Mitchell D C, Niu S L, Litman B J
Section of Fluorescence Studies, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland 20852, USA.
J Biol Chem. 2001 Nov 16;276(46):42801-6. doi: 10.1074/jbc.M105772200. Epub 2001 Sep 5.
The role of membrane composition in modulating the rate of G protein-receptor complex formation was examined using rhodopsin and transducin (G(t)) as a model system. Metarhodopsin II (MII) and MII-G(t) complex formation rates were measured, in the absence of GTP, via flash photolysis for rhodopsin reconstituted in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC) bilayers, with and without 30 mol% cholesterol. Variation in bilayer lipid composition altered the lifetime of MII-G(t) formation to a greater extent than the lifetime of MII. MII-G(t) formation was fastest in 18:0,22:6PC and slowest in 18:0,18:1PC/30 mol% cholesterol. At 37 degrees C and a G(t) to photolyzed rhodopsin ratio of 1:1 in 18:0,22:6PC bilayers, MII-G(t) formed with a lifetime of 0.6 +/- 0.06 ms, which was not significantly different from the lifetime for MII formation. Incorporation of 30 mol% cholesterol slowed the rate of MII-G(t) complex formation by about 400% in 18:0,18:1PC, but by less than 25% in 18:0,22:6PC bilayers. In 18:0,22:6PC, with or without cholesterol, MII-G(t) formed rapidly after MII formed. In contrast, cholesterol in 18:0,18:1PC induced a considerable lag time in MII-G(t) formation after MII formed. These results demonstrate that membrane composition is a critical factor in determining the temporal response of a G protein-coupled signaling system.
以视紫红质和转导蛋白（G(t)）作为模型系统，研究了膜组成在调节G蛋白 - 受体复合物形成速率中的作用。在不存在GTP的情况下，通过闪光光解测量了在1 - 硬脂酰 - 2 - 油酰 - sn - 甘油 - 3 - 磷酸胆碱（18:0,18:1PC）和1 - 硬脂酰 - 2 - 二十二碳六烯酰 - sn - 甘油 - 3 - 磷酸胆碱（18:0,22:6PC）双层膜中重组的视紫红质的变视紫红质II（MII）和MII - G(t)复合物的形成速率，其中有或没有30摩尔％的胆固醇。双层脂质组成的变化对MII - G(t)形成寿命的影响比对MII寿命的影响更大。MII - G(t)在18:0,22:6PC中形成最快，在18:0,18:1PC / 30摩尔％胆固醇中最慢。在37℃且G(t)与光解视紫红质的比例为1:1的18:0,22:6PC双层膜中，MII - G(t)形成的寿命为0.6±0.06毫秒，这与MII形成的寿命没有显著差异。在18:0,18:1PC中加入30摩尔％胆固醇使MII - G(t)复合物的形成速率减慢约400％，但在18:0,22:6PC双层膜中减慢不到25％。在18:0,22:6PC中，无论有无胆固醇，MII - G(t)在MII形成后迅速形成。相比之下，18:0,18:1PC中的胆固醇在MII形成后诱导MII - G(t)形成相当长的延迟时间。这些结果表明，膜组成是决定G蛋白偶联信号系统时间响应的关键因素。
