Measurement of GPCR-G protein activity in living cells

Ratnayake, Kasun; Kankanamge, Dinesh; Senarath, Kanishka; Siripurapu, Praneeth; Weis, Nicole; Tennakoon, Mithila; Payton, John L.; Karunarathne, Ajith

doi:10.1016/bs.mcb.2017.07.008

Cited by 8 publications

(6 citation statements)

References 21 publications

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“…Super-resolution 3D immunohistologies like array tomography (Smith, 2018) and 3D single-molecule methods (Jia et al, 2014; von Diezmann et al, 2017) will enable imaging of dense-core vesicle localization and neuropeptide contents in type-specific network anatomical context. Genetically encoded fluorescent dense-core vesicle cargos will allow real-time detection of neuropeptide secretion (Ding et al, 2019), while genetically encoded sensors of extracellular GPCR ligands (Patriarchi et al, 2018; Sun et al, 2018), GPCR activation (Haider et al, 2019; Hill and Watson, 2018; Livingston et al, 2018; Ratnayake et al, 2017; Stoeber et al, 2018), G-protein mobilization (Ratnayake et al, 2017), cAMP concentration (Hackley et al, 2018; Ma et al, 2018), protein kinase activation (Chen et al, 2014) and protein phosphorylation (Haider et al, 2019) will enable fine dissection of NP dynamics and NP-GPCR signal transduction events (Spangler and Bruchas, 2017). In addition, new caged NP-GPCR ligands (Banghart et al, 2018) and antagonists (Banghart et al, 2013) will provide for precise spatial and temporal control for NP receptor activation.…”

Section: Discussionmentioning

confidence: 99%

Single-cell transcriptomic evidence for dense intracortical neuropeptide networks

Smith

Sümbül

Graybuck

et al. 2019

eLife

134

149

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Seeking new insights into the homeostasis, modulation and plasticity of cortical synaptic networks, we have analyzed results from a single-cell RNA-seq study of 22,439 mouse neocortical neurons. Our analysis exposes transcriptomic evidence for dozens of molecularly distinct neuropeptidergic modulatory networks that directly interconnect all cortical neurons. This evidence begins with a discovery that transcripts of one or more neuropeptide precursor (NPP) and one or more neuropeptide-selective G-protein-coupled receptor (NP-GPCR) genes are highly abundant in all, or very nearly all, cortical neurons. Individual neurons express diverse subsets of NP signaling genes from palettes encoding 18 NPPs and 29 NP-GPCRs. These 47 genes comprise 37 cognate NPP/NP-GPCR pairs, implying the likelihood of local neuropeptide signaling. Here, we use neuron-type-specific patterns of NP gene expression to offer specific, testable predictions regarding 37 peptidergic neuromodulatory networks that may play prominent roles in cortical homeostasis and plasticity.

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Section: Discussionmentioning

confidence: 99%

Single-cell transcriptomic evidence for dense intracortical neuropeptide networks

Smith

Sümbül

Graybuck

et al. 2019

eLife

134

149

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“…YFP-β1 and 2, kappa-opioid receptor, PI3K-CA-CFP and mCh-GPI were kind gifts from Professor N. Gautam's lab, Washington University, St. Louis, MO. Gγ3, Gγ9 and Gγ12 mutants were generated using Gibson assembly (NEB) (49). Parent constructs; mCherry-Gγ3, mCherry-Gγ9, and YFP-Gγ12 were PCR amplified with overhangs containing expected nucleotide mutations.…”

Section: Dna Constructs and Cell Linesmentioning

confidence: 99%

Gγ identity dictates efficacy of Gβγ signaling and macrophage migration

Senarath

Payton

Kankanamge

et al. 2018

Journal of Biological Chemistry

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“…Blue opsin-mTurquoise, GFP-Gγ9, GFP-Gγ3, mCh-Gβ1 and Akt-PH-mCh were kindly provided by Professor N. Gautam's lab, Washington University, St. Louis, MO. Gγ3 and Gγ9 mutants were generated by PCR amplifying the parent constructs in pcDNA3.1 (GFP-Gγ3 and GFP-Gγ9) with overhangs containing expected nucleotide mutations, DpnI (NEB) digestion (to remove the parent construct) followed by Gibson assembly (NEB) 61 . Cell lines: HeLa was originally purchased from the American Tissue Culture Collections (ATCC) and authenticated using a commercial kit to amplify nine unique STR loci.…”

Section: Dna Constructs and Cell Linesmentioning

confidence: 99%

A short C-terminal peptide in Gγ regulates Gβγ signaling efficacy

Tennakoon

Senarath

Kankanamge

et al. 2021

MBoC

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G protein beta-gamma (Gβγ) subunits anchor to the plasma membrane (PM) through the carboxy-terminal (CT) prenyl group in Gγ. This interaction is crucial for the PM localization and functioning of Gβγ, allowing GPCR-G protein signaling to proceed. The diverse Gγ family has twelve members, and we have recently shown that the signaling efficacies of major Gβγ effectors are Gγ-type dependent. This dependency is due to the distinct series of membrane-interacting abilities of Gγ. However, the molecular process allowing for Gβγ subunits to exhibit a discrete and diverse range of Gγ-type dependent membrane affinities is unclear and cannot be explained only using the type of prenylation. The present work explores the unique designs of membrane-interacting CT-residues in Gγ as a major source for this Gγ-type dependent Gβγ signaling. Despite the type of prenylation, results show signaling efficacy at the PM, and associated cell behaviors of Gβγ are governed by crucially located specific amino acids in the 5–6 residue pre-prenylation region of Gγ. The provided molecular picture of Gγ–membrane interactions may explain how cells gain Gγ-type dependent G protein-GPCR signaling as well as how Gβγ elicits selective signaling at various subcellular compartments.

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Measurement of GPCR-G protein activity in living cells

Cited by 8 publications

References 21 publications

Single-cell transcriptomic evidence for dense intracortical neuropeptide networks

Single-cell transcriptomic evidence for dense intracortical neuropeptide networks

Gγ identity dictates efficacy of Gβγ signaling and macrophage migration

A short C-terminal peptide in Gγ regulates Gβγ signaling efficacy

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