Expression cloning of a rat B2 bradykinin receptor.

Abstract

A cDNA encoding a functional bradykinin receptor was isolated from a rat uterus library by a clonal selection strategy using Xenopus laevis oocytes to assay for expression of bradykinin responses. The predicted protein is homologous to the seven transmembrane G protein-coupled superfamily of receptors. Bradykinin and its analogs stimulate a Cl current in oocytes expressing the receptor with the rank order of potency: bradykinin Lys-bradykinin > [Tyr8]-bradykinin >> [Phe6Jbradykinin. This is the rank order of potency observed for these compounds in competitive binding assays on soluble receptor from rat uterus. Des-Arg9-bradykinin (10 pAM) elicits no response when applied to oocytes expressing the receptor; thus, the cDNA encodes a B2 type bradykinin receptor. [This8,DPhe7Jbradykinin, where Thi is I3-(2-thienyl)-alanine, is a very weak partial agonist and inhibits the bradykinin-mediated ion flux, suggesting the cDNA encodes a smooth muscle, rather than a neuronal, B2 receptor subtype. Receptor message has a distribution consistent with previous reports of bradykinin function and/or binding in several tissues and is found in rat uterus, vas deferens, kidney, lung, heart, ileum, testis, and brain. Receptor subtypes are a possibility because several tissues contain two or three message species (4.0, 5.7, and 6.5 kilobases). Southern blot highstringency analysis demonstrated that the rat, guinea pig, and human genomes contain a single gene. As bradykinin is a key mediator of pain, knowledge of the primary structure of this receptor will allow a molecular understanding of the receptor and aid the design of antagonists for pain relief.The nonapeptide bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-ProPhe-Arg) is a mediator of pain, inflammation, vascular permeability, smooth muscle tone in vascular and other tissues, and gastrointestinal function (1-5). Bradykinin can serve as a growth factor (2, 6, 7). Bradykinin binds to G proteincoupled receptors that activate phospholipase C or phospholipase A2 and increases synthesis of inositol trisphosphate or arachadonic acid (8-10).Bradykinin receptors have been classified as two major subtypes, B1 and B2 (1). The bradykinin metabolite des-Arg9-bradykinin is a B1 receptor agonist with potency greater than bradykinin, whereas it is inactive at B2 receptors. B2 receptors have been subdivided into two subtypes, a "neuronal" form, which is fully activated by [Thi58,DPhe7]bradykinin, where Thi is 83-(2-thienyl)-alanine, and a "smooth muscle" form, which is weakly activated by [This'8,DPhe7]bradykinin (11,12). Other subtypes of the B2 receptor have also been suggested (13,14).

A Novel Inhibitory Effect on Prostacyclin Synthesis of Coupling Factor 6 Extracted from the Heart of Spontaneously Hypertensive Rats

“…Because this peptide attenuated the use of endogenous AA in prostacyclin synthesis without affecting the uses of exogenous AA and PGH 2 , its effect appeared to be focused on PLA 2 (36,37). However, BEL has been found to block Mg 2ϩ -dependent phosphatidate phosphohydrolase, a key enzyme in cellular phospholipid metabolism (38).…”

“…Of a number of stimuli, bradykinin (1,2) and arginine vasopressin (3), whose receptors are coupled to GTPbinding proteins, enhance AA release via Ca 2ϩ -dependent translocation of cytosolic phospholipase A 2 (PLA 2 ) (4) and the activation of PLA 2 due to phosphorylation (5,6). In contrast, growth factors (7,8), whose receptors are coupled to tyrosine kinase, activate PLA 2 by the additional mechanism of de novo protein synthesis.…”

Renal Protective Role of Bradykinin B1 Receptor in Stroke-Prone Spontaneously Hypertensive Rats

“…Semi-quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) was performed using Ready-to-Go RT-PCR Beads (Amersham Pharmacia Biotech, Tokyo, Japan) as previously reported (17). Pairs of specific primers for RT-PCR of B1R and β actin were obtained based on previously reported sequences (18,19). Each sample was electrophoresed on 2.0% agarose gel and stained with ethidium bromide.…”

Guanosine Nucleotides Regulate B2 Kinin Receptor Affinity of Agonists But Not of Antagonists: Discussion of a Model Proposing Receptor Precoupling to G Protein

“…Recently the rat, mouse and human B2 kinin receptors, as well as the human B1 kinin receptor, have been cloned (McEachern et al, 1991;Hess et al, 1992;Menke et al, 1994;Webb et al, 1994) and, based on sequence homology, were grouped into the superfamily of the G protein-coupled receptors (GPCRs). These findings were in agreement with previous reports, that BK, via the B2 receptor, is capable of activating phospholipase A and/or C and of inhibiting adenylate cyclase via signal transduction pathways that utilize guanosine nucleotide regulatory proteins (Liebmann and Reissmann, 1990;Gutowski et al, 1991;Liebmann et al, 1991;McEachern et al, 1991;Tippmer et al, 1994). However, both the subtype of the G protein(s) involved, as well as the effect of guanine nucleotides on [ 3 H]BK binding sites, are not completely understood.…”