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


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.
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    • "Increasing amounts of transfected wt GPR64 led to a significant increase in cAMP levels (Fig. 1B). However, no significance was achieved in inositol phosphate (IP) accumulation assay with or without cotransfection of a chimeric Ga qi4 protein, which can redirect Gicoupled receptors to the PLC-b/inositol phosphate pathway [36] (data not shown). When comparing wt to the chimeric P2Y 12 -CTF GPR64 mutant in the same experimental setup, the mutant showed significant increase in cAMP levels and IP accumulation upon cotransfection with the Ga qi4 chimera indicative of a promiscuous coupling to Gs and Gi (Fig. 1C). "
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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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