◽

The Stability ◽

Subtype Selective ◽

G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) selectively couple to specific heterotrimeric G proteins comprised of four subfamilies in order to induce appropriate physiological responses. However, structural determinants in Gα subunits responsible for selective recognition by approximately 800 human GPCRs have remained elusive. Here, we directly compare the influence of subtype-specific Gα structures on the stability of GPCR-G protein complexes and the activation by two Gq-coupled receptors. We used FRET-assays designed to distinguish multiple Go and Gq-based Gα chimeras in their ability to be selectively bound and activated by muscarinic M3 and histaminic H1 receptors. We identify the N-terminus including the αN/β1-hinge, the β2/β3-loop and the α5 helix of Gα to be key selectivity determinants which differ in their impact on selective binding to GPCRs and subsequent activation depending on the specific receptor. Altogether, these findings provide new insights into the molecular basis of G protein-coupling selectivity even beyond the Gα C-terminus.

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