The pharmacological effects of the thermostabilising (m23) mutations and intra and extracellular (β36) deletions essential for crystallisation of the turkey β-adrenoceptor
The pharmacological effects of the thermostabilising (m23) mutations and intra and extracellular (β36) deletions essential for crystallisation of the turkey β-adrenoceptor
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DOI：
10.1007/s00210-011-0648-4
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发表时间：
2011-07-01
影响因子：
3.6
通讯作者：
Tate, Christopher G.
中科院分区：
文献类型：
--
作者：
Baker, Jillian G.;Proudman, Richard G. W.;Tate, Christopher G.
火鸡β-肾上腺素受体的X射线晶体结构最近已被确定。然而，突变被引入到天然受体中，这对结构测定是必不可少的。这些可能导致受体药理学的改变。因此，了解这些突变对受体药理学特性的影响至关重要。本研究检查了m23突变和β 36缺失两者的药理学作用，两者单独使用，然后在用于火鸡β-肾上腺素受体的结晶和结构测定的β 36-m23突变体中组合使用。用每种受体突变体制备稳定的CHO-K1细胞系，并通过H-3-CGP 12177全细胞配体结合、H-3-cAMP积累和CRE-SPAP基因转录测定评估配体的亲和力和功效。m23突变使对激动剂、部分激动剂和中性拮抗剂的亲和力降低约10倍，而单独的β 36缺失对配体亲和力没有影响。这两组变化似乎都减少了受体的激动剂激活。m23和β 36受体都保留了两种活性激动剂诱导的受体构象，类似于原始t β trunc受体的构象。组合的β 36-m23受体结合配体与m23受体具有相似的亲和力;然而，仅用包括儿茶酚胺的少数激动剂观察到激动剂活化。尽管突变的组合严重降低了激活能力，但最终结晶的受体（β 36-m23）仍然是能够结合激动剂和拮抗剂配体并激活细胞内激动剂反应的全功能受体。
The X-ray crystal structure of the turkey beta-adrenoceptor has recently been determined. However, mutations were introduced into the native receptor that was essential for structure determination. These may cause alterations to the receptor pharmacology. It is therefore essential to understand the effects of these mutations on the pharmacological characteristics of the receptor. This study examined the pharmacological effects of both the m23 mutations and the beta 36 deletions, both alone and then in combination in the beta 36-m23 mutant used in the crystallisation and structure determination of the turkey beta-adrenoceptor. Stable CHO-K1 cell lines were made of each of the receptor mutants and the affinity and efficacy of ligands assessed by H-3-CGP 12177 whole cell ligand binding, H-3-cAMP accumulation, and CRE-SPAP gene transcription assays. The m23 mutations reduced affinity for agonists, partial agonists and neutral antagonists by about tenfold whilst the beta 36 deletions alone had no effect on ligand affinity. Both sets of changes appeared to reduce the agonist activation of the receptor. Both the m23 and the beta 36 receptors retained two active agonist-induced receptor conformations similar to that of the original t beta trunc receptor. The combined beta 36-m23 receptor bound ligands with similar affinity to the m23 receptor; however, agonist activation was only observed with a few agonists including the catecholamines. Although the combination of mutations severely reduced the activation ability, the final crystallised receptor (beta 36-m23) was still a fully functional receptor capable of binding agonist and antagonist ligands and activating intracellular agonist responses.