Palmitoylation of the recombinant human A1 adenosine receptor:: enhanced proteolysis of palmitoylation-deficient mutant receptors
Palmitoylation of the recombinant human A1 adenosine receptor:: enhanced proteolysis of palmitoylation-deficient mutant receptors
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DOI：
10.1042/0264-6021:3420387
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发表时间：
1999-09-01
影响因子：
4.1
通讯作者：
Linden, J
中科院分区：
文献类型：
--
作者：
Gao, ZH;Ni, YJ;Linden, J
在HEK-293细胞中表达的重组人A，腺苷受体(A，AR)的棕榈酰化结果表明，从用[H-3]棕榈酸酯代谢标记的细胞中提纯的六氢组氨酸(His(6))/Asp-Tyr-Lys-Asp-Lys(FLAG)(H/F)A(1)ARs将氚掺入38-42 kDa的受体糖蛋白中。细胞与A，AR选择性激动剂N-6-环戊基腺苷(CPA)孵育不影响棕榈酰化量。用中性羟胺处理或通过将Cys-309突变为Ala(C-309->；A)，AR棕榈酰化被取消。根据[S-35]蛋氨酸的蛋白质印迹和脉冲追逐实验，至少90%的野生型受体是棕榈酰化和翻转的，t(1/2)为6.4h。突变型受体中，40%出现类似野生型受体的翻转，t(1/2)为7.1h，60%出现快速切割，形成25 kDa的受体片段，翻转时间为0.8h。在表达类似数量的野生型或C-309、A突变型A(1)Rs的HEK-293细胞中，CPA诱导受体内化(1h)、下调(24h)、抑制Forsklin刺激的cAMP聚集的动力学几乎没有差别。或激活共表达的G蛋白激活的内向整流钾/心脏内向整流钾通道(GIRK1/CIR K+)。同样不受棕榈酰化影响的是鸟苷5‘-[γ-硫代]三磷酸([S]GTP)敏感的结合到膜上的激动剂I-125标记的氨基苯甲基腺苷。结果表明，棕榈酰化对受体-效应器偶联、激动剂诱导的内化或下调几乎没有影响。我们推测棕榈酰化可能使新合成的A(1)ARs偏离导致快速降解的途径。
Palmitoylation of the recombinant human A, adenosine receptor (A,AR) expressed in HEK-293 cells is demonstrated by showing that hexahistidine (His(6))/Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG) (H/F) A(1)ARs, purified to homogeneity from cells metabolically labelled with [H-3]palmitate, incorporate tritium into a 38-42kDa receptor glycoprotein. The amount of palmitoylation is not affected by incubation of cells with the A,AR-selective agonist N-6-cyclopentyladenosine (CPA). A,AR palmitoylation is abolished by treatment with neutral hydroxylamine or by mutation of Cys-309 to Ala (C-309 --> A). Based on Western blotting and pulse-chase experiments with [S-35]methionine, at least 90 % of wild-type receptors are palmitoylated and turn over with a t(1/2) of 6.4 h. Of the C-309 --> A mutated receptors, 40 % appear to turn over like wild-type receptors, with a t(1/2) of 7.1 h, and 60 % appear to be rapidly cleaved to form a 25 kDa receptor fragment that turns over with a t(1/2) of 0.8 h. In HEK-293 cell lines expressing similar numbers of wild-type or C-309, A mutant A(1)Rs, there is little difference in the kinetics of CPA-induced receptor internalization (1 h), down-regulation (24 h), inhibition of forskolin-stimulated cAMP accumulation, or activation of co-transfected G-protein-activated inward rectifier K+/cardiac inward rectifying K+ (GIRK1/CIR K+) channels. Also unaffected by palmitoylation is guanosine 5'-[gamma-thio]triphosphate ([S]GTP)-sensitive binding to membranes by the agonist I-125-labelled aminobenzyladenosine. The results suggest that palmitoylation has little effect on receptor-effector coupling, agonist-induced internalization or down-regulation. We speculate that palmitoylation may divert newly synthesized A(1)ARs from a pathway leading to rapid degradation.