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.[1]References
- The N-terminal amino group of [Tyr8]bradykinin is bound adjacent to analogous amino acids of the human and rat B2 receptor. AbdAlla, S., Jarnagin, K., Müller-Esterl, W., Quitterer, U. J. Biol. Chem. (1996) [Pubmed]
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