Negative Cooperativity in the Human Bradykinin B2Receptor

Abstract

A human kidney bradykinin (BK) B 2 receptor cDNA was transfected in CHO-K1 cells to establish cell lines that express stably and at high density a receptor exhibiting B 2 receptor properties in terms of coupling to cell signaling effectors, desensitization, and internalization. A cell line with a density of 1.3 ؋ 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, B 2 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.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). BK binds to specific receptors that have been classified into B 1 and B 2 receptors according to their relative affinities for des-Arg 9 -BK and BK (2). These two types of receptors belong to the superfamily of seven-transmembrane domain receptors (3, 4). Most of the BK effects described so far are mediated by the B 2 receptor subtype. BK receptors are coupled through pertussis toxin-insensitive G proteins (5, 6) to at least two separate pathways of phospholipid metabolism (7-12), the hydrolysis of inositol phospholipids by phospholipase C (PLC) and the release of arachidonic acid by phospholipase A 2 (PLA 2 ). PLC stimulation produces the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate is likely responsible for the release of Ca 2ϩ from internal stores (13-15), and the physiological effects of BK are thought to be strongly dependent on its ability to mobilize [Ca 2ϩ ] i . BK-induced production of inositol phosphates, release of arachidonic acid, and elevation of [Ca 2ϩ ] i as well as the in vitro physiological responses to BK are of a smaller magnitude in cells or tissues preexposed to BK (7, 13, 16 -19). Agonist-induced phosphorylation of the receptor, resulting in receptor uncoupling and modulation of receptor affinity (20, 21) and loss of cellsurface binding sites (19,22,23) as a consequence of internalization of the ligand-receptor complex (19,23,24), has been proposed to play a role in the decreased responsiveness to BK. The receptor-mediated desensitization can contribute, together with the action of kininases and the triggering of physiological cou...

Bradykinin-induced Internalization of the Human B2Receptor Requires Phosphorylation of Three Serine and Two Threonine Residues at Its Carboxyl Tail

“…To further test this hypothesis, we investigate with the B 2 wt whether PKC is involved in the internalization and phosphorylation processes. It was apparent that [detail (4), this is in contrast with the reported role of PKC in B 2 receptor modulation in other cell types (17, 34 -36) and can be explained by a low PKC content of COS-7 and CHO-K1 cells facing receptor overexpression.…”

“…Then, we choose to stably express in CHO-K1 cells the mutant tY320 bearing the most pronounced structural change and also the largest deleted mutant, del[335-351] lacking the 5 Ser and Thr residues. The B 2 wt CHO cell line was obtained as described previously (4). In these cells (Table II) (Fig.…”

“…Hence deletion mutants tR351, tI334, and tY320 lacked the terminal 13, 30, and 44 amino acid residues, respectively, of the human B 2 17,8, and 9 amino acid residues, respectively; their relative positions within the protein sequence are given in brackets. Point mutations were created by the same procedure to replace Ser and/or Thr residues in the COOH-terminal tail by Ala using the following oligonucleotide primers: 5Ј-GGAGATCCAGGCCGAGAGGAGG-3Ј for the mutant T237A, 5Ј-CGAAAGAAGGCTTGGGAGGTG-3Ј for S316A, 5Ј-GGCTGCAGGGCAGAACCC-3Ј for S331A, 5Ј-GATGGAGA-ACGCCATGGGCAC-3Ј for S339A, 5Ј-CTCCATGGGCGCACTGCGGA-C-3Ј for T342A, 5Ј-GCACACTGCGGGCCGCCATCTCCGTG-3Ј for T345A/S346A, 5Ј-GACCTCCATCGCCGTGGAAC-3Ј for S348A, 5Ј-GG-AGAACGCCATGGGCGCACTGCGG-3Ј for S339A/T342A and 5Ј-GAA-CGCCATGGGCGCACTGCGGGCCGCCATCGCCGTGG-3Ј for B (4). Cells were washed with HBSS before cell surfacebound radioligand was separated from internalized radioactivity by an acidic washing step (27) H]BK challenge, and internalization was followed for 10 min in the continuous presence of the phosphatase inhibitor.…”

Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers

“…Using the 'infinite dilution' method 30,31 as a read-out of the cooperative interaction, we demonstrate an inverse relation between constitutive activity and negative cooperativity in all the three GpHrs tested. Our data provide evidence to support a model in which the TMD tunes the affinity and selectivity of the interaction between the hormone and the ECD.…”

Negative Cooperativity of Glutamate Binding in the Dimeric Metabotropic Glutamate Receptor Subtype 1

“…Extensive biochemical and crystallographic studies have been performed on the bacterial dimeric aspartate receptor, and the mechanism of negative cooperativity on aspartate binding has been elucidated on the basis of the structure (11)(12)(13)(14). Allosteric properties are also inferred in several GPCRs (15,16), however, in these cases, the observed cooperativity seems to result from oligomerization of receptors on the membrane surface. Thus, the allosteric properties in terms of subunit-subunit communication are more obscure in the GPCRs than those in the bacterial aspartate receptor.…”