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, 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 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.

Molecular mechanisms regulating Proteinase‐Activated Receptors (PARs)

“…The E/DR 3.50 Y motif is located at the cytoplasmic interface of TM3 and ICL2, and in Rhodopsin and other GPCRs forms salt bridges with negatively charged residue Glu 6.30 in TM6 [4,44,45]. Ligand occupancy mediated conformational change in TM3 and TM6 disrupts the ionic lock, where mutations disrupting this interaction can result in constitutively active receptors [46]. The DR 3.50 Y motif is DR 3.50 F (D199-F201) in PAR1, QR 3.50 Y (Q172-Y174) in PAR2, NR 3.50 Y (N190-Y192) in PAR3 and DR 3.50 Y (D173-Y175) in PAR4 ( Figure 1).…”

Conformational Flexibility and Structural Dynamics in GPCR-Mediated G Protein Activation: A Perspective

“…However, these data are not meant to imply that the lock is unimportant for function. While the mutation of R in the E/DRY motif of rhodopsin type GPCRs abrogates G protein function 21; 22 , mutation of the conserved Glu in the ERY motif of the bradykinin B2 receptor to either R or A turns agonists into functional antagonists, decreasing phosphoinositol signaling and increasing constitutive internalization of receptors 23 . These types of studies help increase our understanding of processes such as biased agonism and functional selectivity that result in ligand-dependent differences in signaling pathways, through either arrestin binding or through differential signaling to G proteins 24 .…”

Structure versus function—The impact of computational methods on the discovery of specific GPCR–ligands

“…Besides the studies that are directly based on crystal structures, examinations of structural properties by homology models are reported as well. A structural model for the bradykinin B 2 receptor indicated the presence of the ionic lock and showed, in combination with mutational studies, its role for the stabilization of the inactive receptor state [11]. Kling et al [12] used an active-state homology model of the dopamine D 2 receptor-Gα i protein-complex.…”