Serine and alanine mutagenesis of the nine native cysteine residues of the human A(1) adenosine receptor.
Serine and alanine mutagenesis of the nine native cysteine residues of the human A(1) adenosine receptor.
复制标题
人 A(1) 腺苷受体的九个天然半胱氨酸残基的丝氨酸和丙氨酸诱变。
DOI：
10.1016/s0006-2952(00)00474-3
复制
发表时间：
2000
影响因子：
5.8
通讯作者：
Wells,JN
中科院分区：
文献类型：
--
作者：
Scholl,DJ;Wells,JN
为了检查人类 A1 腺苷受体中 9 个天然半胱氨酸残基的重要性，每个半胱氨酸分别突变为丝氨酸和丙氨酸。与 A1 选择性拮抗剂 [3H]DPCPX [8-环戊基-1,3-二(2,3-3H-丙基)黄嘌呤] 的饱和结合导致野生型 Kd 值为 0.92 nM。除 Cys80 和 Cys169 处的丝氨酸/丙氨酸突变外，所有丝氨酸和丙氨酸突变体均具有相似的 Kd 值。这两个半胱氨酸残基在 G 蛋白偶联受体中高度保守，并被假设通过二硫桥连接，表明没有检测到与 [3H]DPCPX 的结合。通过免疫染色可见，残基 Cys80 和 Cys169 处的丝氨酸和丙氨酸突变均导致细胞表面检测不到的受体。通过与拮抗剂茶碱和激动剂 NECA（5'-N-乙基羧酰胺腺苷）和 R-PIA [(R)N6-苯基异丙基腺苷] 的竞争结合，进一步表征了确实结合 [3H]DPCPX 的丝氨酸/丙氨酸突变体。野生型茶碱Ki值为2.41μM，丝氨酸/丙氨酸突变体具有相似的值。野生型 NECA 和 R-PIA Ki 值分别为 0.74 μM 和 97.0 nM。所有突变体的 Ki 值与野生型相似，但 Cys85Ser 突变体除外，其 NECA 和 R-PIA 值分别为 9.30 μM 和 387.3 nM。这些数据表明 Cys80 和 Cys169 对于将受体递送至质膜是绝对必需的。 Cys85Ser 数据表明，虽然该位置不需要半胱氨酸，但该残基可能在配体结合或受体结构中具有重要作用。
To examine the importance of the nine native cysteine residues in the human A1adenosine receptor, each cysteine was individually mutated to both serine and alanine. Saturation binding with the A1selective antagonist [3H]DPCPX [8-cyclopentyl-1,3-di(2,3-3H-propyl)xanthine] resulted in a wild-type Kdvalue of 0.92 nM. All serine and alanine mutants had similar Kdvalues with the exception of serine/alanine mutations at Cys80 and Cys169. These two cysteine residues, which are highly conserved in G protein-coupled receptors and hypothesized to be linked through a disulfide bridge, demonstrated no detectable binding with [3H]DPCPX. Both serine and alanine mutations at residues Cys80 and Cys169 resulted in receptors that were not detectable at the cell surface, as visualized by immunostaining. The serine/alanine mutants that did bind [3H]DPCPX were characterized further through competition binding with the antagonist theophylline and the agonists NECA (5′-N-ethylcarboxamidoadenosine) and R-PIA [(R)N6-phenylisopropyl adenosine]. The wild-type theophylline Kivalue was 2.41 μM, with the serine/alanine mutants having similar values. Wild-type NECA and R-PIA Kivalues were 0.74 μM and 97.0 nM, respectively. All mutants had Kivalues similar to wild-type with the exception of the Cys85Ser mutant, which had NECA and R-PIA values of 9.30 μM and 387.3 nM, respectively. These data show that Cys80 and Cys169 are absolutely required for delivery of the receptor to the plasma membrane. The Cys85Ser data indicate that although a cysteine is not required at this position, this residue may have an important role in ligand binding or for the structure of the receptor.