Beta-Blockers Show Inverse Agonism to a Novel Constitutively Active Mutant of β1-Adrenoceptor

Ahmed, Maruf; Muntasir, Habib Abul; Hossain, Murad; Ishiguro, Masaji; Komiyama, Tomoyoshi; Muramatsu, Ikunobu; Kurose, Hitoshi; Nagatomo, Takafumi

doi:10.1254/jphs.fp0060640

Cited by 7 publications

(11 citation statements)

References 27 publications

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“…43) In competition binding, it is evident that the substitution of Asp at position 104 by Lys decreased significantly (pϽ0.05) the binding affinity to isoproterenol and (Ϫ)-epinephrine in comparison with wild type b 1 -AR. On the other hand, binding affinity of several antagonists were unchanged compared to wild type receptor and resulted in monophasic binding curves with Hill slopes close to Unity, which resembles the previous Asp104Ala mutant data, 39) shown in Table 1 and Fig. 2.…”

Section: Discussionsupporting

confidence: 80%

“…39) Both mutant receptors were expressed in HEK293 cells and assessed for ligand binding affinity and agonist induced cAMP production.…”

Section: Discussionmentioning

confidence: 99%

“…39) In brief, adherent cells in 12-well plates (5ϫ10 5 cells/well) were incubated for 30 min at 37°C in 500 ml/well of Hank's balance salt solution (HBSS) buffered with 25 mM HEPES, 1 mM ascorbic acid and 1 mM isobutyl-methylxanthine (Sigma, St. Louis, U.S.A.), in the presence of agonist or other drugs. The reaction was stopped by washing twice with 1 ml ice cold (Ϫ) phosphate buffered saline and immediate addition of 250 ml 1 N NaOH.…”

Section: Mutagenesis Of Cdna and Transfectionmentioning

confidence: 99%

“…We also showed that mutation of negatively charged aspartic acid to neutral charged alanine exhibited constitutive activity of b 1 -AR. 39) However, the mutation of negatively charged aspartic acid to positively charged lysine is still remained to be ex- amined. At the present study, we have mutated aspartic acid to lysine (Asp104Lys) residue of human b 1 -AR and investigated the ligand binding, functional characteristic and constitutive activation of Asp104Lys mutant of human b 1 -AR.…”

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confidence: 99%

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Mutation of Important Amino Acid Residue of Asp104Lys in Human .BETA.1-Adrenergic Receptor Triggers Functional and Constitutive Inactivation

Hossain

Ahmed

Bhuiyan

et al. 2008

Biological & Pharmaceutical Bulletin

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The b 1 -adrenergic receptor (b 1 -AR) belongs to the largest super family of G-protein-coupled receptors (GPCRs). The b 1 -AR responds to norepinephrine and epinephrine by activating adenylyl cyclase pathway and promotes production of cAMP.1) Activation of cardiac b-adrenergic receptors plays an important role in regulating the physiological responses of the heart to an increase demand.2) The pathophysiology of human b 1 -ARs is associated with several cardiovascular diseases such as dilated cardiomyopathy, congestive heart failure, ventricular arrhythmia and sudden death. [3][4][5] Recently, several GPCRs have exhibited constitutive activity and inactivity in vivo. Naturally occurring amino acid mutations in the luteinizing hormone receptor, 6) parathyroid hormone receptor, 7) melanocyte-stimulating hormone receptor, 8) thyrotropin receptor 9) and rhodopsin receptor 10) result in constitutive activation of second messenger production and are thought to play a role in disease pathophysiology. Constitutively active mutant receptors have been a valuable tool to demonstrate that certain ligands stabilize inactive conformations. Those ligands are known as inverse agonist, given that they have the opposite effect of agonists. Therefore, b-antagonists are used clinically to reduce heart rate and force of contraction in hypertension, angina and acute myocardial infarction by direct blocking endogenous catecholamine activity, [11][12][13] implying that inverse agonists may have preferred therapeutic applications in the pathophysiology of several cardiovascular diseases.In all seven transmembrane helix (TMH) of GPCRs it appears that the general mechanism of activation involves disruption of intramolecular constraints, leading to TMH movement and formation of new interactions, which stabilize the active state of the receptor. [14][15][16][17][18][19][20][21][22][23][24][25][26][27] The aspartic acid at position 104 in TMH II of the human b 1 -AR is conserved in all b-adrenergic 28-31) , a-adrenergic receptor 32) and also muscarinic cholinergic receptors, [33][34][35] which may take part in an ionic interaction with amino group containing catecholamines, whose disruption of interaction makes the receptor active conformation state. It was reported by Chung et al. 36) that aspartic acid residue at position 79 in putative TMH II of b 2 -AR was involved in agonist binding and receptor activation. Substitution of aspartic acid to alanine and asparagine at position 79 did not affect the antagonist binding but significantly decreased the binding affinity of agonists. 37)Moreover, this mutant receptor also affected the functional activity compared to wild type receptor by uncoupling with GPCRs.With the help of molecular modeling, we previously identified the important binding sites of b 1 -AR. We showed that Asp104 in TMH II of b 1 -AR makes strong electrostatic interaction with functional groups of SWR-0342SA.38) Through mutagenesis studies, we suggested that Asp104 is very important site for the ligand binding and functional...

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Section: Discussionsupporting

confidence: 80%

“…39) Both mutant receptors were expressed in HEK293 cells and assessed for ligand binding affinity and agonist induced cAMP production.…”

Section: Discussionmentioning

confidence: 99%

Section: Mutagenesis Of Cdna and Transfectionmentioning

confidence: 99%

mentioning

confidence: 99%

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Mutation of Important Amino Acid Residue of Asp104Lys in Human .BETA.1-Adrenergic Receptor Triggers Functional and Constitutive Inactivation

Hossain

Ahmed

Bhuiyan

et al. 2008

Biological & Pharmaceutical Bulletin

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“…However, the equivalent Asp 2.50 Ala mutation results in constitutive activity for the closely related β 1 AR, suggesting that interpretation of mutagenesis in this region is complex. 86 The common assumption that asparagine is a good mimic of a protonated aspartic acid is also questionable. The polarities of side chain analogues of Asn and (protonated) Asp are quite different, as reflected by the 3 kcal/mol difference in hydration free energy for (neutral) acetic acid and acetamide.…”

Section: ■ Discussionmentioning

confidence: 99%

Insights into the Role of Asp79^2.50 in β₂ Adrenergic Receptor Activation from Molecular Dynamics Simulations

2014

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Achieving a molecular-level understanding of G-protein-coupled receptor (GPCR) activation has been a long-standing goal in biology and could be important for the development of novel drugs. Recent breakthroughs in structural biology have led to the determination of high-resolution crystal structures for the β2 adrenergic receptor (β2AR) in inactive and active states, which provided an unprecedented opportunity to understand receptor signaling at the atomic level. We used molecular dynamics (MD) simulations to explore the potential roles of ionizable residues in β2AR activation. One such residue is the strongly conserved Asp79(2.50), which is buried in a transmembrane cavity and becomes dehydrated upon β2AR activation. MD free energy calculations based on β2AR crystal structures suggested an increase in the population of the protonated state of Asp79(2.50) upon activation, which may contribute to the experimentally observed pH-dependent activation of this receptor. Analysis of MD simulations (in total > 100 μs) with two different protonation states further supported the conclusion that the protonated Asp79(2.50) shifts the conformation of the β2AR toward more active-like states. On the basis of our calculations and analysis of other GPCR crystal structures, we suggest that the protonation state of Asp(2.50) may act as a functionally important microswitch in the activation of the β2AR and other class A receptors.

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Measurement of Inverse Agonism in β-Adrenoceptors

Taira

Monczor

Höcht

2010

Methods in Enzymology

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Beta-Blockers Show Inverse Agonism to a Novel Constitutively Active Mutant of β1-Adrenoceptor

Cited by 7 publications

References 27 publications

Mutation of Important Amino Acid Residue of Asp104Lys in Human .BETA.1-Adrenergic Receptor Triggers Functional and Constitutive Inactivation

Mutation of Important Amino Acid Residue of Asp104Lys in Human .BETA.1-Adrenergic Receptor Triggers Functional and Constitutive Inactivation

Insights into the Role of Asp79^2.50 in β₂ Adrenergic Receptor Activation from Molecular Dynamics Simulations

Measurement of Inverse Agonism in β-Adrenoceptors

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Beta-Blockers Show Inverse Agonism to a Novel Constitutively Active Mutant of β1-Adrenoceptor

Cited by 7 publications

References 27 publications

Mutation of Important Amino Acid Residue of Asp104Lys in Human .BETA.1-Adrenergic Receptor Triggers Functional and Constitutive Inactivation

Mutation of Important Amino Acid Residue of Asp104Lys in Human .BETA.1-Adrenergic Receptor Triggers Functional and Constitutive Inactivation

Insights into the Role of Asp792.50 in β2 Adrenergic Receptor Activation from Molecular Dynamics Simulations

Measurement of Inverse Agonism in β-Adrenoceptors

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Insights into the Role of Asp79^2.50 in β₂ Adrenergic Receptor Activation from Molecular Dynamics Simulations