Arrestin variants display differential binding characteristics for the phosphorylated N-formyl peptide receptor carboxyl terminus
Arrestin variants display differential binding characteristics for the phosphorylated N-formyl peptide receptor carboxyl terminus
复制标题
DOI：
10.1074/jbc.m111086200
复制
发表时间：
2002-03-15
影响因子：
4.8
通讯作者：
Prossnitz, ER
中科院分区：
文献类型：
--
作者：
Potter, RM;Key, TA;Prossnitz, ER
抑制蛋白与G蛋白偶联受体（gpcr）胞质结构域的磷酸化依赖性结合被认为是受体脱敏的关键步骤。在一些GPCR系统中，阻滞也被证明与受体内化、再敏化和信号级联的激活有关。本研究的目的是研究抑制蛋白家族成员与甲酰基肽受体（FPR）的结合相互作用，甲酰基肽受体是GPCR受体家族的一员。合成了代表FPR未磷酸化和磷酸化羧基末端的肽，并通过生物素/链亲和素相互作用结合到聚苯乙烯珠上。利用荧光素偶联的抑制蛋白，用流式细胞术分析了抑制蛋白与头结合的FPR羧基端之间的结合相互作用。Arrestin-2和arrestin-3以磷酸化依赖的方式与fpr羧基末端肽结合，其K-d值在微摩尔范围内。没有观察到高选择性结合视紫红质的视觉抑制素的结合。拦阻蛋白的截短突变体形式的拦阻蛋白2-(1-382)和拦阻蛋白3-(1-393)显示与完整受体的磷酸化无关的结合，也被观察到以磷酸化依赖的方式结合头结合的FPR末端，但比全长拦阻蛋白具有更大的亲和力，在5-50 nm范围内产生Kd值。另外两种抑制蛋白突变体，全长但与完整的gpcr显示磷酸化无关的结合，它们与FPR羧基末端的结合亲和力被评估。单点突变体arrestin-2 R169E表现出与全长arrestin相似的亲和力，而三点突变体arrestin-2 I386A/V387A/F388A表现出与截断形式的arrestin更相似的亲和力。结果表明，抑制蛋白的羧基末端是调控抑制蛋白与gpcr磷酸化结构域结合亲和力的关键决定因素。
The phosphorylation-dependent binding of arrestins to cytoplasmic domains of G protein-coupled receptors (GPCRs) is thought to be a crucial step in receptor desensitization. In some GPCR systems, arrestins have also been demonstrated to be involved in receptor internalization, resensitization, and the activation of signaling cascades. The objective of the current study was to examine binding interactions of members of the arrestin family with the formyl peptide receptor (FPR), a member of the GPCR family of receptors. Peptides representing the unphosphorylated and phosphorylated carboxyl terminus of the FPR were synthesized and bound to polystyrene beads via a biotin/streptavidin interaction. Using fluorescein-conjugated arrestins, binding interactions between arrestins and the bead-bound FPR carboxyl terminus were analyzed by flow cytometry. Arrestin-2 and arrestin-3 bound to the FPRcarboxyl-terminal peptide in aphosphorylation-dependent manner, with K-d values in the micromolar range. Binding of visual arrestin, which binds rhodopsin with high selectivity, was not observed. Arrestin-2-(1-382) and arrestin-3-(1-393), truncated mutant forms of arrestin that display phosphorylation-independent binding to intact receptors, were also observed to bind the bead-bound FPR terminus in a phosphorylation-dependent manner, but with much greater affinity than the full-length arrestins, yielding Kd values in the 5-50 nm range. Two additional arrestin mutants, which are full-length but display phosphorylation-independent binding to intact GPCRs, were evaluated for their binding affinity to the FPR carboxyl terminus. Whereas the single point mutant, arrestin-2 R169E, displayed an affinity similar to that of the full-length arrestins, the triple point mutant, arrestin-2 I386A/V387A/F388A, displayed an affinity more similar to that of the truncated forms of arrestin. The results suggest that the carboxyl terminus of arrestin is a critical determinant in regulating the binding affinity of arrestin for the phosphorylated domains of GPCRs.