Relevant role of Leu265 in helix VI of the angiotensin AT1 receptor in agonist binding and activity

2002 ◽  
Vol 80 (5) ◽  
pp. 426-430 ◽  
Author(s):  
Silvana Aparecida Alves Correa ◽  
Lucimar Pereira França ◽  
Claudio Miguel Costa-Neto ◽  
Laerte Oliveira ◽  
Antonio Cechelli Mattos Paiva ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Transmembrane Domain ◽  
Inositol Phosphate ◽  
Chinese Hamster ◽  
Side Chain ◽  
Maximum Effect ◽  
Agonist Binding

The finding of critical residues for angiotensin II (AII) binding and receptor signalling in helices V and VI led us to assess if, in this region of the receptor, aliphatic side chains might play a role in the agonist-mediated mechanism. Two mutations of the angiotensin AT1 receptor were designed to explore a possible role of a leucine at two positions, Leu265 and Leu268. Thus two mutants, L265D and L268D, were prepared through single substitutions of Leu265, located in the C-terminal region of transmembrane VI (TM-VI), and Leu268, in the adjoining region of the third extracellular loop (EC-3), for an aspartyl residue, and were stably transfected into Chinese hamster ovary (CHO) cells. Ligand-binding experiments and the functional assays determining inositol phosphate (IP) production were performed in these cells expressing these mutants. No significant changes were found in the binding affinity for the ligands, AII, DuP753, and [Sar1Leu8]AII in the mutant L268D. Moreover, the relative potency and the maximum effect on IP production of this mutant were similar to those of the wild-type receptor. In contrast, L265D mutant AT1 receptor, located within the transmembrane domain, markedly decreased binding affinity and ability to stimulate phosphatidylinositol turnover. Our results suggest that the hydrophobic side chain of Leu265, at the C-terminal portion of the AT1's TM-VI, but not Leu268, which belongs to the EC-3 loop, might interact with the AII molecule. On the other side, the aliphatic side chain of Leu265 may be involved in the formation of the ligand binding sites through allosteric effects, thus helping to stabilize the receptor structure around the agonist binding site for full activity.Key words: angiotensin II, AT1 receptor, site-directed mutagenesis.

scholarly journals The Constitutively Active N111G-AT1 Receptor for Angiotensin II Maintains a High Affinity Conformation Despite Being Uncoupled from Its Cognate G Protein Gq/11α

Endocrinology ◽  
2003 ◽  
Vol 144 (12) ◽  
pp. 5277-5284 ◽  
Author(s):  
Mannix Auger-Messier ◽  
Martin Clement ◽  
Pascal M. Lanctot ◽  
Patrice C. Leclerc ◽  
Richard Leduc ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
G Protein ◽  
Binding Affinity ◽  
Transmembrane Domain ◽  
Critical Role ◽  
At1 Receptor ◽  
Agonist Binding ◽  
Wild Type ◽  
High Affinity ◽  
Constitutively Active

Abstract Asn111, localized in the third transmembrane domain of the AT1 receptor for angiotensin II, plays a critical role in stabilizing the inactive conformation of the receptor. We evaluated the functional and G protein-coupling properties of mutant AT1 receptors in which Asn111 was substituted with smaller (Ala or Gly) or larger residues (Gln or Trp). All four mutants were expressed at high levels in COS-7 cells and, except for N111W-AT1, recognized 125I-Ang II with high affinities comparable to that of the wild-type AT1 receptor. In phospholipase C assays, the four mutants encompassed the entire spectrum of functional states, ranging from constitutive activity (without agonist) for N111A-AT1 and N111G-AT1 to a significant loss of activity (upon maximal stimulation) for N111Q-AT1 and a major loss of activity for N111W-AT1. In Ca2+ mobilization studies, N111W-AT1 produced a weak Ca2+ transient and, unexpectedly, N111G-AT1 also produced a Ca2+ transient that was much weaker than that of the wild-type AT1. The agonist binding affinity of N111W-AT1 was not modified in the presence of GTPγ S, suggesting that this receptor is not basally coupled to a G protein. GTPγ S did not modify the high agonist-binding affinity of N111G-AT1 but abolished the coimmunoprecipitation of Gq/11α with this constitutively active mutant receptor. These results are a direct demonstration that the N111G-AT1 receptor maintains a high affinity conformation despite being uncoupled from the G protein Gq/11.

Electron Transport from QA to Thymoquinone in a Synechococcus Oxygen-Evolving Photosystem II Preparation: Role of QB and Binding Affinity of Thymoquinone to the QB Site

1993 ◽  
Vol 48 (3-4) ◽  
pp. 174-178 ◽  
Author(s):  
Kazuhiko Satoh ◽  
Yasuhiro Kashino ◽  
Hiroyuki Koike
Keyword(s):  
Binding Affinity ◽  
Turnover Rate ◽  
Side Chain ◽  
Head Group ◽  
Binding Affinities ◽  
Ps Ii ◽  
Thermophilic Cyanobacteria ◽  
High Concentrations ◽  
Oxygen Evolving

Abstract We have recently shown that binding affinities of benzoquinones can be estimated by two methods in photosystem (PS) II particles (K. Satoh et al., Biochim. Biophys. Acta 1102, 45-52 (1992)). Using these methods we calculated the binding affinity of thymoquinone (2-methyl-5-isopropyl-p-benzoquinone) to the QB site and studied how the quinone accepts electrons in oxygen-evolving PS II particles isolated from the thermophilic cyanobacteria, Synechococcus elongatus and S. vulcanus. The results are as follows: (1) The binding constant of thymoqui­ none to the QB site determined by several methods was around 0.33 mᴍ . (2) At low thymoquinone concentrations the quinone was supposed to accept electrons via QB-plastoquinone, whereas at high concentrations the quinone seemed to bind to the QB site and accept an electron directly from Q-A. Lower rates of photoreduction of the quinone at high concentrations were attributed to a slower turnover rate of the quinone at the QB site than that of endogenous plastoquinone. (3) A model for the function of plastoquinone at the QB site, which can explain all the results, was presented. According to this model, the plastoquinone molecule at the QB site is not replaced by another plastoquinone molecule. Instead, it transfers electrons to pool plastoquinone molecules by turning over its head group but remaining its long side chain bound to the PS II complexes.

Role of Phe308 in the seventh transmembrane domain of the AT2 receptor in ligand binding and signaling

2004 ◽  
Vol 319 (4) ◽  
pp. 1138-1143 ◽  
Author(s):  
Lakshmi Pulakat ◽  
Chirag H Mandavia ◽  
Nara Gavini
Keyword(s):  
Ligand Binding ◽  
Transmembrane Domain ◽  
At2 Receptor

Role of Side-Chain Bioisosteres in Determining the Binding Affinity of Click Chemistry Derived RGD Peptidomimetics to αvβ3Integrin

2014 ◽  
Vol 2014 (34) ◽  
pp. 7595-7604 ◽  
Author(s):  
Pierangelo Fabbrizzi ◽  
Gloria Menchi ◽  
Silvia Raspanti ◽  
Antonio Guarna ◽  
Andrea Trabocchi
Keyword(s):  
Click Chemistry ◽  
Binding Affinity ◽  
Side Chain

The Cytoplasmic Domain of Glycoprotein Ibβ Is Critical to Surface Trafficking of Glycoprotein Ib-IX Complex.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1531-1531
Author(s):  
Xi Mo ◽  
Jose A. Lopez ◽  
Renhao Li
Keyword(s):  
Cho Cells ◽  
Transmembrane Domain ◽  
Complex Structure ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Expression Level ◽  
Platelet Glycoprotein ◽  
Cellular Expression

Abstract Platelet glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to the subendothelium of damaged vessel walls under high shear conditions. Delineating the assembly process of the GP Ib-IX complex will aid our understanding of the complex structure and shed light on the signaling mechanism underlying platelet activation. The GP Ib-IX complex comprises three polypeptides, GP Ibα, GP Ibβ and GP IX, and efficient surface expression of the complex in transfected Chinese hamster ovary (CHO) cells requires all of its three subunits, indicating that the assembly of the complex in the endoplasmic reticulum is required for its subsequent trafficking to the plasma membrane. We recently showed that the interaction between the transmembrane domain of GP Ibβ and the other subunits is critical to efficient surface expression of the GP Ib-IX complex. Here, we have explored the role of the Ibβ cytoplasmic domain in the complex assembly and surface expression. In CHO cells transiently expressing the mutant Ib-IX complex in which the Ibβ cytoplasmic domain was deleted or replaced entirely with the IX counterpart, neither cellular expression of GP Ibα nor surface expression of GP Ibα and IX was detected. In contrast, deletion and/or replacement of the Ibα or IX cytoplasmic domains did not affect significantly the assembly and surface expression of the receptor complex. Furthermore, deletion of the last six residues in the Ibβ cytoplasmic domain did not affect the cellular expression level of GP Ibα but significantly decreased its surface expression level in CHO cells. Intriguingly, further deletion of the Ibβ cytoplasmic domain that removed the binding site for 14-3-3ζ restored surface expression of the GP Ib-IX complex to a level comparable to the wild type construct. Overall, our results demonstrated an important role of the Ibβ cytoplasmic domain in both assembly and trafficking of the GP Ib-IX complex. Critical residues in the Ibβ cytoplasmic domain have been identified and further characterization is currently underway.

scholarly journals Talin activates integrins by altering the topology of the β transmembrane domain

2012 ◽  
Vol 197 (5) ◽  
pp. 605-611 ◽  
Author(s):  
Chungho Kim ◽  
Feng Ye ◽  
Xiaohui Hu ◽  
Mark H. Ginsberg
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Outer Membrane ◽  
Transmembrane Domain ◽  
Side Chain ◽  
Amino Acid Side Chain ◽  
Integrin Activation ◽  
Integrin Αiibβ3 ◽  
Environmentally Sensitive ◽  
Extracellular Side

Talin binding to integrin β tails increases ligand binding affinity (activation). Changes in β transmembrane domain (TMD) topology that disrupt α–β TMD interactions are proposed to mediate integrin activation. In this paper, we used membrane-embedded integrin β3 TMDs bearing environmentally sensitive fluorophores at inner or outer membrane water interfaces to monitor talin-induced β3 TMD motion in model membranes. Talin binding to the β3 cytoplasmic domain increased amino acid side chain embedding at the inner and outer borders of the β3 TMD, indicating altered topology of the β3 TMD. Talin’s capacity to effect this change depended on its ability to bind to both the integrin β tail and the membrane. Introduction of a flexible hinge at the midpoint of the β3 TMD decoupled the talin-induced change in intracellular TMD topology from the extracellular side and blocked talin-induced activation of integrin αIIbβ3. Thus, we show that talin binding to the integrin β TMD alters the topology of the TMD, resulting in integrin activation.

Role of the amino-terminal transmembrane domain of sulfonylurea receptor SUR2B for coupling to KIR6.2, ligand binding, and oligomerization

2011 ◽  
Vol 385 (3) ◽  
pp. 287-298 ◽  
Author(s):  
Marcus Winkler ◽  
Petra Kühner ◽  
Ulrich Russ ◽  
David Ortiz ◽  
Joseph Bryan ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Transmembrane Domain ◽  
Sulfonylurea Receptor ◽  
Amino Terminal

scholarly journals Participation of the Lys313-Ile333 Sequence of the Purinergic P2X4 Receptor in Agonist Binding and Transduction of Signals to the Channel Gate

2006 ◽  
Vol 281 (43) ◽  
pp. 32649-32659 ◽  
Author(s):  
Zonghe Yan ◽  
Zhaodong Liang ◽  
Tomas Obsil ◽  
Stanko S. Stojilkovic
Keyword(s):  
Transmembrane Domain ◽  
Critical Role ◽  
Receptor Function ◽  
Agonist Binding ◽  
Channel Opening ◽  
Channel Gate ◽  
Agonist Concentration ◽  
Opening And Closing ◽  
Kinetics Of

To study the roles of the Lys313-Ile333 ectodomain sequence of the rat P2X4 receptor in ATP binding and transduction of signals to the channel gate, the conserved Lys313, Tyr315, Gly316, Ike317, Arg318, Asp320, Val323, Lys329, Phe330, and Ile333 residues were mutated. Current recordings were done on lifted cells and ATP was applied using an ultrafast solution-switching system. The rates of wild type channel opening and closing in the presence of ATP, but not the rate of washout-induced closing, were dependent on agonist concentration. All mutants other than I317A were expressed in the plasma membrane at comparable levels. The majority of mutants showed significant changes in the peak amplitude of responses and the EC50 values for ATP. When stimulated with the supramaximal (1.4 mm) ATP concentration, mutants also differed in the kinetics of their activation, deactivation, and/or desensitization. The results suggest a critical role of the Lys313 residue in receptor function other than coordination of the phosphate group of ATP and possible contribution of the Tyr315 residue to the agonist binding module. The pattern of changes of receptor function by mutation of other residues was consistent with the operation of the Gly316-Ile333 sequence as a signal transduction module between the ligand binding domain and the channel gate in the second transmembrane domain.

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