Kokkola T, Watson M A, White J, Dowell S, Foord S M, Laitinen J T
Department of Physiology, University of Kuopio, Kuopio, Finland.
Biochem Biophys Res Commun. 1998 Aug 19;249(2):531-6. doi: 10.1006/bbrc.1998.9182.
A yeast functional colorimetric assay was employed to test the effects of site-directed point mutations on the function of the human Mel1a melatonin receptor. Seven mutants were created in transmembrane domains III, V, and VII of the receptor to test the rhodopsin-based model of melatonin recognition. Two mutants in transmembrane domains III and VI were created to investigate the mechanisms of G protein activation in the melatonin receptor. Mutations in transmembrane domain V either potentiated agonist efficiencies (H195A) or totally abolished all responses to tested compounds (V192T+H195A). Mutation N124A in the conserved NRY motif in the end of transmembrane domain III seriously impaired receptor activation. Several mutants were found to have decreased ability to activate functional responses, reflecting the importance of these residues for receptor function. These data also suggest that activation of the receptor involves interaction of the 5-methoxy group of melatonin with the conserved histidine H195 in transmembrane domain V.
采用酵母功能比色法检测定点突变对人Mel1a褪黑素受体功能的影响。在受体的跨膜结构域III、V和VII中创建了七个突变体，以测试基于视紫红质的褪黑素识别模型。在跨膜结构域III和VI中创建了两个突变体，以研究褪黑素受体中G蛋白激活的机制。跨膜结构域V中的突变要么增强了激动剂效率（H195A），要么完全消除了对测试化合物的所有反应（V192T + H195A）。跨膜结构域III末端保守的NRY基序中的突变N124A严重损害了受体激活。发现几个突变体激活功能反应的能力下降，反映了这些残基对受体功能的重要性。这些数据还表明，受体的激活涉及褪黑素的5-甲氧基与跨膜结构域V中保守的组氨酸H195的相互作用。
