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Constitutive Activation of A3 Adenosine Receptors by Site-Directed Mutagenesis

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Abstract

The objective of this study was to create constitutively active mutant human A3 adenosine receptors (ARs) using single amino acid replacements, based on findings from other G protein-coupled receptors. A3 ARs mutated in transmembrane helical domains (TMs) 1, 3, 6, and 7 were expressed in COS-7 cells and subjected to agonist radioligand binding and phospholipase C (PLC) and adenylyl cyclase (AC) assays. Three mutant receptors, A229E in TM6 and R108A and R108K in the DRY motif of TM3, were found to be constitutively active in both functional assays. The potency of the A3 agonist Cl-IB-MECA (1-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide) in PLC activation was enhanced by at least an order of magnitude over wild type (EC50 951 nM) in R108A and A229E mutant receptors. Cl-IB-MECA was much less potent (>10-fold) in C88F, Y109F, and Y282F and mutants or inactive following double mutation of the DRY motif. The degree of constitutive activation was more pronounced for the AC signaling pathway than for the PLC signaling pathway. The results indicated that specific locations within the TMs proximal to the cytosolic region were responsible for constraining the receptor in a G protein-uncoupled conformation.

References (37)

  • A. De Lean et al.

    A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptor

    J. Biol. Chem.

    (1980)
  • P. Samama et al.

    A mutation-induced activated state of the β2-adrenergic receptor. Extending the ternary complex model

    J. Biol. Chem.

    (1993)
  • J.E. Porter et al.

    Activation of the α1b-adrenergic receptor is initiated by disruption of an interhelical salt bridge constraint

    J. Biol. Chem.

    (1996)
  • P. Wonerow et al.

    Deletions in the third intracellular loop of the thyrotropin receptor. A new mechanism for constitutive activation

    J. Biol. Chem.

    (1998)
  • M.J. Zuscik et al.

    Identification of a conserved switch residue responsible for selective constitutive activation of the β2-adrenergic receptor

    J. Biol. Chem.

    (1998)
  • J. Kim et al.

    Site-directed mutagenesis identifies residues involved in ligand recognition in the human A2A adenosine receptor

    J. Biol. Chem.

    (1995)
  • G.J. Gross

    ATP-sensitive potassium channels and myocardial preconditioning

    Basic Res. Cardiol.

    (1995)
  • J. Strickler et al.

    Direct preconditioning of cultured chick ventricular myocytes. Novel functions of cardiac adenosine A2a and A3 receptors

    J. Clin. Invest.

    (1996)

    • Cited by (0)

    1

    On leave from Israel Institute of Biological Research, Ness Ziona, Israel.

    2

    To whom correspondence should be addressed at Molecular Recognition Section, LBC, National Institute of Diabetes, Digestive, and Kidney Diseases, Bldg. 8A, Rm. B1A-19, NIH, Bethesda, MD 20892-0810. Fax: (301) 480-8422. E-mail: [email protected].

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