Human bradykinin B2 receptors isolated by receptor-specific monoclonal antibodies are tyrosine phosphorylated.

Abstract

We report the immunoaffinity isolation of bradykinin B2 receptors in a tyrosine-phosphorylated state from WI-38 human lung fibroblasts. We generated six monoclonal antibodies directed against B2 bradykinin receptor biologic activity mediating prostaglandin E2 production in WI- Bradykinin (BK), Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, is a multifunctional mediator that aids in maintaining tissue homeostasis and plays a key role in events accompanying inflammation. BK is proteolytically generated from kininogen precursors in circulation and interstitial tissue fluids; its local concentration is rapidly increased upon tissue injury. BK receptors belonging to the guanine nucleotide-binding protein (G-protein)-coupled family trigger neuronal transmission of pain or irritant messages, enhancement of vascular permeability, and altered vasomotor tone that are prominent features of the inflammatory process. BK action on these same receptors also regulates mitogenesis and protein production in fibroblasts, initiating repair steps that restore normal tissue form and function following injury (1-4).We (1) previously demonstrated that IMR-90 human lung fibroblasts sequentially mobilize BK B2 subtype receptors of Kd = 2.5-5 nM and 44 nM that mediate prostaglandin E2 (PGE2) production, upon progression through their finite life span in vitro. Further information is needed at the cellular and molecular level to elucidate the nature of BK B2 receptors and the means by which cells can express diverse receptor forms. Although recent cloning of two BK receptors has disclosed their primary sequences (5-7), the molecular structure of the protein as expressed in intact cells remains uncharacterized. We have generated monoclonal antibodies (mAbs) that distinguish the BK receptors of Kd = 5 nM and 44 nM in human lung fibroblasts and have used them to isolate the receptor protein and to demonstrate that it containsThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. phosphorylated tyrosine, which may serve a regulatory function. We also show that tyrosine kinase activity is essential for BK-mediated PGE2 production. These mAbs will allow us to explore the role of BK receptors in the growth and development of fibroblasts and in these cells' participation in onset and/or propagation of the injury/inflammation response. 4°C was covalently crosslinked to receptors with 0.4 mM disuccinyl suberimidate (Pierce) at 4°C for 20 min. After quenching with 150 mM Tris (pH 8), cells were solubilized 2.5 min in 400 ,ul of 1% Triton X-100 or 12 mM 3-[(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/ 0.5% digitonin (CD) in 0.15 M NaCl/15 mM Tris Cl, pH 7.4, containing protease inhibitors (100 ,uM phenylmethanesulfonyl fluoride, 16.8 uM leupeptin, chymostatin at 4 ,ug/ml, 5.8 ,tM pepstatin, 6.6 ;LM antipain, 0.08 trypsin inhibitor unit of aprotinin per ml, 10 mM benzamidine, soybe...

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

“…We were unable to detect Tyr phosphorylation both by phosphoamino acid analysis and by Western blotting using a monoclonal antibody to phosphotyrosine, PY20 (data not shown). This latter finding contrasts with a previous report in which phosphorylation of the B2 receptor from WI-38 human lung fibroblasts was shown to occur on Tyr residue(s) by the PY20 antibody (45). Because the cells were not stimulated by bradykinin, the distinct possibility remains that Tyr phosphorylation was heterologously induced by an unknown ligand.…”

“…Crude membranes were collected by centrifugation at 30,000 ϫ g for 30 min at 4°C and further homogenized by repeated passages through increasingly smaller cannulas. The membranes were layered on top of a stepwise gradient consisting of 3 ml each of 45,35, and 20% (w/w) sucrose in buffer A and centrifuged at 100,000 ϫ g for 90 min at 4°C in a swing-out rotor. The membrane fractions were collected, diluted (1:4) in buffer A, centrifuged (100,000 ϫ g, 20 min, 4°C), and washed with buffer A including 250 mM NaCl and protease inhibitors.…”

Effects of Intracellular Tyrosine Residue Mutation and Carboxyl Terminus Truncation on Signal Transduction and Internalization of the Rat Bradykinin B2 Receptor

“…This sequence contains four serine and two threonine residues and two protein kinase C consensus sequences. We also focused on the two intracellular Tyr residues that have been implicated in signal transduction leading to prostaglandin synthesis (12). Tyr-131, located in the second intracellular loop (i2) within the DRY motif, is within a region that is conserved among the family of G-protein coupled receptors (13).…”

International Union of Pharmacology. XLV. Classification of the Kinin Receptor Family: from Molecular Mechanisms to Pathophysiological Consequences

“…Early studies claimed the tyrosine phosphorylation of the human B 2 receptor (Jong et al, 1993) ) in proximity to the identified palmitoylation site in the C-terminal domain (Soskic et al, 1999). However, ligand-dependent tyrosine phosphorylation of the B 2 receptor has not yet been shown.…”