Negative Cooperativity in the Human Bradykinin B2Receptor

Anne Pizard,Jeannine Marchetti,Jacqueline Allegrini,François Alhenc-Gelas,Rabary M Rajerison

doi:10.1074/jbc.273.3.1309

Abstract

A human kidney bradykinin (BK) B2 receptor cDNA was transfected in CHO-K1 cells to establish cell lines that express stably and at high density a receptor exhibiting B2 receptor properties in terms of coupling to cell signaling effectors, desensitization, and internalization. A cell line with a density of 1.3 x 10(6) receptors/cell allowed us to carry out a detailed study of BK-receptor interaction over a wide range of BK concentrations. A model assuming that BK binds to two receptor affinity states (depending on guanine nucleotide-sensitive coupling) was not sufficient to account for the kinetics of BK binding. Equilibrium kinetic analysis and studies of the effects of receptor occupancy by agonists or antagonists on the kinetics of BK-receptor complex dissociation revealed features typical of negative cooperative binding. The negative cooperativity phenomenon was also observed in isolated membranes in both the presence and absence of guanine nucleotide. Thus, following the interaction with BK, B2 receptor molecules likely interact with each other, resulting in an acceleration of bound ligand dissociation and a decrease in the apparent affinity of the receptor for BK. This phenomenon can participate in the desensitization process.

Highlights

Bradykinin (BK)1 is involved in a variety of physiological and pathological processes, including vasodilation and control of vascular tone, ion transfer in epithelia, and pain [1]
After the obtainment and pharmacological characterization of a recombinant human renal BK B2 receptor that was expressed at high density in CHO-K1 cells, we studied in detail the kinetics of BK interaction with the receptor
This study was performed on stable CHO-K1 cell lines transfected with BK B2 receptor cDNA synthesized from human kidney RNAs

Summary

EXPERIMENTAL PROCEDURES

Cloning of Human BK B2 Receptor and Transfection—One ␮g of total RNA prepared from normal human kidney tissue (CLONTECH, Heidelberg, Germany) was reverse-transcribed and amplified (30 cycles of 1 min at 95 °C, 1.5 min at 55 °C, and 1 min at 72 °C) using two B2 receptor-specific primers, 5Ј-GGAATTCTCACTCACATCCCACTTGAGTC-3Ј and 5Ј-GGAATTCACAGCAGCCCTGCTGGC-3Ј, corresponding to the sequence of the published human fibroblast B2 receptor [4] with an EcoRI site added at the 5Ј-end. Cells were rinsed at 37 °C: three times with 1 ml of phosphate-buffered saline, once with 1 ml of HBSS, and twice for 20 min with 1 ml of HBSS containing 0.1% fatty acid-free BSA. Phospholipase A2 activation experiments were performed in 0.5 ml of HBSS containing 0.1% BSA and protease inhibitors at 37 °C for 10 min and started by adding test compounds or vehicles and stopped by the removal of the incubation medium. Cell-associated radioactivity was determined after lysis with 0.2 ml of 1 N NaOH to express results as the ratio between the medium radioactivity containing the [3H]arachidonic acid released and the sum of the medium plus cell-associated radioactivity. Comparisons between experimental conditions were performed by analysis of variance

RESULTS

IP production with analogue

DISCUSSION