The N-terminal amino group of [Tyr8]bradykinin is bound adjacent to analogous amino acids of the human and rat B2 receptor.
To obtain data of the bradykinin B2 receptor's agonist binding site, we used a combined approach of affinity labeling and "immunoidentification" of receptor fragments generated by cyanogen bromide cleavage. Domain-specific antibodies to the various extracellular receptor domains were applied to detect receptor fragments with covalently attached [125I-Tyr8]bradykinin. As a cross-linker we used the homobifunctional reagent disuccinimidyl tartarate (DST), which reacts preferentially with primary amines. With this technique a [125I-Tyr8]bradykinin-labeled receptor fragment derived from the third extracellular domain was identified. The epsilon-amino group of lysine (Lys172) of the human B2 receptor provides the only primary amino group within this receptor fragment. This strongly suggests that DST attached the N-terminal amino group of [Tyr8]bradykinin to Lys172 of the human B2 receptor. Next we asked whether DST attaches [Tyr8]bradykinin to the analogous residue, Lys174 of the rat B2 receptor, which is 81% identical to the human B2 receptor, and we attempted to label the wild-type rat B2 receptor and a rat B2 receptor mutant where Lys174 had been exchanged for alanine. Affinity labeling of the wild-type rat B2 receptor worked efficiently, whereas DST did not attach detectable amounts of [125I-Tyr8]bradykinin to the K174A rat B2 receptor mutant. Taken together these observations indicate that the N-terminal amino group of [Tyr8]bradykinin is bound to analogous positions of the rat and of the human B2 receptor, i.e. [Tyr8]bradykinin's N terminus is bound adjacent to Lys172 of the human and Lys174 of the rat B2 receptor.
AbdAlla
S
S
Institute of Physiological Chemistry and Pathobiochemistry, University of Mainz, 55099 Germany.
Jarnagin
K
K
Müller-Esterl
W
W
Quitterer
U
U
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001920:Bradykinin / Q000031:analogs & derivatives* / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D003432:Cross-Linking Reagents; D006801:Humans; D007457:Iodine Radioisotopes; D008239:Lysine; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D051381:Rats; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011994:Recombinant Proteins; C102309:bradykinin, Tyr(8)-; D008239:Lysine; D001920:Bradykinin