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Research ArticleCellular and Molecular

Interruption of the Ionic Lock in the Bradykinin B2 Receptor Results in Constitutive Internalization and Turns Several Antagonists into Strong Agonists

Jasmin Leschner, Goeran Wennerberg, Jens Feierler, Marcel Bermudez, Benjamin Welte, Irina Kalatskaya, Gerhard Wolber and Alexander Faussner
Journal of Pharmacology and Experimental Therapeutics January 2013, 344 (1) 85-95; DOI: https://doi.org/10.1124/jpet.112.199190
Jasmin Leschner
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universitaet, Muenchen (J.L., G.W., J.F., B.W., A.F.), Institut für Pharmazie, Pharmazeutische Chemie, Freie Universitaet Berlin, Berlin, Germany (M.B., G.W.); and Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada (I.K.)
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Goeran Wennerberg
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universitaet, Muenchen (J.L., G.W., J.F., B.W., A.F.), Institut für Pharmazie, Pharmazeutische Chemie, Freie Universitaet Berlin, Berlin, Germany (M.B., G.W.); and Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada (I.K.)
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Jens Feierler
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universitaet, Muenchen (J.L., G.W., J.F., B.W., A.F.), Institut für Pharmazie, Pharmazeutische Chemie, Freie Universitaet Berlin, Berlin, Germany (M.B., G.W.); and Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada (I.K.)
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Marcel Bermudez
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universitaet, Muenchen (J.L., G.W., J.F., B.W., A.F.), Institut für Pharmazie, Pharmazeutische Chemie, Freie Universitaet Berlin, Berlin, Germany (M.B., G.W.); and Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada (I.K.)
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Benjamin Welte
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universitaet, Muenchen (J.L., G.W., J.F., B.W., A.F.), Institut für Pharmazie, Pharmazeutische Chemie, Freie Universitaet Berlin, Berlin, Germany (M.B., G.W.); and Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada (I.K.)
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Irina Kalatskaya
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universitaet, Muenchen (J.L., G.W., J.F., B.W., A.F.), Institut für Pharmazie, Pharmazeutische Chemie, Freie Universitaet Berlin, Berlin, Germany (M.B., G.W.); and Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada (I.K.)
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Gerhard Wolber
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universitaet, Muenchen (J.L., G.W., J.F., B.W., A.F.), Institut für Pharmazie, Pharmazeutische Chemie, Freie Universitaet Berlin, Berlin, Germany (M.B., G.W.); and Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada (I.K.)
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Alexander Faussner
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universitaet, Muenchen (J.L., G.W., J.F., B.W., A.F.), Institut für Pharmazie, Pharmazeutische Chemie, Freie Universitaet Berlin, Berlin, Germany (M.B., G.W.); and Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada (I.K.)
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Abstract

The DRY motif with the highly conserved R3.50 is a hallmark of family A G protein-coupled receptors (GPCRs). The crystal structure of rhodopsin revealed a salt bridge between R1353.50 and another conserved residue, E2476.30, in helix 6. This ionic lock was shown to maintain rhodopsin in its inactive state. Thus far, little information is available on how interruption of this ionic bond affects signaling properties of nonrhodopsin GPCRs, because the focus has been on mutations of R3.50, although this residue is indispensable for G protein activation. To investigate the importance of an ionic lock for overall receptor activity in a nonrhodopsin GPCR, we mutated R1283.50 and E2386.30 in the bradykinin (BK) B2 receptor (B2R) and stably expressed the constructs in HEK293 cells. As expected, mutation of R3.50 resulted in lack of G protein activation. In addition, this mutation led to considerable constitutive receptor internalization. Mutation of E6.30 (mutants E6.30A and E6.30R) also caused strong constitutive internalization. Most intriguingly, however, although the two E6.30 mutants displayed no increased basal phosphatidylinositol hydrolysis, they gave a response to three different B2R antagonists that was almost comparable to that obtained with BK. In contrast, swapping of R3.50 and E6.30, thus allowing the formation of an inverse ionic bond, resulted in rescue of the wild type phenotype. These findings demonstrate for the first time, to our knowledge, that interruption of the ionic lock in a family A GPCR can have distinctly different effects on receptor internalization and G protein stimulation, shedding new light on its role in the activation process.

Footnotes

  • ↵This work was supported by the Förderprogramm für Forschung und Lehre of the Medical Faculty of the Ludwig-Maximilians-University of Munich, Munich, Germany (to B.W.).

  • J.L. and G.W. (Goeran Wennerberg) contributed equally to this work.

  • Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

  • dx.doi.org/10.1124/jpet.112.199190.

  • Received August 9, 2012.
  • Accepted October 16, 2012.
  • Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 344 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 344, Issue 1
1 Jan 2013
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Research ArticleCellular and Molecular

Function of Ionic Lock in Bradykinin B2 Receptor Activities

Jasmin Leschner, Goeran Wennerberg, Jens Feierler, Marcel Bermudez, Benjamin Welte, Irina Kalatskaya, Gerhard Wolber and Alexander Faussner
Journal of Pharmacology and Experimental Therapeutics January 1, 2013, 344 (1) 85-95; DOI: https://doi.org/10.1124/jpet.112.199190

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Research ArticleCellular and Molecular

Function of Ionic Lock in Bradykinin B2 Receptor Activities

Jasmin Leschner, Goeran Wennerberg, Jens Feierler, Marcel Bermudez, Benjamin Welte, Irina Kalatskaya, Gerhard Wolber and Alexander Faussner
Journal of Pharmacology and Experimental Therapeutics January 1, 2013, 344 (1) 85-95; DOI: https://doi.org/10.1124/jpet.112.199190
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