Monomers and oligomers of the M2 muscarinic cholinergic receptor purified from Sf9 cells
Monomers and oligomers of the M2 muscarinic cholinergic receptor purified from Sf9 cells
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DOI：
10.1021/bi034491m
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发表时间：
2003-11-11
期刊：
影响因子：
2.9
通讯作者：
Wells, JW
中科院分区：
文献类型：
--
作者：
Park, PSH;Wells, JW
已知G蛋白偶联受体形成寡聚体。为了探索这种聚集体的性质以及单体的作用和流行程度，已从Sf9细胞中分离出M-2受体的表位标记形式作为寡聚体和单体。共表达c-Myc和FLAG标记受体的细胞膜被溶解在洋地黄素-胆酸盐中，通过DEAE-Sepharose、亲和树脂3-(2‘-氨基苯羟氧基)托烷(ABT)-Sepharose和羟基磷灰石连续传代纯化受体。两个表位的免疫共沉淀表明，在纯化的每个阶段都存在低聚物，直到(但不包括)从ABT-Sepharose特异性洗脱出来的部分。因此，亲和纯化的受体似乎是单体。未能检测到免疫共沉淀并不是由于无效的抗体，纯化条件似乎也没有促进解聚。在纯化的各个阶段，受体都与N-[H-3]甲基东莨菪碱和[H-3]奎宁基苯甲酸酯高亲和力结合，但未保留在ABT-Sepharose上的受体的容量仅为用抗M-2抗体探测的免疫印迹密度法所预期的4%。同样，通过在抗c-Myc和抗FLAG免疫亲和柱上连续通过共表达的受体分离得到的寡聚体的活性也很低。因此，M-2受体似乎以活性单体和大部分或全部非活性寡聚体的形式共存于Sf9细胞的溶解提取物中。当混合感染的细胞在增溶之前用喹诺酮基苯甲酸酯处理时，出现了不同的模式，从这些细胞中纯化的ABT受体显示出免疫共沉淀。因此，用拮抗剂处理导致了至少部分组成部位活跃的寡聚体，并被ABT-琼脂糖凝胶保留。
G protein-coupled receptors are known to form oligomers. To probe the nature of such aggregates, as well as the role and prevalence of monomers, epitope-tagged forms of the M-2 muscarinic receptor have been isolated as oligomers and monomers from Sf9 cells. Membranes from cells coexpressing the c-Myc- and FLAG-tagged receptor were solubilized in digitonin-cholate, and the receptor was purified by successive passage through DEAE-Sepharose, the affinity resin 3-(2'-aminobenzhydryloxy)tropane (ABT)-Sepharose, and hydroxyapatite. Coimmunoprecipitation of the two epitopes indicated the presence of oligomers at each stage of the purification up to but not including the fraction eluted specifically from ABT-Sepharose. The affinity-purified receptor therefore appeared to be monomeric. The failure to detect coimmunoprecipitation was not due to an ineffective antibody, nor did the conditions of purification appear to promote disaggregation. Receptor at all stages of purification bound N-[H-3]methylscopolamine and [H-3]quinuclidinylbenzilate with high affinity, but the capacity of receptors that were not retained on ABT-Sepharose was only 4% of that expected from densitometry of western blots probed with an anti-M-2 antibody. Similarly low activity was found with oligomers isolated by successive passage of coexpressed receptor on anti-c-Myc and anti-FLAG immunoaffinity columns. M-2 muscarinic receptors therefore appear to coexist as active monomers and largely or wholly inactive oligomers in solubilized extracts of Sf9 cells. A different pattern emerged when coinfected cells were treated with quinuclidinylbenzilate prior to solubilization, in that ABT-purified receptors from those cells exhibited coimmunoprecipitation. Treatment with the antagonist therefore led to oligomers in which at least some of the constituent sites were active and were retained by ABT-Sepharose.