Hamaguchi N, True T A, Goetz A S, Stouffer M J, Lybrand T P, Jeffs P W
Department of Structural Chemistry, Glaxo Wellcome Research Institute, Research Triangle Park, North Carolina 27709, USA.
Biochemistry. 1998 Apr 21;37(16):5730-7. doi: 10.1021/bi972733a.
Mutational studies in conjunction with ligand binding assays were used to examine the basis of alpha1-adrenergic receptor subtype selectivity for a series of 4-piperidyloxazole antagonists. A set of chimeric alpha 1A receptors were created by systematically substituting individual transmembrane domains from alpha 1D adrenergic receptors. The oxazole antagonists exhibited significant reductions in affinity against the receptor construct alpha 1A/D(TM2), and moderate reductions in affinity versus constructs alpha 1A/D(TM5), alpha 1A/B(TM5), and alpha 1A/D(TM6). Antagonist affinities for these chimeras exceeded those found for wild type alpha 1D and alpha 1B. Site-directed mutagenesis methods were then used to explore the role that individual residues in TM2 and TM5 play in ligand binding affinity and selectivity. These studies revealed that mutations at position 86 in the second transmembrane domain and position 185 in the fifth transmembrane domain of the alpha 1A receptor have a major impact on receptor subtype selectivity.
结合配体结合试验进行的突变研究，用于检验一系列4-哌啶基恶唑拮抗剂对α1-肾上腺素能受体亚型选择性的基础。通过系统地替换α1D肾上腺素能受体的单个跨膜结构域，构建了一组嵌合α1A受体。恶唑拮抗剂对受体构建体α1A/D(TM2)的亲和力显著降低，对构建体α1A/D(TM5)、α1A/B(TM5)和α1A/D(TM6)的亲和力适度降低。这些嵌合体的拮抗剂亲和力超过了野生型α1D和α1B的亲和力。然后使用定点突变方法来探究跨膜结构域2（TM2）和跨膜结构域5（TM5）中单个残基在配体结合亲和力和选择性中所起的作用。这些研究表明，α1A受体第二个跨膜结构域中的第86位和第五个跨膜结构域中的第185位的突变对受体亚型选择性有重大影响。
