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.

The bradykinin (BK) B 2 and B 1 receptors (B 2 R, B 1 R) belong to the rhodopsin-like G protein-coupled receptors (GPCRs) and are involved in (patho)physiological processes such as blood pressure regulation or inflammation. They mediate the effects of the pro-inflammatory peptides bradykinin/kallidin and desArg 9 -BK/desArg 10 -kallidin, respectively. Whereas the B 2 R is constitutively expressed and gets internalized upon activation, the B 1 R is especially induced by inflammatory mediators and responds to stimulation with increased surface receptor numbers. Stimulation of both receptors activates phospholipase Cb (PLCb) and mitogen activated protein kinase (MAPK) signaling. Because inflammatory processes are characterized by heat (fever), we analyzed the effect of increased temperature (418C vs. 378C) on B 1 R and B 2 R signaling in HEK 293 and IMR 90 cells. Our results show that signaling of both receptors is temperature-sensitive, however to a different extent and with regard to the investigated pathways. Comparing PLCb activity and Ca 2q -regulated signals, a temperature-dependent increase was only observed for B 1 R but not for B 2 R activation, whereas MAPK activities were doubled at 418C for both receptors. Taken together, our findings suggest that the observed temperature sensitivity of B 1 R-induced PLCb activation is B 1 R-specific. In contrast, the enhanced stimulation of MAPK activity under hyperthermic conditions appears to be a common phenomenon for GPCRs.