scholarly journals Contributions of Intracellular Loops 2 and 3 of the Lutropin Receptor in Gs Coupling

2008 ◽  
Vol 22 (1) ◽  
pp. 126-138 ◽  
Author(s):  
Krassimira Angelova ◽  
Francesca Fanelli ◽  
David Puett
Keyword(s):  
G Protein ◽  
Hot Spots ◽  
Computational Models ◽  
Structural Changes ◽  
Protein Complexes ◽  
Receptor Activation ◽  
Receptor Sites ◽  
Receptor Recognition ◽  
Intracellular Loops

Abstract A number of amino acids essential for Gs coupling, i.e. hot spots, were identified after in vitro Ala-scanning mutagenesis of the cytosolic extensions of helices 3, 5, and 6 and of intracellular loops 2 and 3 (IL2 and IL3) of the human LH receptor (LHR). Consistent with the results of in vitro experiments involving ligand binding and ligand-mediated signaling in transiently transfected human embryonic kidney 293 cells, computational modeling of the isolated receptor and of the receptor-G protein complexes suggests an important role of the cytosolic extension of helix 3 and the N-terminal portion of the IL2 in Gsα interaction, whereas the contribution of IL3 is marginal. Mapping the hot spots into the computational models of LHR and the LHR-Gs complexes allowed for a distinction between receptor sites required for intramolecular structural changes (i.e. I460, T461, H466, and I549) and receptor sites more likely involved in G protein recognition (i.e. R464, T467, I468, Y470, Y550, and D564). The latter sites include the highly conserved arginine of the (E/D)R(Y/W) motif, which is therefore likely to be a receptor recognition point for Gs rather than a switch of receptor activation. The results of in vitro and in silico experiments carried out in this study represent the first comprehensive delineation of functionality of the individual residues in the intracellular domains of LHR and establish potential switches of receptor activation as well as a map of the primary receptor recognition sites for Gs. A novel way to consider constitutively active mutants was inferred from this study, i.e. receptor states with improved complementarity for the G protein compared to the wild-type receptor.

scholarly journals Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132

2018 ◽  
Vol 115 (37) ◽  
pp. 9252-9257 ◽  
Author(s):  
Jamie L. Lahvic ◽  
Michelle Ammerman ◽  
Pulin Li ◽  
Megan C. Blair ◽  
Emma R. Stillman ◽  
...  
Keyword(s):  
Fatty Acids ◽  
G Protein ◽  
Cell Lines ◽  
Saturated Fatty Acids ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Hydroxy Fatty Acids ◽  
High Affinity ◽  
G Protein Coupled

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based β-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure–activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

scholarly journals Dissecting the allosteric networks governing agonist efficacy and potency in G protein-coupled receptors

2021 ◽  
Author(s):  
Franziska Marie Heydenreich ◽  
Maria Marti-Solano ◽  
Manbir Sandhu ◽  
Brian K Kobilka ◽  
Michel Bouvier ◽  
...  
Keyword(s):  
G Protein ◽  
Conformational Changes ◽  
Structural Changes ◽  
Receptor Activation ◽  
Residue Level ◽  
G Protein Coupled Receptors ◽  
Maximal Response ◽  
Pharmacological Properties ◽  
Receptor Ligand ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) translate binding of extracellular ligands into intracellular responses through conformational changes. Ligand properties are described by the maximum response (efficacy) and the agonist concentration at half-maximal response (potency). Integrating structural changes with pharmacological properties remains challenging and has not yet been performed at the resolution of individual amino acids. We use epinephrine and β2-adrenergic receptor as a model to integrate residue-level pharmacology data with intramolecular residue contact data describing receptor activation. This unveils the allosteric networks driving ligand efficacy and potency. We provide detailed insights into how structural rearrangements are linked to fundamental pharmacological properties at single-residue level in a receptor-ligand system. Our approach can be used to determine such pharmacological networks for any receptor-ligand complex.

Characterization of a gastrin-type receptor on rabbit gastric parietal cells using L365,260 and L364,718

1991 ◽  
Vol 260 (2) ◽  
pp. G182-G188
Author(s):  
S. Roche ◽  
J. P. Bali ◽  
J. C. Galleyrand ◽  
R. Magous
Keyword(s):  
Acid Secretion ◽  
Parietal Cell ◽  
Inositol Phosphates ◽  
Secretory Activity ◽  
Receptor Activation ◽  
Parietal Cells ◽  
Receptor Sites ◽  
Gastric Parietal Cells ◽  
Type Receptor

Previous studies have demonstrated that gastrin and the COOH-terminal octapeptide of cholecystokinin (CCK-8) stimulated in vitro acid secretion from isolated rabbit gastric parietal cells. Both peptides bind to receptor sites located on these cells and induce an increase in phosphoinositide turnover and an uptake of [14C]aminopyrine ([14C]AP) with the same efficacy and potency. In the present study, we used the 3-(benzoylamino)-benzodiazepine analogue L365,260 and the 3-(acylamino)-benzodiazepine analogue L364,718 to investigate what type of receptor (gastrin type or CCK-A type) is involved in the regulation of the H+ secretory activity of the rabbit parietal cell. Neither L365,260 nor L364,718 alone caused stimulation of [3H]inositol phosphates ([3H]InsP) production. Each analogue inhibited 125I-labeled gastrin or 125I-CCK-8 binding to parietal cells and gastrin- or CCK-8-induced [3H]InsP production and [14C]AP accumulation. In all cases, L365,260 was approximately 70-100 times more potent than L364,718 (IC50 approximately 2-4 nM for L365,260 and approximately 0.2-0.4 microM for L364,718). Nevertheless, each antagonist displayed the same potency to inhibit the effects of gastrin or CCK-8. These results demonstrate that gastrin and CCK-8 interact with the same "gastrin-type" receptor on parietal cells. Moreover, L365,260 behaves as a competitive antagonist of the action of gastrin on parietal cells. Gastrin induces a rise in the levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] within the first seconds after parietal cell stimulation. The fact that L365,260 (10 nM) totally suppressed the gastrin-induced formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 suggests the involvement of these isomers in the mediation of acid secretion through gastrin receptor activation.

scholarly journals Mu and IS1 Transpositions Exhibit Strong Orientation Bias at the Escherichia coli bgl Locus

2001 ◽  
Vol 183 (11) ◽  
pp. 3328-3335 ◽  
Author(s):  
Dipankar Manna ◽  
Xiuhua Wang ◽  
N. Patrick Higgins
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Hot Spots ◽  
Protein Complexes ◽  
Hot Spot ◽  
High Affinity Site ◽  
Mu Transposition ◽  
Orientation Bias

ABSTRACT The region upstream of the Escherichia coli bgloperon is an insertion hot spot for several transposons. Elements as distantly related as Tn1, Tn5, and phage Mu home in on this location. To see what characteristics result in a high-affinity site for transposition, we compared in vivo and in vitro Mu transposition patterns near the bgl promoter. In vivo, Mu insertions were focused in two narrow zones of DNA nearbgl, and both zones exhibited a striking orientation bias. Five hot spots upstream of the bgl cyclic AMP binding protein (CAP) binding site had Mu insertions exclusively with the phage oriented left to right relative to the direction of bgl transcription. One hot spot within the CAP binding domain had the opposite (right-to-left) orientation of phage insertion. The DNA segment lying between these two Mu hot-spot clusters is extremely A/T rich (80%) and is an efficient target for insertion sequences during stationary phase. IS1 insertions that activate the bgl operon resulted in a decrease in Mu insertions near the CAP binding site. Mu transposition in vitro differed significantly from the in vivo transposition pattern, having a new hot-spot cluster at the border of the A/T-rich segment. Transposon hot-spot behavior and orientation bias may relate to an asymmetry of transposon DNA-protein complexes and to interactions with proteins that produce transcriptionally silenced chromatin.

scholarly journals Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

Cell Research ◽  
2021 ◽  
Author(s):  
Nicolas A. Heyder ◽  
Gunnar Kleinau ◽  
David Speck ◽  
Andrea Schmidt ◽  
Sarah Paisdzior ◽  
...  
Keyword(s):  
G Protein ◽  
Energy Homeostasis ◽  
Protein Complexes ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Binding Modes ◽  
Intracellular Loop ◽  
Melanocortin 4 Receptor ◽  
Biased Signaling ◽  
Gs Protein

AbstractThe melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R–Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor–Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.

Sensitivity of norepinephrine-evoked vasoconstriction to pertussis toxin in the old rat

1998 ◽  
Vol 274 (6) ◽  
pp. R1604-R1612 ◽  
Author(s):  
Alain Robert ◽  
Nguyen N. P. Tran ◽  
Philippe Giummelly ◽  
Jeffrey Atkinson ◽  
Christine Capdeville-Atkinson
Keyword(s):  
Intracellular Calcium ◽  
G Protein ◽  
Pertussis Toxin ◽  
Receptor Activation ◽  
Plasma Norepinephrine ◽  
Adrenoceptor Antagonist ◽  
Vasoconstrictor Response ◽  
High K ◽  
Male Wistar Rats

In male Wistar rats, the in vitro vasoconstrictor response of the perfused tail artery elicited by norepinephrine or serotonin decreased with age (24 mo old vs. 3 mo old), whereas the fluorescent signal (fura 2) produced by intracellular calcium ([Formula: see text]) mobilization increased. Both vasoconstriction and the increase in intracellular calcium concentration elicited by a high-K+, depolarizing solution were unaffected by aging. Pertussis toxin, a G protein inhibitor, had no effect on vasoconstriction induced by high K+ but diminished vasoconstrictor responses to norepinephrine in 3- and 12-mo-old animals but not in 24-mo-old animals. Pertussis toxin had no effect on[Formula: see text] mobilization. The sensitivity of receptor activation to pertussis toxin in tail arteries from 24-mo-old animals was restored by pretreatment with the α-adrenoceptor antagonist nicergoline. Nicergoline had no effect on vasoconstriction induced by high K+. Plasma norepinephrine concentration rose with age; nicergoline had no effect on this rise. We suggest that aging leads to a decrease in the intracellular G protein-modulated amplification of vasoconstriction produced by receptor activation and that this could be linked to the hyperadrenergic state. Ca2+sensitivity can be restored by chronic treatment with an α-adrenoceptor antagonist.

A universal technology for monitoring G‐protein‐coupled receptor activation in vitro and noninvasively in live animals

2007 ◽  
Vol 21 (14) ◽  
pp. 3819-3826 ◽  
Author(s):  
Georges Degenfeld ◽  
Tom S. Wehrman ◽  
Mark M. Hammer ◽  
Helen M. Blau
Keyword(s):  
G Protein ◽  
Receptor Activation ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals Functional Microswitches of Mammalian G Protein-Coupled Bitter-Taste Receptors

2020 ◽  
Author(s):  
Jérémie Topin ◽  
Cédric Bouysset ◽  
Yiseul Kim ◽  
MeeRa Rhyu ◽  
Sébastien Fiorucci ◽  
...  
Keyword(s):  
G Protein ◽  
Bitter Taste ◽  
Current Knowledge ◽  
Receptor Activation ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Taste Receptors ◽  
Bitter Taste Receptors ◽  
G Protein Coupled

AbstractBitter taste receptors (TAS2Rs) are a poorly understood subgroup of G protein-coupled receptors (GPCR). No experimental structure of these receptors is available and key-residues controlling their function remain mostly unknown. Here, we have identified the functional microswitches that encode agonist sensing and downstream signaling mechanisms within TAS2Rs sequences. We thoroughly re-aligned the amino-acid sequences of the 25 human TAS2Rs considering residue conservations and all the experimental data from the literature as constraints. As a test case, an accurate homology model of TAS2R16 was constructed and examined by site-directed mutagenesis and in vitro functional assays. Conserved motifs acting as microswitches during agonist-sensing and receptor activation were pinpointed by comparison with the current knowledge on class A GPCRs. Unravelling these sequence – function relationships is of utmost importance to streamline how TAS2Rs functions are encrypted in their sequence.

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