Conway S, Canning S J, Barrett P, Guardiola-Lemaitre B, Delagrange P, Morgan P J
Molecular Neuroendocrinology Unit, Rowett Research Institute, Aberdeen, Scotland, United Kingdom.
Biochem Biophys Res Commun. 1997 Oct 20;239(2):418-23. doi: 10.1006/bbrc.1997.7482.
Site-directed mutagenesis was used to study two residues, valine 208 and histidine 211, in transmembrane domain 5 of the ovine Mel1a beta melatonin receptor. A series of 4 mutants were constructed (V208A, V208L, H211F, H211L), and each engineered to contain a FLAG-epitope. Immunocytochemistry demonstrated that all the mutants were expressed in COS-7 cells at levels comparable to the FLAG-epitope tagged wild-type Mel1a beta receptor (approximately 120 fmol/mg protein). Ligand binding revealed however that all mutants had reduced affinities for 2-[125I]-iodomelatonin (Kd wild-type 139 pM, Kd mutants 320 to 989 pM). Competition studies, with a series of melatonin analogues, identified a probable interaction between histidine 211 and the 5-methoxy group of melatonin. The wild-type receptor and both valine 208 mutants displayed a dose-dependent melatonin mediated inhibition of cyclic AMP levels in HEK293 cells, with IC50 values in the same rank-order as their melatonin binding affinities. Both H211F and H211L, however, did not display any melatonin mediated effects and may suggest that histidine 211 is critical for melatonin mediated receptor activation.
采用定点诱变技术研究了绵羊褪黑素受体Mel1aβ跨膜结构域5中的两个残基，缬氨酸208和组氨酸211。构建了一系列4个突变体（V208A、V208L、H211F、H211L），并使其均含有一个FLAG表位。免疫细胞化学表明，所有突变体在COS-7细胞中的表达水平与带有FLAG表位标签的野生型Mel1aβ受体相当（约120 fmol/mg蛋白）。然而，配体结合实验表明，所有突变体对2-[125I] - 碘褪黑素的亲和力均降低（野生型Kd为139 pM，突变体Kd为320至989 pM）。通过一系列褪黑素类似物进行的竞争研究确定了组氨酸211与褪黑素的5 - 甲氧基之间可能存在相互作用。野生型受体以及两个缬氨酸208突变体在HEK293细胞中均表现出褪黑素介导的对环磷酸腺苷水平的剂量依赖性抑制作用，其IC50值与其褪黑素结合亲和力处于相同的排序。然而，H211F和H211L均未表现出任何褪黑素介导的效应，这可能表明组氨酸211对褪黑素介导的受体激活至关重要。
