title: Interruption of the ionic lock in the bradykinin B2 receptor results in constitutive internalization and turns several antagonists into strong agonists.
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 R135(3.50) and another conserved residue, E247(6.30), 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 R128(3.50) and E238(6.30) in the bradykinin (BK) B(2) receptor (B(2)R) 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 B(2)R 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.
authors.affiliation: Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universitaet, Pettenkoferstrasse 9, D-80336 Muenchen, Germany.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Leschner
authors.forename: Goeran
authors.initials: G
authors.lastname: Wennerberg
authors.forename: Jens
authors.initials: J
authors.lastname: Feierler
authors.forename: Marcel
authors.initials: M
authors.lastname: Bermudez
authors.forename: Benjamin
authors.initials: B
authors.lastname: Welte
authors.forename: Irina
authors.initials: I
authors.lastname: Kalatskaya
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wolber
authors.forename: Alexander
authors.initials: A
authors.lastname: Faussner
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D019921:Biotinylation; D001920:Bradykinin / Q000378:metabolism; D065094:Bradykinin B2 Receptor Antagonists; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000378:metabolism; D007477:Ions / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011728:Pyridones / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D043782:Receptor, Bradykinin B2 / Q000819:agonists / Q000187:drug effects*; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects / Q000378:metabolism; D013696:Temperature
chemical_list: C578193:1-(4-methyl-3-(2-methyl-4-(4-methyl-2H-pyrazol-3-yl)-quinolin-8-yloxymethyl)pyridin-2-ylmethyl)-3-trifluoromethyl-1H-pyridin-2-one; D000596:Amino Acids; D065094:Bradykinin B2 Receptor Antagonists; D007295:Inositol Phosphates; D007477:Ions; D011728:Pyridones; D011804:Quinolines; D043782:Receptor, Bradykinin B2; D043562:Receptors, G-Protein-Coupled; D019204:GTP-Binding Proteins; D001920:Bradykinin