Reconstitution of beta-adrenergic receptors in lipid vesicles: affinity chromatography-purified receptors confer catecholamine responsiveness on a heterologous adenylate cyclase system.
Reconstitution of beta-adrenergic receptors in lipid vesicles: affinity chromatography-purified receptors confer catecholamine responsiveness on a heterologous adenylate cyclase system.
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脂质囊泡中β-肾上腺素能受体的重建：亲和层析纯化的受体赋予异源腺苷酸环化酶系统儿茶酚胺反应性。
DOI：
10.1073/pnas.80.16.4899
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发表时间：
1983
影响因子：
11.1
通讯作者：
Lefkowitz,RJ
中科院分区：
文献类型：
--
作者：
Cerione,RA;Strulovici,B;Benovic,JL;Strader,CD;Caron,MG;Lefkowitz,RJ
纯化的受体的结合功能可以用放射性配体进行评估，但受体与其生化效应物的相互作用还不能直接研究。为此，已经开发了用于直接证明纯化的β-肾上腺素能受体制剂的功能性的程序。通过亲和色谱法将来自青蛙红细胞或大鼠肺的毛地黄皂苷增溶的β-肾上腺素能受体纯化约100- 5，000倍，并在辛基葡糖苷存在下插入青蛙红细胞脂质和二肉豆蔻酰磷脂酰胆碱的混合物中。β-肾上腺素能受体结合的重建通常为25-50%，并且在辛基葡糖苷存在下也可以用大豆磷脂酰胆碱实现。然后将重建的β-肾上腺素能受体与非洲爪蟾红细胞融合，非洲爪蟾红细胞含有前列腺素E1敏感性腺苷酸环化酶[ATP焦磷酸裂解酶（环化），EC 4.6.1.1]，但几乎没有β-肾上腺素能受体和很少或没有儿茶酚胺敏感性腺苷酸环化酶。重组受体与非洲爪蟾红细胞的融合建立了β-激动剂异丙肾上腺素对杂合腺苷酸环化酶的实质性（2- 10倍）刺激。刺激的程度取决于加入的重建β-肾上腺素能受体的量，可被普萘洛尔阻断，并在重建前通过煮沸β-肾上腺素能受体消除。纯化的受体与异源腺苷酸环化酶的成功偶联为研究受体结构-功能关系开辟了道路。
The binding function of purified receptors can be assessed with radioligands, but the interaction of receptors with their biochemical effectors has not been amenable to direct study. Toward this end, procedures have been developed for directly demonstrating functionality of purified beta-adrenergic receptor preparations. Digitonin-solubilized beta-adrenergic receptors from frog erythrocytes or rat lung were purified approximately equal to 100- to 5,000-fold by affinity chromatography and inserted into a mixture of frog erythrocyte lipids and dimyristoyl phosphatidylcholine in the presence of octyl glucoside. Reconstitution of beta-adrenergic receptor binding was typically 25-50% and could also be effected with soybean phosphatidylcholine in the presence of octyl glucoside. The reconstituted beta-adrenergic receptors were then fused with Xenopus laevis erythrocytes, which contain prostaglandin E1-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] but few beta-adrenergic receptors and little or no catecholamine-sensitive adenylate cyclase. Fusion of reconstituted receptor with Xenopus laevis erythrocytes establishes a substantial (2- to 10-fold) stimulation of the hybrid adenylate cyclase by the beta-agonist isoproterenol. The extent of stimulation depends on the amount of reconstituted beta-adrenergic receptor added, is blocked by propranolol, and is eliminated by boiling the beta-adrenergic receptor prior to reconstitution. The successful coupling of a purified receptor to a heterologous adenylate cyclase opens the way to the study of receptor structure---function relationships.