GPCR structure and function relationship: identification of a biased apelin receptor mutant

2018 ◽  
Vol 475 (23) ◽  
pp. 3813-3826 ◽  
Author(s):  
Ting Ban ◽  
Xun Li ◽  
Xiaochuan Ma ◽  
Hui Yang ◽  
Yunpeng Song ◽  
...  
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Molecular Mechanism ◽  
Transmembrane Domain ◽  
Hydrophobic Interactions ◽  
Double Mutation ◽  
Mutant Receptor ◽  
Therapeutic Benefits ◽  
Biased Signaling ◽  
Apelin Receptor

Biased ligands of G protein-coupled receptors (GPCRs) may have improved therapeutic benefits and safety profiles. However, the molecular mechanism of GPCR biased signaling remains largely unknown. Using apelin receptor (APJ) as a model, we systematically investigated the potential effects of amino acid residues around the orthosteric binding site on biased signaling. We discovered that a single residue mutation I109A (I1093.32) in the transmembrane domain 3 (TM3) located in the deep ligand-binding pocket was sufficient to convert a balanced APJ into a G protein signaling biased receptor. APJ I109A mutant receptor retained full capabilities in ligand binding and G protein activation, but was defective in GRK recruitment, β-arrestin recruitment, and downstream receptor-mediated ERK activation. Based on molecular dynamics simulations, we proposed a molecular mechanism for biased signaling of I109A mutant receptor. We postulate that due to the extra space created by I109A mutation, the phenyl group of the last residue (Phe-13) of apelin rotates down and initiates a cascade of conformational changes in TM3. Phe-13 formed a new cluster of hydrophobic interactions with the sidechains of residues in TM3, including F1103.33 and M1133.36, which stabilizes the mutant receptor in a conformation favoring biased signaling. Interruption of these stabilizing interactions by double mutation F110A/I109A or M113A/I109A largely restored the β-arrestin-mediated signaling. Taken together, we describe herein the discovery of a biased APJ mutant receptor and provide detailed molecular insights into APJ signaling selectivity, facilitating the discovery of novel therapeutics targeting APJ.

scholarly journals Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor

2003 ◽  
Vol 371 (2) ◽  
pp. 443-449 ◽  
Author(s):  
Frank NEUSCHÄFER-RUBE ◽  
Eva ENGEMAIER ◽  
Sina KOCH ◽  
Ulrike BÖER ◽  
Gerhard P. PÜSCHEL
Keyword(s):  
Amino Acids ◽  
Ligand Binding ◽  
G Protein ◽  
Transmembrane Domain ◽  
Sequence Similarity ◽  
Membrane Receptors ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Receptor Subtypes ◽  
Plasma Membrane Receptors

Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand-binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF2α and PGD2. Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF2α-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation.

scholarly journals A structural basis for how ligand binding site changes can allosterically regulate GPCR signaling and engender functional selectivity

2020 ◽  
Vol 13 (617) ◽  
pp. eaaw5885 ◽  
Author(s):  
Marta Sanchez-Soto ◽  
Ravi Kumar Verma ◽  
Blair K. A. Willette ◽  
Elizabeth C. Gonye ◽  
Annah M. Moore ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
G Protein ◽  
Structural Basis ◽  
Protein Signaling ◽  
Ligand Binding Site ◽  
Intracellular Loop ◽  
Gpcr Signaling ◽  
Biased Signaling ◽  
Dynamics Simulations

Signaling bias is the propensity for some agonists to preferentially stimulate G protein–coupled receptor (GPCR) signaling through one intracellular pathway versus another. We previously identified a G protein–biased agonist of the D2 dopamine receptor (D2R) that results in impaired β-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with β-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired β-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the β2-adrenergic receptor (β2R) to build β2R-WT and β2R-Y1995.38A models in complex with the full β2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in β2R-Y1995.38A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in β2R-Y1995.38A, which is predicted to affect its interactions with β-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling.

scholarly journals High Throughput Quantitation of cAMP Production Mediated by Activation of Seven Transmembrane Domain Receptors

1999 ◽  
Vol 4 (1) ◽  
pp. 27-32 ◽  
Author(s):  
Ilona Kariv ◽  
Michelle E. Stevens ◽  
Davette L. Behrens ◽  
Kevin R. Oldenburg
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Transmembrane Domain ◽  
Receptor Function ◽  
Camp Production ◽  
Ligand Interaction ◽  
G Protein Coupled ◽  
Native Ligand

Impairment of G protein—coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate™ (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within ≤20 μm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein—coupled receptor function as measured by cAMP production and is suitable for high throughput screening.

scholarly journals A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled α-Factor Receptor

2004 ◽  
Vol 24 (5) ◽  
pp. 2041-2051 ◽  
Author(s):  
Jennifer C. Lin ◽  
Ken Duell ◽  
James B. Konopka
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Transmembrane Domains ◽  
Consecutive Series ◽  
Subsequent Step ◽  
G Protein Coupled ◽  
Specific Reagent

ABSTRACT The α-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.

scholarly journals Elucidation of Molecular Mechanism of a Selective PPARα Modulator, Pemafibrate, through Combinational Approaches of X-ray Crystallography, Thermodynamic Analysis, and First-Principle Calculations

2020 ◽  
Vol 21 (1) ◽  
pp. 361 ◽  
Author(s):  
Mayu Kawasaki ◽  
Akira Kambe ◽  
Yuta Yamamoto ◽  
Sundaram Arulmozhiraja ◽  
Sohei Ito ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Molecular Mechanism ◽  
Structural Changes ◽  
Molecular Level ◽  
Hydrophobic Interactions ◽  
Binding Pocket ◽  
Titration Calorimetry ◽  
X Ray ◽  
X Ray Crystallography ◽  
Steroid Receptor Coactivator

The selective PPARα modulator (SPPARMα) is expected to medicate dyslipidemia with minimizing adverse effects. Recently, pemafibrate was screened from the ligand library as an SPPARMα bearing strong potency. Several clinical pieces of evidence have proved the usefulness of pemafibrate as a medication; however, how pemafibrate works as a SPPARMα at the molecular level is not fully known. In this study, we investigate the molecular mechanism behind its novel SPPARMα character through a combination of approaches of X-ray crystallography, isothermal titration calorimetry (ITC), and fragment molecular orbital (FMO) analysis. ITC measurements have indicated that pemafibrate binds more strongly to PPARα than to PPARγ. The crystal structure of PPARα-ligand binding domain (LBD)/pemafibrate/steroid receptor coactivator-1 peptide (SRC1) determined at 3.2 Å resolution indicates that pemafibrate binds to the ligand binding pocket (LBP) of PPARα in a Y-shaped form. The structure also reveals that the conformation of the phenoxyalkyl group in pemafibrate is flexible in the absence of SRC1 coactivator peptide bound to PPARα; this gives a freedom for the phenoxyalkyl group to adopt structural changes induced by the binding of coactivators. FMO calculations have indicated that the accumulation of hydrophobic interactions provided by the residues at the LBP improve the interaction between pemafibrate and PPARα compared with the interaction between fenofibrate and PPARα.

scholarly journals Molecular Mechanism of Biased Signaling in a Prototypical G-protein-coupled Receptor

2020 ◽  
Vol 118 (3) ◽  
pp. 162a
Author(s):  
Carl-Mikael Suomivuori ◽  
Naomi R. Latorraca ◽  
Laura M. Wingler ◽  
Stephan Eismann ◽  
Matthew C. King ◽  
...  
Keyword(s):  
G Protein ◽  
Molecular Mechanism ◽  
G Protein Coupled Receptor ◽  
Biased Signaling ◽  
G Protein Coupled

scholarly journals Identification of Serine 348 on the Apelin Receptor as a Novel Regulatory Phosphorylation Site in Apelin-13-induced G Protein-independent Biased Signaling

2014 ◽  
Vol 289 (45) ◽  
pp. 31173-31187 ◽  
Author(s):  
Xiaoyu Chen ◽  
Bo Bai ◽  
Yanjun Tian ◽  
Hui Du ◽  
Jing Chen
Keyword(s):  
G Protein ◽  
Phosphorylation Site ◽  
Regulatory Phosphorylation ◽  
Biased Signaling ◽  
Apelin Receptor

scholarly journals Cell surface delivery and structural re-organization by pharmacological chaperones of an oligomerization-defective α1b-adrenoceptor mutant demonstrates membrane targeting of GPCR oligomers

2008 ◽  
Vol 417 (1) ◽  
pp. 161-172 ◽  
Author(s):  
Meritxell Canals ◽  
Juan F. Lopez-Gimenez ◽  
Graeme Milligan
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Ligand Binding ◽  
Transmembrane Domain ◽  
Early Stage ◽  
Mutant Cell ◽  
Critical Role ◽  
Wild Type ◽  
Pharmacological Chaperones ◽  
Mutant Receptor

Many G-protein-coupled receptors, including the α1b-adrenoceptor, form homo-dimers or oligomers. Mutation of hydrophobic residues in transmembrane domains I and IV alters the organization of the α1b-adrenoceptor oligomer, with transmembrane domain IV playing a critical role. These mutations also result in endoplasmic reticulum trapping of the receptor. Following stable expression of this α1b-adrenoceptor mutant, cell surface delivery, receptor function and structural organization were recovered by treatment with a range of α1b-adrenoceptor antagonists that acted at the level of the endoplasmic reticulum. This was accompanied by maturation of the mutant receptor to a terminally N-glycosylated form, and only this mature form was trafficked to the cell surface. Co-expression of the mutant receptor with an otherwise wild-type form of the α1b-adrenoceptor that is unable to bind ligands resulted in this wild-type variant also being retained in the endoplasmic reticulum. Ligand-induced cell surface delivery of the mutant α1b-adrenoceptor now allowed co-recovery to the plasma membrane of the ligand-binding-deficient mutant. These results demonstrate that interactions between α1b-adrenoceptor monomers occur at an early stage in protein synthesis, that ligands of the α1b-adrenoceptor can act as pharmacological chaperones to allow a structurally compromised form of the receptor to pass cellular quality control, that the mutated receptor is not inherently deficient in function and that an oligomeric assembly of ligand-binding-competent and -incompetent forms of the α1b-adrenoceptor can be trafficked to the cell surface by binding of a ligand to only one component of the receptor oligomer.

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