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 ␤-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 helixbreaking 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 ␤-arrestin2 as shown by co-immunoprecipitation. Recruitment of ␤-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 ␤-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.The human bradykinin B 2 receptor (B 2 R) 2 belongs to the family A (rhodopsin/␤-adrenergic-like) of G protein-coupled receptors (GPCRs). The B 2 R is ubiquitously expressed in many cells and tissues, and its activation results in a variety of physiological effects that range from vasodilatation and increased vascular permeability to hyperalgesia and natriuresis (1). Recent studies with B 2 R knock-out mice also point to a protective role of the B 2 R in the process of aging and in diabetes (2). After extracellular binding of its endogenous agonists, of the nona-peptide bradykinin (BK), or of kallidin (Lys-BK), the B 2 R undergoes conformational changes that turn it into a guanine nucleotide exchange factor for the G proteins G q/11 and G i , thus leading to the activation of G protein-dependent signaling cascades. Among other events, this results in phosphatidylinositol hydrolysis and activation of MAPK pathways. As reported for many GPCRs, desensitization of the B 2 R comes along with phosphorylation of serine/threonine residues in its C terminus by G protein-coupled receptor kinases (GRKs) or second messenger kinases as well as recruitment of ␤-arrestins and ends with the sequestration of the receptor into intracellular compartments (1). Upon short term stimulation the receptor gets recycled to the plasma membrane, whereas long term stimulation leads to the down-...

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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.

Equilibrium binding, dissociation and competition studies with various B2 receptor ligands and [ 3 H]-JSM10292 were performed at 4°C and 37°C. The experiments were carried out using HEK293 cells stably (over)expressing wild-type and mutant B2 receptors of human and animal origin. H]-BK showed a different affinity profile for the wild-type B2 receptor in different species (man, cynomolgus, rabbit, mouse, rat, dog, pig, guinea pig). Characterization of B2 receptor mutants and species orthologues combined with homology modelling, using the CXCR4 as template, suggests that the binding site of JSM10292 is different from that of BK but overlaps with that of MEN16132, another small non-peptide B2 receptor ligand. KEY RESULTS CONCLUSIONS AND IMPLICATIONS[ 3 H]-JSM10292 is a novel, cell membrane-permeant, high-affinity B2 receptor antagonist that allows direct in detail studies of active, surface and intracellularly located wild-type and mutant B2 receptors. AbbreviationsAA, acetic acid; B2 receptor, bradykinin B2

The human bradykinin B 2 receptor (B 2 R) mediates the effects of the nonapeptide bradykinin (BK) and of kallidin (lysyl-BK). B 2 R has been reported to play a role in a number of physiological and pathophysiological situations. Its activation causes vasodilation and hypotension, increased vascular permeability and edema, or generation of pain via C fibers [1]. B 2 R, which is expressed constitutively in many tissues and cultured cells, is a prototypical member of family A (rhodopsin ⁄ b-adrenergic-like receptors) of the membrane-bound 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 fi AAA, retained the ability to specifically bind [ 3 H]bradykinin. The binding affinities at 4 or 37°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.Abbreviations B 2 Rwt, bradykinin B 2 receptor wild-type; BK, bradykinin; CMV, cytomegalovirus; EC 50, half-maximal effective concentration; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HA, hemagglutinin; HEK 293, human embryonic kidney cells; ICL-1, ICL-2, ICL-3, first, second and third intracellular loops; IP, inositol phosphate; PAO, phenylarsine oxide.