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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"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  . 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)    , 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  . 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 , highlighting the importance of the lipid environment for GPCR stability. "
"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  (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). "
"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  . 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  . "
[Show abstract][Hide abstract] 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.
Full-text · Article · Dec 2014 · Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics