Mini-G proteins: Novel tools for studying GPCRs in their active conformation

Nehmé, Rony; Carpenter, Byron; Singhal, Ankita; Strege, Annette; Edwards, Patricia C.; White, Courtney F.; Du, Haijuan; Grisshammer, Reinhard; Tate, Christopher G.

doi:10.1371/journal.pone.0175642

Cited by 232 publications

(304 citation statements)

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“…To increase Gs coupling, membrane of cells expressing Cys‐CRF1R mutants were treated with recombinant mini‐Gs; a truncated variant of Gαs . It has been shown that GPCRs bound to mini‐Gs proteins adopt similar active‐state conformations as those of Gs‐protein bound receptors . Indeed, yields of crosslinking on ClAc‐ligand/Cys‐CRF1R pairs increased by increasing the concentration of mini‐Gs in the probes (Figure A).…”

Section: Resultssupporting

confidence: 73%

Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions

et al. 2019

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Pairwise crosslinking is a powerful technique to characterize interactions between G protein coupled receptors and their ligands in the live cell. In this work, the “thiol trapping” method, which exploits the proximity‐enhanced reaction between haloacetamides and cysteine, is examined to identify intermolecular pairs of vicinal positions. By incorporating cysteine into the corticotropin‐releasing factor receptor and either α‐chloro‐ or α‐bromoacetamide groups into its ligands, it is shown that thiol trapping provides highly reproducible signals and a low background, and represents a valid alternative to classical “disulfide trapping”. The method is advantageous if reducing agents are required during sample analysis. Moreover, it can provide partially distinct spatial constraints, thus giving access to a wider dataset for molecular modeling. Finally, by applying recombinant mini‐Gs, GTPγS, and Gαs‐depleted HEK293 cells to modulate Gs coupling, it is shown that yields of crosslinking increase in the presence of elevated levels of Gs.

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Section: Resultssupporting

confidence: 73%

Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions

et al. 2019

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“…In this work we used a construct of A 2A R that contained thioredoxin at the N-terminus of the receptor 26 . This was originally designed with a rigid linker between the thioredoxin and the receptor to generate a large hydrophilic surface to A 2A R to improve crystallisation, although this proved unsuccessful.…”

Section: Resultsmentioning

confidence: 99%

“…The presence of thioredoxin did not significantly affect the pharmacology of A 2A R, as assessed by determination of its apparent K D for the inverse agonist ZM241385 or in agonist shift assays (Figure 1). It could also be purified to homogeneity and coupled effectively to both mini-G S 26 and to the heterotrimer containing mini-G S , β 1 , γ 2 and Nb35 (Figure 1). Detergent-solubilised A 2A R coupled to the heterotrimer had a molecular weight (excluding the detergent micelle of LMNG) of approximately 130 kDa 26 .…”

Section: Resultsmentioning

confidence: 99%

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Cryo-EM structure of the adenosine A_2A receptor coupled to an engineered heterotrimeric G protein

Tate

García-Nafría

Lee

et al. 2018

Preprint

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34The adenosine A 2A receptor (A 2A R) is a prototypical G protein-coupled receptor (GPCR) that 35 couples to the heterotrimeric G protein G S . Here we determine the structure by electron cryo-36 microscopy (cryo-EM) of A 2A R at pH 7.5 bound to the small molecule agonist NECA and 37 coupled to an engineered heterotrimeric G protein, which contains mini-G S , the βγ subunits 38 and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. 39 Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-G S 40 are virtually identical and that the density of the side chains and ligand are of comparable 41 quality. However, the cryo-EM density map also indicates regions that are flexible in 42 comparison to the crystal structures, which unexpectedly includes regions in the ligand 43 binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the 44 β subunit of the G protein was observed. 45 46 47 48 49The adenosine A 2A receptor (A 2A R) is an archetypical Class A G protein-coupled 50 receptor (GPCR) 1 . A 2A R is activated by the endogenous agonist adenosine and plays a 51 prominent role in cardiac function, the immune system and central nervous system, including 52 the release of the major excitatory neurotransmitter glutamate 2,3 . Given the widespread tissue 53 distribution and physiological relevance of A 2A R, it is a validated drug target for many 54 disorders 4 , including Parkinson's disease 5 and cancer 6 . A 2A R is one of the most stable GPCRs 55 and structures have been determined of A 2A R in an inactive state bound to inverse agonists 7-56 13 , an active intermediate state bound to agonists 14-16 and the fully active state bound to an 57 agonist and coupled to an engineered G protein, mini-G S 17 . In addition, structure-based drug 58 design has been applied to inactive state structures of A 2A R to develop potent and subtype 59 specific inverse agonists with novel scaffolds 9 and these are currently in clinical trials. 60Comparison of the structures has led to an understanding of the molecular determinants for 61 an inverse agonist compared to an agonist 15 , the conformational changes induced by agonist 62 binding to convert the inactive state to the active intermediate state 18 , and the role of the G 63 protein in stabilising the fully active state 17 . The active state was determined by crystallizing 64 the receptor coupled solely to mini-G S , an engineered G protein with eight point mutations 65 and three deletions, including the whole of the α-helical domain 19 . Although 66 pharmacologically mini-G S recapitulates the ability of a heterotrimeric G protein to increase 67 the affinity of agonist binding to the receptor 17 , the roles for the βγ subunits could not be 68 described. In terms of the interactions between a heterotrimeric G protein and a Class A 69 GPCR, the vast majority of interactions are made by the α subunit, in particular the C-70 terminal α5 helix 20 . However, there was an interaction be...

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“…Thus we focused on mini-G (mG) proteins, engineered versions of the Ras-like domain of G protein alpha subunits which bind directly to activated GPCRs but are not known to engage other cellular proteins 31,34 . We assessed binding to receptors in intact cells by redistribution of fluorescently labeled Nb or mG fusion proteins from the cytoplasm to the plasma membrane ( Figure 1B).…”

Section: Comparative Detection Of Direct Protein Recruitment By Opioimentioning

confidence: 99%

Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells

Stoeber

Jullié

et al. 2019

Preprint

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G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric 'bias' into the signaling system. However, the core underlying hypothesisthat different agonists drive GPCRs to engage different cytoplasmic proteins in living cellsremains untested due to the complexity of downstream readouts through which receptor-proximal interactions are typically inferred. Here we describe a scalable cell-based assay to overcome this challenge, based on the use of engineered GPCR-interacting proteins as orthogonal biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between protein probe recruitment produced by chemically distinct opioid ligands in living cells. We then show how the selective recruitment applies to GRK2, a biologically relevant opioid receptor regulator protein, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.

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Mini-G proteins: Novel tools for studying GPCRs in their active conformation

Cited by 232 publications

References 50 publications

Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions

Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions

Cryo-EM structure of the adenosine A_2A receptor coupled to an engineered heterotrimeric G protein

Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells

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Mini-G proteins: Novel tools for studying GPCRs in their active conformation

Cited by 232 publications

References 50 publications

Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions

Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions

Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein

Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells

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Cryo-EM structure of the adenosine A_2A receptor coupled to an engineered heterotrimeric G protein