Expression of two human beta-adrenergic receptors in Escherichia coli: functional interaction with two forms of the stimulatory G protein.
Expression of two human beta-adrenergic receptors in Escherichia coli: functional interaction with two forms of the stimulatory G protein.
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两种人类 β-肾上腺素能受体在大肠杆菌中的表达：与两种形式的刺激性 G 蛋白的功能相互作用。
DOI：
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发表时间：
1991
影响因子：
11.1
通讯作者：
A. Strosberg
中科院分区：
文献类型：
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作者：
M. Freissmuth;E. Selzer;S. Marullo;W. Schütz;A. Strosberg
当在大肠杆菌中表达时，人β 1-和β 2-肾上腺素能受体保留其配体结合特异性。通过分析受体-鸟嘌呤核苷酸结合调节（G）蛋白偶联，研究了它们的功能完整性，所述偶联通过使用在E. coli（rGs α-S和rGs α-L）和从牛脑中纯化的G蛋白的β γ亚基。在用（-）-[125 I]碘氰基吲哚酚和（-）-异丙肾上腺素的竞争结合实验中，rGs α-S. β γ和rGs α-L. β γ重建了具有可比亲和力的鸟嘌呤核苷酸敏感性高亲和力激动剂结合，而rGs α PT，具有改变的羧基末端的rGs α-L的突变体，和抑制性G蛋白的α亚基的重组亚型，rGi α-1，效力分别降低了约20倍和约200倍。β 1和β 2肾上腺素能受体在大肠杆菌中表达的比较。大肠杆菌与S49鼠淋巴瘤细胞膜中β 2受体的比较显示rGs α-S和rGs α-L对重组和天然受体的亲和力相似。rGs α-S. β-γ稳定掺入E.大肠杆菌膜，受体-G蛋白偶联也通过测定异丙肾上腺素介导的鸟嘌呤5 '-[γ-[35 S]硫代]三磷酸结合速率的加速来验证。这些结果表明：（1）受体-G蛋白偶联在E.大肠杆菌中使用重组成分和（ii）这种方法可以更普遍地用于评估受体和G-蛋白亚基的定义的分子种类之间的耦合偏好。
When expressed in Escherichia coli, the human beta 1- and beta 2-adrenergic receptors retain their ligand binding specificity. Their functional integrity was investigated by analyzing receptor-guanine nucleotide-binding regulatory (G) protein coupling by using two splice variants of the alpha subunit of the stimulatory G protein Gs synthesized in E. coli (rGs alpha-S and rGs alpha-L) and the beta gamma subunits of G protein purified from bovine brain. In competition binding experiments with (-)-[125I]iodocyanopindolol and (-)-isoproterenol, rGs alpha-S.beta gamma and rGs alpha-L.beta gamma reconstituted guanine nucleotide-sensitive high-affinity agonist binding with comparable affinities, whereas rGs alpha PT, a mutant of rGs alpha-L with an altered carboxyl terminus, and a recombinant subtype of the alpha subunit of the inhibitory G protein, rGi alpha-1, were approximately 20- and approximately 200-fold less potent, respectively. A comparison of the beta 1- and beta 2-adrenergic receptor expressed in E. coli with the beta 2-receptor in S49 murine lymphoma cyc- cell membranes revealed a similar affinity of rGs alpha-S and rGs alpha-L for the recombinant and native receptors. After stable incorporation of rGs alpha-S.beta gamma into E. coli membranes, receptor-G protein coupling was also verified by determining the isoproterenol-mediated acceleration of the rate for guanine 5'-[gamma-[35S]thio]triphosphate binding. These results show that (i) receptor-G protein coupling can be reconstituted in E. coli using recombinant components and that (ii) such an approach may be more generally used to evaluate coupling preferences between defined molecular species of receptors and G-protein subunits.