Mutagenic analysis of platelet thromboxane receptor cysteines - Roles in ligand binding and receptor-effector coupling
Mutagenic analysis of platelet thromboxane receptor cysteines - Roles in ligand binding and receptor-effector coupling
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DOI：
10.1074/jbc.271.11.6233
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发表时间：
1996-03-15
影响因子：
4.8
通讯作者：
Dorn, GW
中科院分区：
文献类型：
--
作者：
DAngelo, DD;Eubank, JJ;Dorn, GW
人血小板血栓素 A(2) 受体是 G 蛋白偶联受体超家族的成员。先前的药理学研究检查了生化还原、氧化或巯基烷基化对血栓素受体的影响，表明半胱氨酸在确定受体结合特性中发挥着作用。为了表征单个半胱氨酸的作用，我们采用定点诱变在整个人 K562 血栓素受体的七个位置上用丝氨酸替换半胱氨酸，并分析突变受体放射性配体 ([1S-(1 α,2 β(5Z),3 α-(1E,3S),4 α]-7-[3-(3-羟基-4-(对碘苯氧基)-1-丁烯基)-7-氧杂双环-[2.2.1]庚烷-2-基]-5-庚烯酸)结合和钙信号传导。替换氨基末端的半胱氨酸(氨基酸位置11)和跨膜结构域2和6(位置68和257)对血栓素受体结合或信号传导几乎没有影响。第一个（位置 105）或第二个（位置 183）胞外环中的半胱氨酸消除了血栓素受体结合，这与这些位置之间存在关键二硫键一致。胞外环一（位置 102）中第二个半胱氨酸的突变导致受体的结合亲和力降低且结合能力低，仅转导低幅度的钙信号，表明该位置处的游离巯基参与了受体-配体相互作用。位于细胞内环三的位置 223 的半胱氨酸的突变导致受体具有正常配体结合特征，但不转导钙信号。该受体区域中的一些额外氨基酸取代（Cys-223 --> Ala、Thr-221 --> Met）导致受体具有正常结合但转导低幅度钙信号，而同一区域中的其他突变（His-224 --> Arg 和 His-227）。 --> Arg) 表现出正常的结合和钙信号传导特征。这些发现表明，细胞外环 1 和细胞外环 2 中的半胱氨酸有助于配体与血栓素受体的正确结合，并显示了第三个细胞内环中的离散氨基酸序列，尤其是半胱氨酸 223，在血栓素受体-效应器偶联中的重要性。
The human platelet thromboxane A(2) receptor is a member of the G-protein-coupled superfamily of receptors. Previous pharmacologic studies examining the effects of biochemical reduction, oxidation, or sulfhydryl alkylation on thromboxane receptors have suggested a role for cysteines in determining receptor binding characteristics. To characterize the roles of individual cysteines, we employed site-directed mutagenesis to substitute serines for cysteines at seven positions throughout the human K562 thromboxane receptor and analyzed mutant receptor radioligand ([1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3S),4 alpha]-7-[3-(3- hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptane-2-yl]-5-heptenoic acid) binding and calcium signaling. Replacing cysteines in the amino terminus (amino acid position 11), and transmembrane domains two and six (positions 68 and 257) had little effect on thromboxane receptor binding or signaling. Introduction of serines for cysteines in the first (position 105) or the second (position 183) extracellular loop eliminated thromboxane receptor binding, consistent with the existence of a critical disulfide bond between these positions. Mutation of a second cysteine in extracellular loop one (position 102) resulted in a receptor with decreased binding affinity and low binding capacity that transduced only a low amplitude calcium signal, suggesting the involvement of a free sulfhydryl group at this location in receptor-ligand interactions. Finally, mutation of the cysteine at position 223, located in intracellular loop three, resulted in a receptor with normal ligand binding characteristics, but which did not transduce a calcium signal. Some additional amino acid substitutions in this region of the receptor (Cys-223 --> Ala, Thr-221 --> Met) resulted in receptors that had normal binding but transduced low amplitude calcium signals, while other mutations in the same region (His-224 --> Arg and His-227 --> Arg) exhibited normal binding and calcium signaling characteristics. These findings demonstrate that cysteines in extracellular loops one and two contribute to proper ligand binding to thromboxane receptors and show the importance of discrete amino acid sequences in the third intracellular loop, especially cysteine 223, in thromboxane receptor-effector coupling.