Campbell Adrian P, MacDougall Iain J A, Griffith Renate, Finch Angela M
Department of Pharmacology, School of Medical Sciences, University of New South Wales, Kensington, NSW 2052, Australia.
School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia.
Eur J Pharmacol. 2014 Jun 15;733:90-6. doi: 10.1016/j.ejphar.2014.03.034. Epub 2014 Mar 29.
The extracellular loops of the adrenoceptors present a potential therapeutic target in the design of highly selective adrenergic drugs. These regions are less conserved than the orthosteric binding site but have to date not been implicated in activation of adrenoceptors. A previously generated homology model identified an extracellular residue, D191, as a potential regulator of agonist binding. We have generated mutants of the α1B adrenoceptor replacing the charged aspartate, D191, as well as a potential interaction partner, K331, with uncharged alanines to observe effects on ligand binding and receptor activation. Significant 4-6 fold reductions in affinity for the endogenous agonists, epinephrine and norepinephrine were observed for receptors with the D191A mutation in the second extracellular loop. While changes in EC50 were observed, operational analysis yielded no apparent change in receptor activation. Based on these findings, we suggest that D191, in the second extracellular loop of the α1B adrenoceptor, acts as a 'point of first contact' for the receptor's endogenous agonists. Implication of the non-conserved extracellular regions of the receptor in agonist binding makes it a potential target for the design of highly selective drugs.
肾上腺素能受体的细胞外环是设计高选择性肾上腺素能药物的潜在治疗靶点。这些区域的保守性低于正构结合位点，但迄今为止尚未发现其与肾上腺素能受体的激活有关。先前生成的同源模型确定细胞外残基D191是激动剂结合的潜在调节剂。我们构建了α1B肾上腺素能受体的突变体，将带电荷的天冬氨酸D191以及一个潜在的相互作用伙伴K331替换为不带电荷的丙氨酸，以观察对配体结合和受体激活的影响。在第二个细胞外环中具有D191A突变的受体，对内源性激动剂肾上腺素和去甲肾上腺素的亲和力显著降低了4至6倍。虽然观察到了EC50的变化，但操作分析显示受体激活没有明显变化。基于这些发现，我们认为α1B肾上腺素能受体第二个细胞外环中的D191作为该受体内源性激动剂的“首次接触点”。受体非保守细胞外区域参与激动剂结合表明它是设计高选择性药物的潜在靶点。
