Substitution of three amino acids switches receptor specificity of Gq to that of Giα. Nature

Department of Pharmacology, University of California, San Francisco 94143.
Nature (Impact Factor: 41.46). 06/1993; 363(6426):274-6. DOI: 10.1038/363274a0
Source: PubMed

ABSTRACT Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement of GDP bound to the alpha-subunit by GTP. mutations in the C terminus of the alpha-subunit, its covalent modification by pertussis toxin-catalysed ribosylation of ADP, peptide-specific antibodies directed against it, and peptides mimicking C-terminal sequences, all inhibit receptor-mediated activation of G proteins. The logical prediction--that specific amino-acid residues at the C-termini of alpha-subunits can determine the abilities of individual G proteins to discriminate among specific subsets of receptors--has so far not been tested experimentally. Different hormone receptors specifically activate Gq or Gi, whose alpha-subunits (alpha q or alpha i) stimulate phosphatidylinositol-specific phospholipase C or inhibit adenylyl cyclase, respectively. Here we replace C-terminal amino acids of alpha q with the corresponding residues of alpha i2 to create alpha q/alpha i2 chimaeras that can mediate stimulation of phospholipase C by receptors otherwise coupled exclusively to Gi. A minimum of three alpha i2 amino acids, including a glycine three residues from the C terminus, suffices to switch the receptor specificity of the alpha q/alpha i2 chimaeras. We propose that a C-terminal turn, centered on this glycine, plays an important part in specifying receptor interactions of G proteins in the Gi/Go/Gz family.

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Available from: Bruce Conklin, Mar 26, 2014
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    • "The cellular response depends on the G protein subtype, and specific GPCRs can couple through one or more G protein subtypes, which are typically classified by the α subunit of the heterotrimer, with four families identified to date: G s , G i , G q , and G 12 [2] [3]. The Gα subunit directly interacts with the receptor through interactions with the transmembrane (TM) core (TM3, 5, and 6) and intracellular loops (IC2 and 3) [4] [5] [6] [7], which leads to a large conformational change in the G protein allowing the exchange of GDP for GTP in the nucleotide binding pocket, and initiating downstream signalling, through the α subunit and the βγ heterodimer [4] [8]. Part of the recent crystallographic success which has advanced our understanding of GPCR activation can be attributed to the use of lipidic mesophases for crystallisation [9], highlighting the importance of the lipid environment for GPCR stability. "
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    ABSTRACT: Upon binding of extracellular ligands, G protein coupled-receptors (GPCRs) initiate signalling cascades by activating heterotrimeric G proteins through direct interactions with the α subunit. While the lipid dependence of ligand binding has previously been studied for one class A GPCR, the neurotensin receptor 1 (NTS1), the role the lipid environment plays in the interaction of activated GPCRs with G proteins is less well understood. It is therefore of interest to understand the balance of lipid interactions required to support both ligand binding and G protein activation, not least since some receptors have multiple locations, and may experience different membrane environments when signalling in the plasma membrane or during endocytosis. Here, using the sensitive biophysical technique of microscale thermophoresis in conjunction with nanodisc lipid bilayer reconstitution, we show that in more native lipid environments rich in phosphatidyl ethanolamine (PE), the Gαi1 subunit has a ~4-fold higher affinity for NTS1 than in the absence of native lipids. The G protein-receptor affinity was further shown to be dependent on the ligand-binding state of the receptor, with potential indication of biased signalling for the known antagonist SR142948A. Gαi1 also showed preferential interaction with empty nanodiscs of native lipid mixtures rich in PE by around 2- to 4-fold over phosphatidyl choline (PC)/phosphatidyl glycerol (PG) lipid mixtures. The lipid environment may therefore play a role in creating favourable micro-environments for efficient GPCR signalling. Our approach combining nanodiscs with microscale thermophoresis will be useful in future studies to elucidate further the complexity of the GPCR interactome. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 08/2015; 1848(11 Pt A). DOI:10.1016/j.bbamem.2015.08.004 · 4.66 Impact Factor
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    • "By monitoring downstream events of the GPCR signaling transduction cascade, such as second messenger activity and transcription of target genes, many different strategies have been developed for the detection of GPCR activation in mammalian cells [183] [184]. Many of the cell-based assays rely on the expression of promiscuous G-protein α subunits or chimeric G-proteins, which renders them applicable to all GPCRs regardless of the endogenous G-protein coupling and consequently eliminates the need for prior knowledge of the interacting G-protein [185] [186]. "
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    ABSTRACT: Neuropeptides are key messengers in almost all physiological processes. They originate from larger precursors and are extensively processed to become bioactive. Neuropeptidomics aims to comprehensively identify the collection of neuropeptides in an organism, organ, tissue or cell. The neuropeptidome of several invertebrates is thoroughly explored since they are important model organisms (and models for human diseases), disease vectors and pest species. The charting of the neuropeptidome is the first step towards understanding peptidergic signaling. This review will first discuss the latest developments in exploring the neuropeptidome. The physiological roles and modes of action of neuropeptides can be explored in two ways, which are largely orthogonal and therefore complementary. The first way consists of inferring the functions of neuropeptides by a forward approach where neuropeptide profiles are compared under different physiological conditions. Second is the reverse approach were neuropeptide collections are used to screen for receptor-binding. This is followed by localization studies and functional tests. This review will focus on how these different functional screening methods contributed to the field of invertebrate neuropeptidomics and expanded our knowledge of peptidergic signaling. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 12/2014; 1854(7). DOI:10.1016/j.bbapap.2014.12.011 · 2.75 Impact Factor
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    • "Together with residues from the lower parts of the α5-helix (Q370, R371, H373 and R375 in Gαs and I344 and N347 in Gαi), which interact with amino acids from the N-terminal part of IL3 of the receptors, they constitute, in general, the main determinant of coupling selectivity of G-proteins. The importance of these regions is supported by mutational studies [37]–[40]. In agreement with functional experiments with artificial model proteins indicating the importance of the N-terminal part of IL3 for D2R coupling [41], [42], the selectivity-determining areas of the D2R-Gαi-complexes were found to be located in the intracellular TM5/N-terminal IL3-region of D2R and the C-terminal part of Gαi. "
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    ABSTRACT: Based on the recently described crystal structure of the β2 adrenergic receptor - Gs-protein complex, we report the first molecular-dynamics simulations of ternary GPCR complexes designed to identify the selectivity determinants for receptor-G-protein binding. Long-term molecular dynamics simulations of agonist-bound β2AR-Gαs and D2R-Gαi complexes embedded in a hydrated bilayer environment and computational alanine-scanning mutagenesis identified distinct residues of the N-terminal region of intracellular loop 3 to be crucial for coupling selectivity. Within the G-protein, specific amino acids of the α5-helix, the C-terminus of the Gα-subunit and the regions around αN-β1 and α4-β6 were found to determine receptor recognition. Knowledge of these determinants of receptor-G-protein binding selectivity is essential for designing drugs that target specific receptor/G-protein combinations.
    PLoS ONE 06/2013; 8(6):e67244. DOI:10.1371/journal.pone.0067244 · 3.23 Impact Factor
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