Knudsen S M, Tams J W, Wulff B S, Fahrenkrug J
Department of Clinical Biochemistry, Bispebjerg Hospital University of Copenhagen, Copenhagen NV, Denmark.
FEBS Lett. 1997 Jul 21;412(1):141-3. doi: 10.1016/s0014-5793(97)00714-x.
The importance of two highly conserved cysteines in the human vasoactive intestinal peptide receptor I (hVIPR 1) was examined. By site-directed mutagenesis each Cys residue was converted into Ala or Ser. The mutant and wild-type genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215-Ala/Ser and Cys285-Ala/Ser showed at least a 10-fold decrease in binding affinity and receptor potency when compared to the wild type. In contradiction to the wild-type receptor, both mutations were insensitive to dithiothreitol (DTT). The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilising the receptor in the correct conformation for ligand binding and activation.
研究了人血管活性肠肽受体I（hVIPR 1）中两个高度保守的半胱氨酸的重要性。通过定点诱变，将每个半胱氨酸残基转化为丙氨酸或丝氨酸。将突变型和野生型基因转染到HEK293细胞中，并测试其结合血管活性肠肽（VIP）和激活环磷酸腺苷（cAMP）生成的能力。与野生型相比，半胱氨酸215突变为丙氨酸/丝氨酸和半胱氨酸285突变为丙氨酸/丝氨酸的突变体在结合亲和力和受体效能上至少降低了10倍。与野生型受体不同，这两种突变对二硫苏糖醇（DTT）均不敏感。结果表明半胱氨酸215和半胱氨酸285之间存在二硫键，这对于将受体稳定在正确的构象以进行配体结合和激活非常重要。
