Structure of the Bradykinin B₂Receptors' Amino Terminus

Abstract

The peptide hormone bradykinin exerts important biological functions by binding to and activating bradykinin B2 receptors. B2 receptors belong to the seven transmembrane domain (7TM) receptor family. Cloning of the cDNA sequences for the rat, human, and mouse bradykinin B2 receptor revealed several in-frame AUG triplets as potential initiation sites for translation. Due to "Kozak-like" consensus nucleotides, the AUG codon closest to transmembrane domain 1 was assumed the preferred initiation site for translation, but the real amino terminus of the B2 receptor protein was unknown. The amino terminus of several 7TM receptors has been shown to be essentially involved in receptor activation and/or ligand binding. Therefore we determined the amino terminus of the human and of the rat B2 receptor using domain-specific antipeptide antibodies, amino acid sequence analysis, and in vitro transcription/translation. We report that the human and rat B2 receptor protein start with the methionine which is translated from the first in-frame AUG. This start site extends the known amino terminus of the human and rat B2 receptors by 27 or 30 amino acid residues, respectively. Antibodies raised against a peptide of the initial 27 amino acids of the human B2 receptor stained B2 receptors on intact cells. This finding excludes the existence of a signal sequence for this receptor.

Review The kinin B2 receptor gene structure, product processing and expression in adult and fetal rats: evidence for gene evolution

“…This exon, not predicted for mice or for the human kinin B 2 receptor gene, was suggested based on evidence from amplification of reverse-transcriptase products from total RNA (Pesquero et al, 1994). Results obtained with domain-specific anti-peptide antibodies, amino acid sequence analysis and in vitro transcription and translation suggested a first translation initiation codon in the human and rat "exon-X" (AbdAlla et al, 1996) rather than in exon-4, as previously thought. This would imply a four-exon structure for the rat and human kinin B 2 receptor gene.…”

“…The latter configuration suggested a four-exon, three-intron gene structure for the rat gene. Others (AbdAlla et al, 1996), based on in vitro studies, reported that the "exon-X" transcripts, containing an AUG initiation codon anteceding an ORF in the purported "exon-X", would add about 27 amino acids to the N-terminal end of the human and rat kinin receptor proteins. The reasons for "exon-X" to be mistakenly identified as a constituent of the mature B 2 kinin receptor mRNA (Pesquero et al, 1994) are hinged on two non-converging aspects apart from the misleading published information (McEachern et al, 1991).…”

“…The predicted N-terminal ~27 residue related to the 211-bp rat kinin B 2 receptor gene "exon-X" did not match published amino acid sequences (Genbank, accession number P25023). Curiously, the predicted alternative N-terminus of human, rat and mouse proteins (AbdAlla et al, 1996) displayed negligible homology with each other. Figure 5A shows the rat kinin B 2 receptor gene structure and Figure 5B shows the respective encoding mature mRNA and exon alignments to the mouse and human genome.…”

Involvement of the Amino Terminus of the B2 Receptor in Agonist-induced Receptor Dimerization

“…Cells-To further analyze the role of the amino terminus of the B 2 receptor in receptor dimerization, we created two rat B 2 receptor variants with truncated amino termini, B 2 53 , starting with amino acid 53 after the conserved cysteine 52 (17), and B 2 65 , lacking the entire amino terminus. The indicated positions of the amino acids are derived from the rat B 2 receptor sequence with extended amino terminus (17).…”

“…The indicated positions of the amino acids are derived from the rat B 2 receptor sequence with extended amino terminus (17). The wild-type B 2 receptor and the two B 2 receptor variants were expressed in HEK293 cells.…”