Comparison of the responses of B1 and B2 kinin receptors to agonist stimulation.

The human B2 kinin receptor (B2KR), stably expressed in Chinese hamster ovary cells, responded to bradykinin stimulation with rapid (within minutes) ligand internalization and loss of cell surface receptors (sequestration). By contrast, B1 kinin receptors (B1KR) showed almost no ligand internalization or receptor sequestration upon stimulation with des-Arg10-Kallidin (DAK). The ability of the B2KR to internalize and sequester is conferred by information in the cytoplasmic tail of the receptor. It is normally impossible to determine receptor affinity at 37 degrees C because of internalization and sequestration processes. We created a mutant B2KR, truncated at K315 of the cytoplasmic tail, that was no longer able to internalize or sequester, and compared the affinity of this mutant, and of the B1KR, at 0 degrees C and 37 degrees C. The B1KR receptor showed the same affinity (Kd = 0.4 nM) at both 0 degrees C and 37 degrees C. By contrast, the K315 mutant of the B2KR showed a lower affinity (Kd = 2.9 nM) at 37 degrees C than at 0 degrees C (Kd = 1.4 nM), indicating more rapid ligand dissociation at 37 degrees C. After ligand exposure, clones expressing B1KR exhibited a very slow dissociation of DAK, even at 37 degrees C. Although both kinin receptor subtypes induce the generation of inositol phosphates, functional responses showed clear differences. The response to stimulation of the B2KR comprises a rapid loss of functional responses, receptor sequestration, and ligand dissociation, and, upon long term stimulation, downregulation. By contrast, ligand stimulation of the B1KR, once this receptor is expressed de novo under pathological conditions, results in persistent signaling due to lack of ligand dissociation, desensitization and receptor sequestration. Moreover, long term stimulation of this receptor actually leads to increased expression.