Cell Signalling, School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom
Cell Signalling, School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom.
Mol Pharmacol. 2015 Jan;87(1):103-20. doi: 10.1124/mol.114.095364. Epub 2014 Oct 16.
Salmeterol is a long-acting β2-agonist, widely used as an inhaled treatment of asthma and chronic obstructive pulmonary disease. It has very high β2-affinity (log KD -8.95) and is very selective for the β2-adrenoceptor (1000-fold selectivity over the β1-adrenoceptor). This study used a mutagenesis approach to determine the exact amino acids in the human β2-adrenoceptor responsible for this very high selectivity. Wild-type β2- and β1-adrenoceptors, chimeric β2/β1-adrenoceptors, and receptors with single-point mutations were transfected into Chinese hamster ovary-K1 cells, and affinity and function were studied using [(3)H]CGP 12177 [(-)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one] whole-cell binding and [(3)H]cAMP accumulation. Extracellular loop 3 (and specifically amino acid K305) had the largest single effect by reducing salmeterol's affinity for the β2-adrenoceptor by 31-fold. H296 in transmembrane 6 also had a major effect (18-fold reduction in salmeterol affinity). Combining these, in the double mutant β2-H296K-K305D, reduced salmeterol's affinity by 275-fold, to within 4-fold of that of the β1-adrenoceptor, without affecting the affinity or selectivity of other β2-agonists (salbutamol, formoterol, fenoterol, clenbuterol, or adrenaline). Another important amino acid was Y308 in transmembrane 7, although this also affected the affinity and selectivity of other agonists. F194 in extracellular loop 2 and R304 in extracellular loop 3 also had minor effects. None of these mutations (including the double mutant β2-H296K-K305D) affected the efficacy or duration of action of salmeterol. This suggests that the high affinity and selectivity of salmeterol are due to specific amino acids within the receptor itself, but that the duration of action is at least in part due to other factors, for example lipophilicity.
沙美特罗是一种长效β2-激动剂，广泛用于治疗哮喘和慢性阻塞性肺疾病的吸入治疗。它对β2-受体具有非常高的亲和力（log KD-8.95），并且对β2-肾上腺素能受体具有非常高的选择性（比β1-肾上腺素能受体高 1000 倍）。本研究使用诱变方法确定负责这种高选择性的人β2-肾上腺素能受体的确切氨基酸。野生型β2-和β1-肾上腺素能受体、β2/β1-嵌合肾上腺素能受体和单点突变受体被转染到中国仓鼠卵巢-K1 细胞中，并使用[（3）H]CGP 12177[（-）-4-（3-叔丁基氨基-2-羟基丙氧基）-苯并咪唑-2-酮]全细胞结合和[（3）H]cAMP 积累研究亲和力和功能。细胞外环 3（特别是氨基酸 K305）的影响最大，使沙美特罗对β2-肾上腺素能受体的亲和力降低了 31 倍。跨膜 6 中的 H296 也有很大的影响（沙美特罗亲和力降低 18 倍）。将这两个突变组合在一起，在双突变体β2-H296K-K305D 中，使沙美特罗的亲和力降低了 275 倍，与β1-肾上腺素能受体的亲和力相差 4 倍，而不影响其他β2-激动剂（沙丁胺醇、福莫特罗、非诺特罗、克伦特罗或肾上腺素）的亲和力或选择性。跨膜 7 中的另一个重要氨基酸是 Y308，尽管这也影响了其他激动剂的亲和力和选择性。细胞外环 2 中的 F194 和细胞外环 3 中的 R304 也有较小的影响。这些突变（包括双突变体β2-H296K-K305D）都不影响沙美特罗的疗效或作用持续时间。这表明沙美特罗的高亲和力和选择性是由于受体本身的特定氨基酸引起的，但作用持续时间至少部分是由于其他因素引起的，例如亲脂性。
