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Molecular Pharmacology

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Research ArticleArticle

Receptor Conformations Involved in Dopamine D2L Receptor Functional Selectivity Induced by Selected Transmembrane-5 Serine Mutations

J. Corey Fowler, Supriyo Bhattacharya, Jonathan D. Urban, Nagarajan Vaidehi and Richard B. Mailman
Molecular Pharmacology June 2012, 81 (6) 820-831; DOI: https://doi.org/10.1124/mol.111.075457
J. Corey Fowler
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Supriyo Bhattacharya
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Jonathan D. Urban
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Nagarajan Vaidehi
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Richard B. Mailman
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Abstract

Although functional selectivity is now widely accepted, the molecular basis is poorly understood. We have studied how aspects of transmembrane region 5 (TM5) of the dopamine D2L receptor interacts with three rationally selected rigid ligands (dihydrexidine, dinapsoline, and dinoxyline) and the reference compounds dopamine and quinpirole. As was expected from homology modeling, mutation of three TM5 serine residues to alanine (S5.42A, S5.43A, S5.46A) had little effect on antagonist affinity. All three mutations decreased the affinity of the agonist ligands to different degrees, S5.46A being somewhat less affected. Four functions [adenylate cyclase (AC), extracellular signal-regulated kinase 1/2 phosphorylation (MAPK), arachidonic acid release (AA), and guanosine 5′-O-(3-thio)triphosphate binding (GTPγS)] were assessed. The intrinsic activity (IA) of quinpirole was unaffected by any of the mutations, whereas S5.42A and S5.46A mutations abolished the activity of dopamine and the three rigid ligands, although dihydrexidine retained IA at MAPK function only with S5.42A. Remarkably, S5.43A did not markedly affect IA for AC and MAPK for any of the ligands and eliminated AA activity for dinapsoline and dihydrexidine but not dinoxyline. These data suggest that this mutation did not disrupt the overall conformation or signaling ability of the mutant receptors but differentially affected ligand activation. Computational studies indicate that these D2 agonists stabilize multiple receptor conformations. This has led to models showing the stabilized conformations and interhelical and receptor-ligand contacts corresponding to the different activation pathways stabilized by various agonists. These data provide a basis for understanding D2L functional selectivity and rationally discovering functionally selective D2 drugs.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported by the National Institutes of Health National Institute of Mental Health [Grants MH082441; MH040537]; and by a Pennsylvania Keystone Innovation Grant.

  • The intellectual property for some of the compounds used in this study were assigned by R.B.M. to the University of North Carolina and licensed to Biovalve Technologies Inc., which was acquired by Valeritas.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    http://dx.doi.org/10.1124/mol.111.075457.

  • ABBREVIATIONS:

    CHO
    Chinese hamster ovary
    DNS
    dinapsoline
    AC
    adenylate cyclase
    AA
    arachidonic acid release
    MAPK
    mitogen-activated protein kinase
    AA
    arachidonic acid
    DNX
    dinoxyline
    DHX
    dihydrexidine
    DA
    dopamine
    GPCR
    G protein-coupled receptor
    PMSF
    phenylmethylsulfonyl fluoride
    TM
    transmembrane
    WT
    wild type
    GTPγS
    guanosine 5′-O-(3-thio)triphosphate
    BSA
    bovine serum albumin
    PBS
    phosphate-buffered saline
    nH
    Hill coefficient
    K0.5
    apparent affinity constant (nH < 1.0), equals KI when nH = 1
    HD2L
    human dopamine D2L receptor
    BE
    binding energy
    HB
    hydrogen bond
    PDB
    protein data bank
    ANOVA
    analysis of variance.

  • Received September 1, 2011.
  • Accepted March 13, 2012.
  • Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 81 (6)
Molecular Pharmacology
Vol. 81, Issue 6
1 Jun 2012
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Research ArticleArticle

Mutation-Induced Changes in Functional Selectivity

J. Corey Fowler, Supriyo Bhattacharya, Jonathan D. Urban, Nagarajan Vaidehi and Richard B. Mailman
Molecular Pharmacology June 1, 2012, 81 (6) 820-831; DOI: https://doi.org/10.1124/mol.111.075457

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Research ArticleArticle

Mutation-Induced Changes in Functional Selectivity

J. Corey Fowler, Supriyo Bhattacharya, Jonathan D. Urban, Nagarajan Vaidehi and Richard B. Mailman
Molecular Pharmacology June 1, 2012, 81 (6) 820-831; DOI: https://doi.org/10.1124/mol.111.075457
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