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 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.

Helix 8 plays a crucial role in bradykinin B(2) receptor trafficking and signaling.

Upon activation the human bradykinin B(2) receptor (B(2)R) acts as guanine nucleotide exchange factor for the G proteins G(q/11) and G(i). Thereafter, it gets phosphorylated by G protein-coupled receptor kinases (GRKs) and recruits ß-arrestins, which block further G protein activation and promote B(2)R internalization via clathrin-coated pits. As for most G protein-coupled receptors of family A, an intracellular helix 8 after transmembrane domain 7 is also predicted for the B(2)R. We show here that disruption of helix 8 in the B(2)R by either C-terminal truncation or just by mutation of a central amino acid (Lys-315) to a helix-breaking proline resulted in strong reduction of surface expression. Interestingly, this malfunction could be overcome by the addition of the membrane-permeable B(2)R antagonist JSM10292, suggesting that helix 8 has a general role for conformational stabilization that can be accounted for by an appropriate antagonist. Intriguingly, an intact helix 8, but not the C terminus with its phosphorylation sites, was indispensable for receptor sequestration and for interaction of the B(2)R with GRK2/3 and ß-arrestin2 as shown by co-immunoprecipitation. Recruitment of ß-arrestin1, however, required the presence of the C terminus. Taken together, our results demonstrate that helix 8 of the B(2)R plays a crucial role not only in efficient trafficking to the plasma membrane or the activation of G proteins but also for the interaction of the B(2)R with GRK2/3 and ß-arrestins. Additional data obtained with chimera of B(2)R with other G protein-coupled receptors of family A suggest that helix 8 might have similar functions in other GPCRs as well.

Fever-like temperature modification differentially affects in vitro signaling of bradykinin B(1) and B(2) receptors.

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 Cß (PLCß) and mitogen activated protein kinase (MAPK) signaling. Because inflammatory processes are characterized by heat (fever), we analyzed the effect of increased temperature (41°C vs. 37°C) 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 PLCß activity and Ca(2+)-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 41°C for both receptors. Taken together, our findings suggest that the observed temperature sensitivity of B(1)R-induced PLCß 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.

A cysteine-type carboxypeptidase, cathepsin X, generates peptide receptor agonists.

The kallikrein-kinin system and the renin-angiotensin system interact at different levels and are linked by various molecules such as angiotensin-converting enzyme which degrades bradykinin into inactive peptides. Here we report that a cysteine-type carboxypeptidase, cathepsin X, is able to modulate the kallikrein-kinin system through carboxyterminal processing of the small peptide hormones bradykinin and kallidin. Both peptides are thereby converted from bradykinin B(2) receptor ligands to bradykinin B(1) receptor specific ligands. Cathepsin X, which has previously been recognized as an inflammatory marker may therefore act as a type I kininase. In addition, we have identified cathepsin X as an alternative possible link between the kallikrein-kinin system and the renin-angiotensin system in that it not only cleaves kinins C-terminally, but also converts angiotensin I to angiotensin II.

Alanine screening of the intracellular loops of the human bradykinin B receptor--effects on receptor maintenance, G protein activation and internalization.

The bradykinin B(2) receptor is coupled to G protein G(q/11) and becomes sequestered into intracellular compartments after activation. To more closely define the receptor sequences involved in these processes and their functions, we systematically mutated all three intracellular loops (ICLs), either as point mutations or in groups of three to five amino acids to Ala, obtaining a total of 14 mutants. All constructs were stably expressed in HEK 293 cells and, with the exception of triple mutant DRY --> AAA, retained the ability to specifically bind [(3)H]bradykinin. The binding affinities at 4 or 37 degrees C of several mutants differed considerably from those determined for the wild-type receptor, indicating an allosteric connection between the conformation of the binding site and that of the ICLs. Mutations in ICL-1 strongly reduced surface expression without affecting G protein signaling or [(3)H]bradykinin internalization. Two cluster mutants in the middle of ICL-2 containing basic residues displayed considerably reduced potencies, whereas two mutations in ICL-3 resulted in receptor conformations that were considered to be semi-active, based on the observation that they responded with phosphoinositide hydrolysis to compounds normally considered to be antagonists. This, and the fact that a cluster mutant at the C-terminal end of ICL-3 was signaling incompetent, hint at the involvement of ICL-2 and ICL-3 in G(q/11) activation, albeit with different functions. None of the mutants displayed reduced ligand-induced receptor internalization, indicating that the loops are not essential for this process. No conclusion could be drawn, however, with regard to the role of the DRY sequence, as the corresponding triplet mutation lacked binding capability.