Cloning, sequencing and functional expression of a guinea pig lung bradykinin B2 receptor

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Abstract

Kinin receptors are classified as B1 and B2 based upon agonist and antagonist potencies and cloning and expression studies. Using sequences from human and rat bradykinin B2 receptors, polymerase chain reaction (PCR) was utilized to isolate cDNA from guinea pig lung. The receptor obtained is predicted to have 372 amino acids and shares >80% sequence homology with human, rat, rabbit and mouse B2 receptors. In competition binding experiments in Chinese hamster ovary (CHO-K1) cells in which the guinea pig cDNA was expressed, [3H]bradykinin was displaced by kinin receptor ligands with an order of potency consistent with a B2 subtype. In CHO cells expressing the guinea pig receptor, bradykinin caused a concentration 45Ca2+ efflux. A B1 receptor agonist, desArg9-bradykinin, also caused 45Ca2+ efflux but with a potency several orders of magnitude lower than bradykinin. Curiously, several B1 and B2 receptor antagonists induced 45Ca2+ efflux, indicating that this receptor may be coupled differently in CHO cells than in native tissues.

Introduction

Receptors for bradykinin and related kinins are divided into B1 and B2 subtypes based on agonist and antagonist potency ratios in various tissues (Farmer and Burch, 1992; Hall and Morton, 1997). Cloning and sequencing studies have confirmed the existence of bradykinin B2 receptors in rats (McEachern et al., 1991), humans (Eggerickx et al., 1992; Hess et al., 1992; Powell et al., 1993), mice (McIntyre et al., 1993; Hess et al., 1994; Borkowski et al., 1995) and rabbits (Bachvarov et al., 1995). Bradykinin B1 receptors from the mouse (McIntyre et al., 1993; Hess et al., 1996; Pesquero et al., 1996), rabbit (MacNeil et al., 1995), and human (Menke et al., 1994; Webb et al., 1994; Bachvarov et al., 1996; Chai et al., 1996) have also been cloned, and their distinct sequence and pharmacology from B2 receptors confirmed. The deduced amino acid sequences from the four species' B2 receptors show extensive similarity (>80%) and they all belong to the G protein-coupled, seven transmembrane domained receptor superfamily. Recently, an avian kinin receptor was cloned. It shows 31% and 49% sequence identity, respectively, to human B1 and B2 receptors and is a G protein-coupled receptor (Schroeder et al., 1997).

The genomic organization of the bradykinin B2 receptor has been studied for the human, rat and mouse genes. The 5′ untranslated regions are interrupted by introns (Ma et al., 1994; Pesquero et al., 1994) and the intron/exon boundaries are highly conserved. There is also evidence for alternative splicing of the 5′ untranslated region of the rat B2 receptor gene (Pesquero et al., 1994). Numerous pharmacological studies in guinea pigs indicate that B2 receptors are expressed ubiquitously in tissues from this species (Farmer and Burch, 1992; Hall and Morton, 1997). Although there is no evidence for guinea pig B1 receptors, pharmacological studies in airway tissues from this species have suggested another subtype of bradykinin receptor, termed `B3' (Farmer et al., 1989b, Farmer et al., 1991; Pyne and Pyne, 1993; Farmer and DeSiato, 1994). The existence of the B3 receptor, however, is controversial (Trifilieff et al., 1991; Field et al., 1992; Da Silva et al., 1995; Scherrer et al., 1995). Evidence for bradykinin B3 receptors, as well as the `unusual' nature of guinea pig B2 receptors compared with those from other species, was reviewed extensively by Hall and Morton (1997). To date, there are no reports on the cloning or sequencing of any guinea pig kinin receptors. In the present paper, we report the isolation, sequencing and functional expression of a guinea pig lung bradykinin receptor.

Section snippets

Isolation and characterization of a guinea pig lung bradykinin receptor

RNA was isolated from whole lung tissue using guanidinium isothiocyanate as described elsewhere (Chirgwin et al., 1979). RACE-PCR (rapid amplification of cDNA ends-polymerase chain reaction) (Frohman et al., 1988; Frohman and Martin, 1990) was utilized to isolate both 5′ and 3′ ends of the guinea pig lung bradykinin receptor cDNA. To isolate the 3′ end of the cDNA, synthesis was carried out with superscript reverse transcriptase (Gibco BRL, Paisley, Scotland) using 5 μg total RNA, and the RACE

Isolation and characterization of a guinea pig lung bradykinin receptor

The 5′ and 3′ fragments of cloned guinea pig lung bradykinin receptor were isolated by RACE-PCR. Direct sequence analysis revealed that the 5′ product was from (−)137 to 601, and the 3′ product was from position 457 to 1198 (Fig. 1), with an overlap of 144 base pairs. A cDNA containing the whole coding sequence, corresponding to positions 1 to 1176 and containing the whole coding sequence, was isolated by reverse transcription (RT)-PCR. Sequences obtained from several independent full-length

Discussion

We report for the first time the cloning, sequencing and functional expression of a guinea pig lung bradykinin receptor. This receptor shares >80% sequence homology with kinin B2 receptors from human, rat, mouse and rabbit. Furthermore, in binding the rank order of potency of competing ligands in the cloned guinea pig receptor is consistent with it being a B2 subtype (Farmer and Burch, 1992; Hall and Morton, 1997). As noted earlier, there are many reports that guinea pig bradykinin B2 receptors

Acknowledgements

We thank Dave Holland for oligonucleotide synthesis.

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