Kinins are some of the most potent inflammatory and nociceptive mediators. They act on two subtypes of G protein-coupled receptor, B1 and B2 bradykinin receptors. The first set of bioactive kinins generated in response to insult, bradykinin and Lys-bradykinin or kallidin, activates the B2 receptor. This receptor is ubiquitously and constitutively expressed in many cell types and is thought to mediate acute inflammatory responses. The metabolites of the first set of kinins, desArg⁹bradykinin and desArg¹⁰kallidin, become a second set of bioactive kinins and activate the B1 receptor. This receptor is normally not expressed but becomes induced by inflammatory signals. Induction of B1 receptor results in the co-existence of both receptor subtypes at many inflammatory sites. The B2 receptor has been intensively characterized in several cell systems and animal models in terms of the mechanisms of the receptor activation and physiological roles. On the other hand, the activation and cellular mechanisms of the B1 receptor is not completely understood due in part to its absence under normal condition. In this study, the molecular mechanisms of the B1 receptor activation and the functional consequence of B1 and B2 receptor co-expression have been investigated using transiently transfected HEK293 cells.

Using B1 receptors tagged in the N-terminus with either FLAG or hemaglutinin epitope it was found that the human B1 receptor forms homo-oligomeric complexes. Co-expression with a truncated receptor construct, B1stop135, which remains intracellular and competes for homo-oligomeric B1 receptor complexes, showed that trafficking of B1 receptors to the plasma membrane, and possibly receptor folding requires oligomerization of the receptor. Once the receptor reaches the plasma membrane, it becomes a functional unit for agonist binding and signaling and exhibits a high level of ligand-independent activity and agonist-dependent coupling to G_q/11-mediated phospholipase C?, which was identified by an increase in basal cellular phosphoinositide hydrolysis as a function of the density of the receptors.

The ligand-independent activity of the B1 receptor is further increased by an alanine point mutation of Asn¹²¹ (B1A¹²¹) in the third transmembrane domain. This mutant mimics the agonist-preferred receptor state since the agonist exhibits increased affinity and decreased efficacy for this receptor. Substitution of the fourth intracellular domain (IC-IV) of the B2 receptor, which exhibits minimal ligand-independent activity, in the B1 receptor to make the chimeric receptor B1(B2ICIV) caused a dramatic loss of both ligand-independent and agonist-dependent activity. The activity was partially recovered by a subsequent point mutation of a cluster of Ser and Thr residues in IC-IV of B1(B2ICIV), which is important for the B2 receptor phosphorylation and desensitization. Thus, the high ligand-independent activity of B1 receptor seems to at least in part be due to the lack of critical epitopes in IC-IV that regulate receptor activity.

The structural basis for the more efficacious coupling of the B1 receptor relative to the B2 receptor to G_q/11-mediated phospholipase Cb was evaluated by making a series of C-terminally truncated B1 and B2 receptor constructs. Truncation of the B1 receptor showed that this receptor does not directly depend on IC-IV for efficient coupling, although coupling is dramatically augmented by residues in the membrane-distal portion downstream from Tyr³²⁷. On the other hand, coupling of the B2 receptor is absolutely dependent on a membrane-proximal epitope in the C-terminal domain upstream from Lys³¹⁵. This epitope is adjacent to a basic residue, Arg³¹¹, which is inhibitory in B2 receptor G_q/11 coupling. Therefore, IC-IV participates in the efficacy of B1 and B2 receptor coupling to G_q/11 by contributing both positive and negative regulatory epitopes.

Co-expression of differentially tagged B1 and B2 kinin receptors revealed that these receptors spontaneously hetero-oligomerize. Also co-expression resulted in degradation of the B2 receptor while the B1 receptor remained intact. Interestingly, the hetero-oligomerized B2 receptor consisted exclusively of the degraded product, and this proteolytic complex was found to be located on the plasma membrane. Furthermore, co-expression of B1 and B2 receptors resulted in enhanced receptor mediated signaling, both in terms of ligand-independent and agonist-dependent phosphoinositide hydrolysis. These mechanisms may contribute to the transition from acute inflammatory signaling to chronic inflammatory signaling and the adaptation of the kinin response from a B2-type to a B1-type during chronic insult.