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Volume 271, Number 44, Issue of November 1, 1996 pp. 27382-27387
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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The N-terminal Amino Group of [Tyr⁸]Bradykinin Is Bound Adjacent to Analogous Amino Acids of the Human and Rat B₂ Receptor

(Received for publication, July 2, 1996, and in revised form, August 14, 1996)

Said AbdAlla , Kurt Jarnagin ^§ , Werner Müller-Esterl and Ursula Quitterer ^¶

From the  Institute of Physiological Chemistry and Pathobiochemistry, University of Mainz, 55099 Germany, the ^§ Inflammatory Diseases Unit, Roche Bioscience, Palo Alto, California 94304, and the ^¶ Institute of Pharmacology and Toxicology, University of Würzburg, 97078 Würzburg, Germany

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 -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.

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