Phosphorylation modulates the affinity of light-activated rhodopsin for G protein and arrestin
Phosphorylation modulates the affinity of light-activated rhodopsin for G protein and arrestin
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DOI：
10.1021/bi991857f
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发表时间：
2000-05-16
期刊：
影响因子：
2.9
通讯作者：
Liebman, PA
中科院分区：
文献类型：
--
作者：
Gibson, SK;Parkes, JH;Liebman, PA
抑制蛋白的降低的效应子活性和结合是广泛接受的GPCR磷酸化的结果。然而，受体多磷酸化对G蛋白活化和抑制蛋白结合参数的影响以前没有被定量研究。我们已经发现受体磷酸化改变G蛋白和抑制蛋白结合常数的光激活视紫红质的比例磷酸化化学计量。将含有不同平均受体磷酸化化学计量的杆盘膜与G蛋白或抑制蛋白结合，并用一系列短暂的闪光滴定。G（t）或抑制蛋白与活化视紫红质的结合通过将MII稳定为MII.G或MII. Arr来增强390 nm MII光吸收信号。使用滴定数据的非线性最小二乘（单纯形）反应模型分析，测定活性抑制蛋白或G（t）的浓度和各自与MII的结合常数。磷酸化的MII对G1的结合亲和力降低，而对arrestin的结合亲和力随着每个添加的磷酸盐而增加。在磷酸化水平小于或等于每个视紫红质两个磷酸盐时，G（t）与Mn结合更紧密;在更高的磷酸化水平下，arrestin结合是有利的。然而，发现在所有磷酸化水平下，抑制蛋白的结合比G（t）慢得多，这可能使得在抑制蛋白加帽视紫红质之前，磷酸化有时间逐渐减少受体-G蛋白相互作用，结合常数对离子强度的敏感性表明，在G（t）的减少和G（t）的减少中，结合以及抑制蛋白结合随视紫红质磷酸化增加而增加。
Reduced effector activity and binding of arrestin are widely accepted consequences of GPCR phosphorylation. However, the effect of receptor multiphosphorylation on G protein activation and arrestin binding parameters has not previously been quantitatively examined. We have found receptor phosphorylation to alter both G protein and arrestin binding constants for light-activated rhodopsin in proportion to phosphorylation stoichiometry. Rod disk membranes containing different average receptor phosphorylation stoichiometries were combined with G protein or arrestin, and titrated with a series of brief light flashes. Binding of G(t) or arrestin to activated rhodopsin augmented the 390 nm MII optical absorption signal by stabilizing MII as MII.G or MII.Arr. The concentration of active arrestin or G(t) and the binding constant of each to MII were determined using a nonlinear least-squares (Simplex) reaction model analysis of the titration data. The binding affinity of phosphorylated MII for G, decreased while that for arrestin increased with each added phosphate, G(t) binds more tightly to Mn at phosphorylation levels less than or equal to two phosphates per rhodopsin; at higher phosphorylation levels, arrestin binding is favored. However, arrestin was found to bind much more slowly than G(t) at all phosphorylation levels, perhaps allowing time for phosphorylation to gradually reduce receptor-G protein interaction before arrestin capping of rhodopsin, Sensitivity of the binding constants to ionic strength suggests that a strong membrane electrostatic component is involved in both the reduction of G(t) binding and the increase of arrestin binding with increasing rhodopsin phosphorylation.