Potenza M N, Graminski G F, Lerner M R
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Anal Biochem. 1992 Nov 1;206(2):315-22. doi: 10.1016/0003-2697(92)90372-e.
As an increasing number of medically important receptors that couple to stimulatory guanine nucleotide (Gs) proteins are isolated and cloned, there is an equally escalating need for methods to rapidly and reproducibly evaluate potential ligands for their properties as agonists or antagonists. Recently, a bioassay that can quickly and accurately determine the effects of numerous chemicals on a beta 1-like adrenergic receptor (AR) endogenous to melanophores derived from Xenopus laevis was developed. Here, the general utility of the melanophore-based pigment dispersion assay is demonstrated by employing it to evaluate the effects of drugs on a human beta 2 AR. Melanophores were both transiently and stably transfected with a plasmid encoding a beta 2 AR. Stimulation of recombinant cells expressing the beta 2 AR, but not wild-type cells, with beta 2-selective agonists induced pigment dispersion and concomitant elevations in intracellular cAMP. Using a microtiter plate reader, it was straightforward to construct reproducible dose-response curves and rapidly determine rank-order potency and EC50 and IC50 values for agonists and antagonists, respectively. The demonstration of functional expression of a human beta 2 AR in the melanophore-based bioassay suggests that the system may be used for the rapid pharmacological characterization of ligands upon any specific Gs-linked receptor for which a cDNA clone is available.
随着越来越多与刺激性鸟嘌呤核苷酸（Gs）蛋白偶联的具有医学重要性的受体被分离和克隆，对于快速且可重复地评估潜在配体作为激动剂或拮抗剂性质的方法的需求也在同样地不断增加。最近，一种生物测定法被开发出来，它能够快速且准确地确定多种化学物质对源自非洲爪蟾的黑素细胞内源性类β1肾上腺素能受体（AR）的影响。在此，通过用基于黑素细胞的色素分散测定法来评估药物对人β2肾上腺素能受体的影响，证明了该测定法的普遍实用性。用编码β2肾上腺素能受体的质粒对黑素细胞进行瞬时和稳定转染。用β2选择性激动剂刺激表达β2肾上腺素能受体的重组细胞，而非野生型细胞，可诱导色素分散并伴随细胞内cAMP升高。使用酶标仪，构建可重复的剂量反应曲线并分别快速确定激动剂和拮抗剂的效价顺序以及EC50和IC50值是很直接的。在基于黑素细胞的生物测定法中人类β2肾上腺素能受体功能表达的证明表明，该系统可用于对任何有cDNA克隆可用的特定Gs偶联受体的配体进行快速药理学表征。
