Interruption of the ionic lock in the bradykinin B2 receptor results in constitutive internalization and turns several antagonists into strong agonists

The DRY motif with the highly conserved R3.50 is a hallmark of family A 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. So far, little information is available on how interruption of this ionic bond affects signaling properties of non-rhodopsin GPCRs, as 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 non-rhodopsin 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, E6.30R) also caused strong constitutive internalization. Most intriguingly, however, although the two E6.30 mutants displayed no increased basal phosphatidyl inositol 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 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.