Stable Expression of the Human Kinin B1 Receptor in Chinese Hamster Ovary Cells

Abstract

To delineate ligand binding and functional characteristics of the human B1 kinin receptor, a stable clone of Chinese hamster ovary cells expressing a single class of binding sites for [3H]des-Arg10-lysylbradykinin with a Kd of 0.3 nM and a Bmax of 38 fmol/mg protein ( approximately 40,000 receptors/cell) was isolated. Studies with peptide analogs showed that a lysine residue at position 1 (based on the lysylbradykinin sequence) of ligands was essential for high affinity binding to the human B1 receptor. In marked contrast to cloned Chinese hamster ovary cells expressing the human kinin B2 receptor, which internalized approximately 80% of the ligand within 5 min upon exposure to 2 nM [3H]bradykinin, exposure of cells expressing the B1 receptor to 1 nM [3H]des-Arg10-lysylbradykinin resulted in minimal ligand internalization. Stimulation of the B1 receptor led to inositol phosphate generation and transient increases in intracellular calcium, confirming coupling to phospholipase C, while immunoprecipitation of photoaffinity-labeled G-proteins from membranes indicated specific coupling of the receptor to Galphaq/11 and Galphai1,2. The B1, unlike the B2, receptor does not desensitize (as demonstrated by continuous phosphoinositide hydrolysis), enhancing the potential role of this receptor during inflammatory events.

Up-Regulation of Functional Kinin B1 Receptors in Allergic Airway Inflammation

“…BK has been reported to activate the MAPK ERK in a variety of cells (27,33). Although des-Arg-BK has also been reported to activate ERK in human embryonic kidney 293 cells overexpressing B1 receptors (34), the significance of this observation is uncertain because des-Arg-BK is not an effective agonist for the human B1 receptor (11,12).…”

“…The specificity of their agonist activity at the concentrations used have been carefully documented. BK and Lys-BK (kallidin) are equally effective agonists for the B2 receptor; however, Lys-BK, but not BK, shows some agonist activity for the B1 receptor (11,12). Their carboxypeptidase metabolites (desArg-BK and Lys-des-Arg-BK) have very different B1 receptor agonist activities; Lys-des-Arg-BK is a potent agonist for the human B1 receptor, while des-Arg-BK is at least 1000-fold less potent (11,12).…”

“…In humans, cloned gene sequences of the receptors have confirmed distinct B2 and B1 receptors, each belonging to the seven transmembrane G protein-coupled superfamily (10,11). BK and Lys-BK (kallidin) are equally effective agonists for the B2 receptor; however, the carboxypeptidase metabolite of Lys-BK, Lys-des-Arg-BK, is the only known potent agonist for the human B1 receptor (11,12). B2 receptors are constitutively expressed on many cell types (13) including human airway epithelial cells (14), whereas B1 receptors are expressed at low levels in normal tissues but can be induced in response to pathophysiological stimuli (15)(16)(17).…”

Non‐competitive pharmacological antagonism at the rabbit B1 receptor

“…Non-equilibrium antagonism at the rabbit B 1 receptor exhibits several interesting features that should orient future molecular investigations: the drugs are charged peptides of relatively high molecular weight, making unlikely the interference with non-receptor intracellular sites; unlike the B 2 receptor, the B 1 receptor is not believed to undergo important agonist-induced phosphorylation and internalization (Austin et al, 1997;Faussner et al, 1998). Finally, the irreversible or slowly reversible binding of B-9858 can be exploited to demonstrate receptor upregulation by inactivating a pre-existing receptor population, thus supporting studies of the dynamic regulation of this receptor type.…”

Fever-like temperature modification differentially affects in vitro signaling of bradykinin B1 and B2 receptors

“…Moreover, unlike B 2 R, B 1 R does not become desensitized and responds in some cell types to activation with an increase in surface receptor number rather than internalization, e.g., in IMR 90 cells (Phagoo et al, 2000;Leeb-Lundberg et al, 2005). As both kinin receptors participate in inflammatory processes by mediating the release of proinflammatory cytokines and recruitment of immune cells (McLean et al, 2000;Leeb-Lundberg et al, 2005;Ehrenfeld et al, 2006), the properties of the B 1 R indicate sustained signaling and thus a role in the prolonged phase of the immune response with amplification of inflammatory processes (Blaukat et al, 1996;Austin et al, 1997;Marceau et al, 1998;Faussner et al, 1999;Phagoo et al, 2000;Marceau et al, 2002;Leeb-Lundberg et al, 2005). Inflammation is characterized by five cardinal symptoms: pain, redness, swelling, loss of function, and heat (Elliott et al, 1960).…”