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

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010801117 ◽

2020 ◽

Vol 117 (48) ◽

pp. 30755-30762

Author(s):

Wonjo Jang ◽

C. Elizabeth Adams ◽

Heng Liu ◽

Cheng Zhang ◽

Finn Olav Levy ◽

...

Keyword(s):

G Protein ◽

G Proteins ◽

Ternary Complex ◽

Dynamic Range ◽

Protein Complexes ◽

Binary Complex ◽

Agonist Binding ◽

Inactive State ◽

Stimulus Response ◽

The Impact

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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Heterotrimeric G proteins promote FLS2 protein accumulation through inhibition of FLS2 autophagic degradation

10.1101/438135 ◽

2018 ◽

Author(s):

Jimi C. Miller ◽

Stacey A. Lawrence ◽

Nicole K. Clay

Keyword(s):

Plasma Membrane ◽

G Protein ◽

G Proteins ◽

Protein Complex ◽

Heterotrimeric G Proteins ◽

Immune Signaling ◽

Heterotrimeric G Protein ◽

Protein Levels ◽

Autophagic Degradation ◽

Protein Components

ABSTRACTFLAGELLIN-SENSITIVE 2 (FLS2) is a plant immune receptor that binds bacterial flagellin to activate immune signaling. This immune signal is transduced by a heterotrimeric G protein complex at the plasma membrane and activates downstream signaling. However, it is unknown whether the heterotrimeric G proteins have functions at other subcellular locations away from the plasma membrane. Here, we show that components of the heterotrimeric G protein complex stabilize FLS2 protein levels by inhibiting the autophagic degradation of FLS2. Using genetic analysis, we determined that mutations of G protein components resulted in reduced immune signaling in part due to decreased FLS2 protein levels. Furthermore, reduction of FLS2 protein levels was caused by elevated proteasomal and autophagic degradation of FLS2. Genetic inhibition of autophagy in G protein mutants rescued FLS2 levels and immunity. Our findings suggest that the heterotrimeric G protein components, in addition to being part of the heterotrimeric G protein complex that transduces signals at the plasma membrane, also function away from the plasma membrane to control FLS2 protein levels. These results expand the functional capacity of the heterotrimeric G protein complexes in plant immunity.

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Complexes between photoactivated rhodopsin and transducin: progress and questions

Biochemical Journal ◽

10.1042/bj20100270 ◽

2010 ◽

Vol 428 (1) ◽

pp. 1-10 ◽

Cited By ~ 32

Author(s):

Beata Jastrzebska ◽

Yaroslav Tsybovsky ◽

Krzysztof Palczewski

Keyword(s):

G Protein ◽

Conformational Changes ◽

Structural Changes ◽

Protein Complexes ◽

G Protein Coupled Receptors ◽

Heterotrimeric G Proteins ◽

Α Subunit ◽

Rapid Responses ◽

G Protein Α Subunit ◽

G Protein Coupled

Activation of GPCRs (G-protein-coupled receptors) leads to conformational changes that ultimately initiate signal transduction. Activated GPCRs transiently combine with and activate heterotrimeric G-proteins resulting in GTP replacement of GDP on the G-protein α subunit. Both the detailed structural changes essential for productive GDP/GTP exchange on the G-protein α subunit and the structure of the GPCR–G-protein complex itself have yet to be elucidated. Nevertheless, transient GPCR–G-protein complexes can be trapped by nucleotide depletion, yielding an empty-nucleotide G-protein–GPCR complex that can be isolated. Whereas early biochemical studies indicated formation of a complex between G-protein and activated receptor only, more recent results suggest that G-protein can bind to pre-activated states of receptor or even couple transiently to non-activated receptor to facilitate rapid responses to stimuli. Efficient and reproducible formation of physiologically relevant, conformationally homogenous GPCR–G-protein complexes is a prerequisite for structural studies designed to address these possibilities.

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Every Detail Matters. That Is, How the Interaction between Gα Proteins and Membrane Affects Their Function

Membranes ◽

10.3390/membranes11030222 ◽

2021 ◽

Vol 11 (3) ◽

pp. 222

Author(s):

Agnieszka Polit ◽

Paweł Mystek ◽

Ewa Błasiak

Keyword(s):

Signal Transduction ◽

G Protein ◽

G Proteins ◽

Lipid Membranes ◽

Signaling Proteins ◽

Heterotrimeric G Proteins ◽

Membrane Attachment ◽

The Individual ◽

Multicellular Organisms ◽

G Protein Coupled

In highly organized multicellular organisms such as humans, the functions of an individual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increasing interest in studying the accompanying protein–lipid interactions, which have been demonstrated to not only provide the environment but also regulate proper and efficient signal transduction. The mode of interaction between the cell membrane and G proteins is well known. Despite this, the recognition mechanisms at the molecular level and how the individual G protein-membrane attachment signals are interrelated in the process of the complex control of membrane targeting of G proteins remain unelucidated. This review focuses on the mechanisms by which mammalian Gα subunits of G proteins interact with lipids and the factors responsible for the specificity of membrane association. We summarize recent data on how these signaling proteins are precisely targeted to a specific site in the membrane region by introducing well-defined modifications as well as through the presence of polybasic regions within these proteins and interactions with other components of the heterocomplex.

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Signaling from G Protein-coupled Receptors to ERK5/Big MAPK 1 Involves Gαqand Gα12/13Families of Heterotrimeric G Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m002410200 ◽

2000 ◽

Vol 275 (28) ◽

pp. 21730-21736 ◽

Cited By ~ 67

Author(s):

Shigetomo Fukuhara ◽

Maria Julia Marinissen ◽

Mario Chiariello ◽

J. Silvio Gutkind

Keyword(s):

G Protein ◽

G Proteins ◽

G Protein Coupled Receptors ◽

Heterotrimeric G Proteins ◽

G Protein Coupled

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Physiological Regulation of G Protein-Linked Signaling

Physiological Reviews ◽

10.1152/physrev.1999.79.4.1373 ◽

1999 ◽

Vol 79 (4) ◽

pp. 1373-1430 ◽

Cited By ~ 307

Author(s):

Andrew J. Morris ◽

Craig C. Malbon

Keyword(s):

G Protein ◽

G Proteins ◽

Chemokine Receptors ◽

Transcriptional Control ◽

Heterotrimeric G Proteins ◽

Protein Prenylation ◽

Physiological Control ◽

Physiological Regulation ◽

Surface Receptors ◽

Genes Encoding

Heterotrimeric G proteins in vertebrates constitute a family molecular switches that transduce the activation of a populous group of cell-surface receptors to a group of diverse effector units. The receptors include the photopigments such as rhodopsin and prominent families such as the adrenergic, muscarinic acetylcholine, and chemokine receptors involved in regulating a broad spectrum of responses in humans. Signals from receptors are sensed by heterotrimeric G proteins and transduced to effectors such as adenylyl cyclases, phospholipases, and various ion channels. Physiological regulation of G protein-linked receptors allows for integration of signals that directly or indirectly effect the signaling from receptor→G protein→effector(s). Steroid hormones can regulate signaling via transcriptional control of the activities of the genes encoding members of G protein-linked pathways. Posttranscriptional mechanisms are under physiological control, altering the stability of preexisting mRNA and affording an additional level for regulation. Protein phosphorylation, protein prenylation, and proteolysis constitute major posttranslational mechanisms employed in the physiological regulation of G protein-linked signaling. Drawing upon mechanisms at all three levels, physiological regulation permits integration of demands placed on G protein-linked signaling.

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Characterisation of Antibody Interactions with the G Protein of Vesicular Stomatitis Virus Indiana Strain and Other Vesiculovirus G Proteins

10.1101/330910 ◽

2018 ◽

Cited By ~ 1

Author(s):

Altar M Munis ◽

Maha Tijani ◽

Mark Hassall ◽

Giada Mattiuzzo ◽

Mary K Collins ◽

...

Keyword(s):

G Protein ◽

G Proteins ◽

Vesicular Stomatitis Virus ◽

Conformational Changes ◽

Lentiviral Vectors ◽

Medicinal Products ◽

Vaccine Vectors ◽

Receptor Binding Site ◽

Advanced Therapy ◽

Vesicular Stomatitis

ABSTRACTVesicular stomatitis virus Indiana strain G protein (VSVind.G) is the most commonly used envelope glycoprotein to pseudotype lentiviral vectors (LV) for experimental and clinical applications. Recently, G proteins derived from other vesiculoviruses (VesG), for example Cocal virus, have been proposed as alternative LV envelopes with possible advantages compared to VSVind.G. Well-characterised antibodies that recognise VesG will be useful for vesiculovirus research, development of G protein-containing advanced therapy medicinal products (ATMPs), and deployment of VSVind-based vaccine vectors. Here we show that one commercially available monoclonal antibody, 8G5F11, binds to and neutralises G proteins from three strains of VSV as well as Cocal, and Maraba viruses, whereas the other commercially available monoclonal anti-VSVind.G antibody, IE9F9, binds to and neutralises only VSVind.G. Using a combination of G protein chimeras and site-directed mutations, we mapped the binding epitopes of IE9F9 and 8G5F11 on VSVind.G. IE9F9 binds close to the receptor binding site and competes with soluble low-density lipoprotein receptor (LDLR) for binding to VSVind.G, explaining its mechanism of neutralisation. In contrast, 8G5F11 binds close to a region known to undergo conformational changes when the G protein moves to its post-fusion structure, and we propose that 8G5F11 cross-neutralises VesGs by inhibiting this.IMPORTANCEVSVind.G is currently regarded as the gold-standard envelope to pseudotype lentiviral vectors. However, recently other G proteins derived from vesiculoviruses have been proposed as alternative envelopes. Here, we investigated two anti-VSVind.G monoclonal antibodies for their ability to cross-react with other vesiculovirus G proteins, and identified the epitopes they recognise, and explored the mechanisms behind their neutralisation activity. Understanding how cross-neutralising antibodies interact with other G proteins may be of interest in the context of host-pathogen interaction and co-evolution as well as providing the opportunity to modify the G proteins and improve G protein-containing medicinal products and vaccine vectors.

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Class Frizzled GPCRs (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

IUPHAR/BPS Guide to Pharmacology CITE ◽

10.2218/gtopdb/f25/2019.4 ◽

2019 ◽

Vol 2019 (4) ◽

Author(s):

Elisa Arthofer ◽

Jacomijn Dijksterhuis ◽

Belma Hot ◽

Pawe&lstrok; Kozielewicz ◽

Matthias Lauth ◽

...

Keyword(s):

G Proteins ◽

Conformational Changes ◽

Hedgehog Signaling ◽

Cell Signalling ◽

Resonance Energy Transfer ◽

Rho Kinase ◽

Density Lipoprotein ◽

Resonance Energy ◽

Scaffolding Protein ◽

Heterotrimeric G Proteins

Receptors of the Class Frizzled (FZD, nomenclature as agreed by the NC-IUPHAR subcommittee on the Class Frizzled GPCRs [156]), are GPCRs originally identified in Drosophila [17], which are highly conserved across species. While SMO shows structural resemblance to the 10 FZDs, it is functionally separated as it mediates effects in the Hedgehog signaling pathway [156]. FZDs are activated by WNTs, which are cysteine-rich lipoglycoproteins with fundamental functions in ontogeny and tissue homeostasis. FZD signalling was initially divided into two pathways, being either dependent on the accumulation of the transcription regulator β-catenin or being β-catenin-independent (often referred to as canonical vs. non-canonical WNT/FZD signalling, respectively). WNT stimulation of FZDs can, in cooperation with the low density lipoprotein receptors LRP5 (O75197) and LRP6 (O75581), lead to the inhibition of a constitutively active destruction complex, which results in the accumulation of β-catenin and subsequently its translocation to the nucleus. β-Catenin, in turn, modifies gene transcription by interacting with TCF/LEF transcription factors. β-Catenin-independent FZD signalling is far more complex with regard to the diversity of the activated pathways. WNT/FZD signalling can lead to the activation of heterotrimeric G proteins [28, 159, 135], the elevation of intracellular calcium [164], activation of cGMP-specific PDE6 [2] and elevation of cAMP as well as RAC-1, JNK, Rho and Rho kinase signalling [48]. Novel resonance energy transfer-based tools have allowed the study of the GPCR-like nature of FZDs in greater detail. Upon ligand stimulation, FZDs undergo conformational changes and signal via heterotrimeric G proteins [213, 214]. Furthermore, the phosphoprotein Dishevelled constitutes a key player in WNT/FZD signalling. Importantly, FZDs exist in at least two distinct conformational states that regulate the pathway selection [214]. As with other GPCRs, members of the Frizzled family are functionally dependent on the arrestin scaffolding protein for internalization [19], as well as for β-catenin-dependent [12] and -independent [80, 13] signalling. The pattern of cell signalling is complicated by the presence of additional ligands, which can enhance or inhibit FZD signalling (secreted Frizzled-related proteins (sFRP), Wnt-inhibitory factor (WIF), sclerostin or Dickkopf (DKK)), as well as modulatory (co)-receptors with Ryk, ROR1, ROR2 and Kremen, which may also function as independent signalling proteins.

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Heterotrimeric G-Proteins Are Indispensable for FLT3-ITD autophosphorylation and Oncogenic Function

Blood ◽

10.1182/blood.v128.22.2712.2712 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2712-2712

Author(s):

Maike Rehage ◽

Susanne Wingert ◽

Nadine Haetscher ◽

Sabrina Bothur ◽

Hubert Serve ◽

...

Keyword(s):

Tyrosine Kinase ◽

G Protein ◽

G Proteins ◽

B Cell Lymphoma ◽

Physical Interaction ◽

Heterotrimeric G Proteins ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Oncogenic Function

Abstract Heterotrimeric G-proteins transmit signals of G-protein coupled receptors and regulate many basic cellular functions. However, their role in normal and malignant hematopoietic stem cells remains obscure. Activating mutations in the heterotrimeric G-protein Gaq were found in various cancers and its expression is enhanced in diffuse large B-cell lymphoma and T-ALL. Our previous data suggested the involvement of heterotrimeric G-proteins in Flt3-ITD-mediated leukemic transformation. FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD) is a frequent oncoprotein in acute myeloid leukemia causing constitutive active STAT5 signaling. Here, we investigated a novel role of Gaq in Flt3-ITD-induced leukemic transformation. We could show that Gaq is indispensable for aberrant FLT3-ITD activation and oncogenic function as Gaq activity is necessary to maintain the autophosphorylation of FLT3-ITD. Gaq abrogation resulted in diminished cell proliferation and colony formation as well as delayed leukemogenesis in vivo of Flt3-ITD leukemic cells. Importantly, the growth inhibition could be rescued by addition of IL3 and did not occur in the presence of FLT3 ligand-activated FLT3 wildtype receptor, demonstrating the specificity of Gaq requirement for FLT3-ITD oncogenic signaling. Interestingly, co-immunoprecipitations revealed a direct physical interaction between FLT3-ITD and Gaq which did not require phosphorylation of the receptor tyrosine kinase. Hence, FLT3-ITD hyperphosphorylation seems to be rather a consequence of the interaction than a prerequisite. Flt3-ITD-induced transformation of murine hematopoietic stem/progenitor cells (HSPCs) strictly depended on the presence of Gaq, and the ablation of Gaq/11 in transplanted Flt3-ITD-transduced HSPCs from conditional Gaq/11 double knock-out mice delayed leukemic burden. These findings of an unexpected, yet critical, role of Gaq place the molecule as an important target for antileukemic strategies. Disclosures No relevant conflicts of interest to declare.

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