Fathy D B, Mathis S A, Leeb T, Leeb-Lundberg L M
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760, USA.
J Biol Chem. 1998 May 15;273(20):12210-8. doi: 10.1074/jbc.273.20.12210.
In order to identify agonist- and antagonist-binding epitopes in the human B1 and B2 bradykinin (BK) receptors, we exploited the ability of these receptors to discriminate between peptide ligands that differ only by the absence (B1) and presence (B2) of a C-terminal Arg. This was done by constructing chimeric proteins in which specific domains were exchanged between these receptors as recently described by us (Leeb, T., Mathis, S. A., and Leeb-Lundberg, L. M. F. (1997) J. Biol. Chem. 272, 311-317). The constructs were then expressed in HEK293 and A10 cells and assayed by radioligand binding and by agonist-stimulated inositol phospholipid hydrolysis and intracellular Ca2+ mobilization. Substitution of the third transmembrane domain (TM-III) of the B1 receptor in the B2 receptor (B2(B1III)) dramatically reduced the affinities of B2-selective peptide ligands including both the agonist BK and the antagonist NPC17731. High affinity binding of both ligands to B2(B1III) was fully regained when one residue, Lys111, in TM-III of this chimera was replaced with the corresponding wild-type (WT) B2 receptor residue, Ser (B2(B1IIIS111)). Replacement of Ser111 with Lys in the WT B2 receptor decreased the affinities of BK and NPC17731 and increased the affinity of the B1-selective des-Arg10 analog of NPC17731, NPC18565. The results show that the C-terminal residue of peptide agonists and antagonists when bound to the B2 receptor is adjacent to Ser111 in the receptor. A Lys at this position, as is the case in the WT B1 receptor, provides a positive charge that repels the C-terminal Arg in B2-selective peptides and attracts the negative charge of the C terminus of B1-selective peptides, which lack the C-terminal Arg. Therefore, the residues at this one single position are crucial in determining the peptide selectivity of B1 and B2 BK receptors.
为了确定人B1和B2缓激肽（BK）受体中激动剂和拮抗剂结合表位，我们利用了这些受体区分仅因C末端精氨酸缺失（B1）和存在（B2）而不同的肽配体的能力。这是通过构建嵌合蛋白来实现的，其中特定结构域在这些受体之间进行交换，正如我们最近所描述的（Leeb, T., Mathis, S. A., and Leeb-Lundberg, L. M. F. (1997) J. Biol. Chem. 272, 311 - 317）。然后将构建体在HEK293和A10细胞中表达，并通过放射性配体结合、激动剂刺激的肌醇磷脂水解和细胞内Ca2+动员进行测定。将B1受体的第三个跨膜结构域（TM-III）替换到B2受体中（B2(B1III)），显著降低了B2选择性肽配体的亲和力，包括激动剂BK和拮抗剂NPC17731。当该嵌合体TM-III中的一个残基Lys111被相应的野生型（WT）B2受体残基Ser取代时（B2(B1IIIS111)），两种配体与B2(B1III)的高亲和力结合完全恢复。在WT B2受体中将Ser111替换为Lys会降低BK和NPC17731的亲和力，并增加B1选择性的NPC17731去-Arg10类似物NPC18565的亲和力。结果表明，肽激动剂和拮抗剂与B2受体结合时的C末端残基与受体中的Ser111相邻。在这个位置的一个Lys，如WT B1受体中的情况，提供了一个正电荷，排斥B2选择性肽中的C末端精氨酸，并吸引缺乏C末端精氨酸的B1选择性肽C末端的负电荷。因此，这个单一位置的残基对于确定B1和B2 BK受体的肽选择性至关重要。
