Ligand-induced Phosphorylation/Dephosphorylation of the Endogenous Bradykinin B2 Receptor from Human Fibroblasts

Abstract

The nonapeptide bradykinin (NH 2 -Arg-Pro-Pro-Gly-PheSer-Pro-Phe-Arg-COOH), a prototypic member of the kinin family, mediates important biological processes such as hypotension, edema formation, pain sensations, smooth muscle contraction, and cell growth (1). Kinins are locally released on the surface of target cells due to limited proteolysis of their parental molecules, kininogens, by the kallikreins (2). The broad spectrum of their (patho)physiological activities is mediated by cognate kinin receptors that classify pharmacologically as B1 and B2 subtypes (3). B1 receptors respond preferentially to des-Arg 10 -kallidin, whereas B2 receptors are stimulated by bradykinin and kallidin (lysylbradykinin). The multiplicity of biological effects elicited by the kinins is reflected by the complexity of their signaling pathways. B2 receptors couple to various G proteins such as G q , thereby triggering the inositol trisphosphate/Ca 2ϩ pathway via phospholipase C-␤ and/or the arachidonic acid/prostaglandin pathway via phospholipase A 2 (4). Recent findings indicate that B2 receptors might also couple to the cAMP pathway via G s (5). In human foreskin fibroblasts, bradykinin induces, via phospholipase C, a transient rise in [Ca 2ϩ ] i that is counteracted by Ca 2ϩ extrusion (6). The increased [Ca 2ϩ ] i activates the nitric oxide/cGMP pathway and, together with diacylglycerol, drives the translocation and activation of protein kinase C, specifically of its isoforms ␣, ⑀, and (7).Given the remarkable pharmacological profile of these substances, it is evident that the activity of these peptides demands a careful control. Elaborate mechanisms exist that direct the kininogens to the surface of their target cells and allow kinin release at or next to its site of action (8). The liberated kinins are rapidly degraded in vivo by peptidases such as angiotensin-converting enzyme (kininase II), carboxypeptidase N (kininase I), and aminopeptidase P, which truncate and thereby inactivate the kinin peptides (9); the half-life of bradykinin in the plasma is Ͻ15 s (10). At the level of their receptors, the actions of kinins are restricted with respect to time and space by mechanisms involving receptor desensitization (11), internalization of the receptor-ligand complex (12, 13), loss of extracellular ligand-binding sites (14), and modulation of receptor affinity (11). Although tachyphylaxis and redistribution are well documented for the B2 receptor, the molecular mechanisms underlying these phenomena are not well understood. We hypothesized that reversible phosphorylation of kinin receptors might contribute to these phenomena.Here we have set out to investigate the agonist-induced phosphorylation of the bradykinin B2 receptor in a nontransformed human cell line (HF-15 fibroblasts) that endogenously expresses a high copy number of the B2 receptor. Using an anti-peptide antibody cross-reactive with the native receptor, we demonstrate the ligand-induced phosphorylation on Ser and Thr residues located in the carboxyl-terminal domain of th...

Mutation of Tyrosine in the Conserved NPXXY Sequence Leads to Constitutive Phosphorylation and Internalization, but Not Signaling, of the Human B2 Bradykinin Receptor

“…6, upper panel, left). Activation with 1 M BK resulted in appearance of three additional spots which correspond to peptides comprising Ser(P) 339 , Ser(P) 346 , and Thr(P) 342 , as their relative intensities and distribution were in full agreement with the results reported for human fibroblasts (21). Surprisingly, however, even without stimulation, the Y305A mutant exhibited a phosphorylation pattern similar to that observed in the wt B 2 R only after ligand stimulation (Fig.…”

“…The B 2 R displayed distinct phosphorylation even in the absence of a stimulus (Fig. 5), as has been reported before in human fibroblasts (21). When stimulated for 5 min with a saturating concentration of 1 M BK at 37°C, however, the wt B 2 R responded with a marked increase (2.5 Ϯ 0.5-fold over basal) in phosphorylation.…”

“…The lysate was centrifuged at 6.240 ϫ g for 20 min at 4°C. The supernatant (0.5 ml with ϳ1.5 mg of total protein) was incubated with 35 l of protein G-agarose and 2.5 l of antiserum AS346 (21,22) for 3 h at 4°C. The mixture was then washed twice with RIPA buffer and once with distilled water, resuspended in 30 l of Laemmli buffer, and incubated 6 min at 95°C.…”

Participation of kinins in the captopril‐induced inhibition of intimal hyperplasia caused by interruption of carotid blood flow in the mouse

“…Kinins are vasoactive peptides released by kallikreins from the precursor kininogens. Binding of kinins to speci®c 7 trans-membrane domain, G-protein-coupled B 1 and B 2 receptors expressed on vascular endothelial cells (ECs) stimulates the formation of nitric oxide (NO) and prostacyclin, thus leading to activation of cyclic GMP and cyclic AMP pathways (Blaukatt et al, 1996;D'Orleans-Juste et al, 1989). Di erently from the constitutively expressed B 2 receptors, B 1 are inducible receptors, i.e.…”

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).…”