Variable G protein determinants of GPCR coupling selectivity

Okashah, Najeah; Wan, Qingwen; Ghosh, Soumadwip; Sandhu, Manbir; Inoue, Asuka; Vaidehi, Nagarajan; Lambert, Nevin A.

doi:10.1073/pnas.1905993116

Cited by 127 publications

(154 citation statements)

References 37 publications

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“…A similar effect has been described for inverse-agonist-bound 5-HT 7 receptors (18), although the underlying mechanism is unclear. Forskolin binds directly to AC, but its actions are highly synergistic with Gα s (19,20), and G s is required for potent forskolin-induced AC activation in HEK 293 cells (21,22). Therefore, inhibition of forskolin action is consistent with efficient sequestration of G s heterotrimers by inactive 5-HT 7 receptors.…”

Section: Resultsmentioning

confidence: 99%

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

Jang

Adams

Liu

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Resultsmentioning

confidence: 99%

“…A plasmid encoding 5-HT 7 -SmBit was derived from unlabeled 5-HT 7 by standard subcloning into a SmBit vector. A plasmid encoding LgBit-Gγ 2 was kindly provided by Stephen (22,37,38). All plasmid constructs were verified by Sanger sequencing.…”

Section: Methodsmentioning

confidence: 99%

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

Jang

Adams

Liu

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…The Glosensor-22F cAMP plasmid (E2301) was obtained from Promega. Plasmids encoding Gα s Δ10, Venus-Kras, Venus-1-155-Gγ 1 , and Venus-155-239-Gβ 1 GPCR-luciferase constructs, and p115RhoGEF-Rluc8 have been described previously (6,15,36). Plasmids encoding rGFP-CAAX, rGFP-FYVE, and V 2 R-RlucII have been described previously (16).…”

Section: Methodsmentioning

confidence: 99%

“…Because receptor-G protein complexes are transient it can be difficult to detect agonist-induced BRET signals between receptors and G proteins when guanine nucleotides are present (6). For example, arginine vasopressin (AVP) did not detectably increase BRET between vasopressin V 2 receptors (V 2 R) and G s heterotrimers in the presence of GDP (Fig.…”

Section: Significancementioning

confidence: 99%

Agonist-induced formation of unproductive receptor-G ₁₂ complexes

Okashah

Wright

Kawakami

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2 receptors (V2R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gs complexes, V2R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V2R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

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“…amino acid sequence, polarity, charge, steric properties) to their selectivity for their cognate G-protein. The notion of a singular cognate G-protein for each receptor is also one that is challenged by evidence of receptor promiscuity to several Gα classes (23, 24) including melanopsin promiscuously activating transducin in vitro (25). GPCR regions important for G-protein binding selectivity include the 2 nd and 3 rd intracellular loops, and cytoplasmic extensions of transmembrane helices 5 and 6, which form critical contacts with helices 4 and 5 on Gα (26).…”

Section: Introductionmentioning

confidence: 99%

The C-terminus and Third Cytoplasmic Loop Cooperatively Activate Mouse Melanopsin Phototransduction

Valdez-Lopez

Petr

Donohue³

et al. 2020

Preprint

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27Melanopsin, an atypical vertebrate visual pigment, mediates non-image forming light responses 28 including circadian photoentrainment and pupillary light reflexes, and contrast detection for image 29formation. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), are 30 characterized by sluggish activation and deactivation of their light responses. The molecular determinants 31 of mouse melanopsin's deactivation have been characterized (i.e. C-terminal phosphorylation and β-32 arrestin binding), but a detailed analysis of melanopsin's activation is lacking. We propose that an 33 extended 3 rd cytoplasmic loop is adjacent to the proximal C-terminal region of mouse melanopsin in the 34 inactive conformation which is stabilized by ionic interaction of these two regions. This model is 35supported by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy of 36 melanopsin, the results of which suggests a high degree of steric freedom at the 3 rd cytoplasmic loop, 37which is increased upon C-terminus truncation, supporting the idea that these two regions are close in 3-38 dimensional space in wild-type melanopsin. To test for a functionally critical C-terminal conformation, 39 calcium imaging of melanopsin mutants including a proximal C-terminus truncation (at residue 365) and 40 proline mutation of this proximal region (H377P, L380P, Y382P) delayed melanopsin's activation rate. 41Mutation of all potential phosphorylation sites, including a highly conserved tyrosine residue (Y382), into 42 alanines also delayed the activation rate. A comparison of mouse melanopsin with armadillo 43 melanopsin-which has substitutions of various potential phosphorylation sites and a substitution of the 44 conserved tyrosine-indicates that substitution of these potential phosphorylation sites and the tyrosine 45 residue result in dramatically slower activation kinetics, a finding that also supports the role of 46 phosphorylation in signaling activation. We therefore propose that melanopsin's C-terminus is proximal 47 to intracellular loop 3 and C-terminal phosphorylation permits the ionic interaction between these two 48 regions, thus forming a stable structural conformation that is critical for initiating G-protein signaling. 49 50 STATEMENT OF SIGNIFICANCE 51 Melanopsin Structure and Activation 3 Melanopsin is an important visual pigment in the mammalian retina that mediates non-image 52forming responses such as circadian photoentrainment and pupil constriction, and supports contrast 53 detection for image formation. In this study, we detail two critical structural features of mouse 54 melanopsin-its 3 rd cytoplasmic loop and C-terminus-that are important in the activation of 55 melanopsin's light responses. Furthermore, we propose that these two regions directly participate in 56 coupling mouse melanopsin to its G-protein. These findings contribute to further understanding of GPCR-57 G-protein coupling, and given recent findings suggesting flexibility of melanopsin signal trans...

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Variable G protein determinants of GPCR coupling selectivity

Cited by 127 publications

References 37 publications

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

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

Agonist-induced formation of unproductive receptor-G ₁₂ complexes

The C-terminus and Third Cytoplasmic Loop Cooperatively Activate Mouse Melanopsin Phototransduction

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Variable G protein determinants of GPCR coupling selectivity

Cited by 127 publications

References 37 publications

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

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

Agonist-induced formation of unproductive receptor-G 12 complexes

The C-terminus and Third Cytoplasmic Loop Cooperatively Activate Mouse Melanopsin Phototransduction

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Agonist-induced formation of unproductive receptor-G ₁₂ complexes