Inhibition of WNT signaling by G protein-coupled receptor (GPCR) kinase 2 (GRK2).
Inhibition of WNT signaling by G protein-coupled receptor (GPCR) kinase 2 (GRK2).
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DOI：
10.1210/me.2009-0084
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发表时间：
2009-09
影响因子：
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通讯作者：
Liming Wang;D. Gesty‐Palmer;T. Fields;R. Spurney
中科院分区：
文献类型：
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作者：
Liming Wang;D. Gesty‐Palmer;T. Fields;R. Spurney
Wnt 信号通路的激活导致转录调节因子 β-连环蛋白从由轴蛋白和腺瘤性结肠息肉病 (APC) 蛋白（典型信号通路）组成的破坏复合物中释放并稳定。 APC 和轴蛋白中 G 蛋白信号传导 (RGS) 结构域调节因子之间的蛋白质-蛋白质相互作用促进了该复合物的组装。由于 G 蛋白偶联受体激酶 2 (GRK2) 具有与轴蛋白中的 RGS 结构域密切相关的 RGS 结构域，因此我们确定了 GRK2 是否调节经典信号传导。我们发现 GRK2 抑制 Wnt1 诱导的报告构建体激活，并减少 Wnt3a 依赖性稳定和 β-连环蛋白的核转位。这种负调节效应需要 GRK2 酶活性，并且使用小干扰 RNA 消耗内源性 GRK2 会增强经典信号传导。 GRK2 依赖性经典信号传导抑制与成骨细胞 (OB) 生物学相关，因为 GRK2 的过度表达会减弱颅骨 OB 中的 Wnt/β-连环蛋白信号传导。免疫共沉淀研究发现：1）GRK2结合APC； 2）GRK2酶活性促进GRK2-APC相互作用； 3) GRK2 中 RGS 结构域的缺失阻止了 GRK2-APC 相互作用和 GRK2 依赖性的经典信号传导抑制。这些数据表明：1）GRK2 负向调节 Wnt 信号传导； 2) GRK2依赖性的经典信号传导抑制需要GRK2中的RGS结构域和APC之间的蛋白质-蛋白质相互作用； 3) 酶活性促进 GRK2-APC 相互作用，并且是对经典信号传导产生负调节作用所必需的。我们推测抑制成骨 OB 中的 GRK2 活性可能是增加骨量的有效治疗策略。
Activation of Wnt signaling pathways causes release and stabilization of the transcription regulator beta-catenin from a destruction complex composed of axin and the adenomatous polyposis coli (APC) protein (canonical signaling pathway). Assembly of this complex is facilitated by a protein-protein interaction between APC and a regulator of G protein signaling (RGS) domain in axin. Because G protein-coupled receptor kinase 2 (GRK2) has a RGS domain that is closely related to the RGS domain in axin, we determined whether GRK2 regulated canonical signaling. We found that GRK2 inhibited Wnt1-induced activation of a reporter construct as well as reduced Wnt3a-dependent stabilization and nuclear translocation of beta-catenin. GRK2 enzymatic activity was required for this negative regulatory effect, and depletion of endogenous GRK2 using small interfering RNA enhanced canonical signaling. GRK2-dependent inhibition of canonical signaling is relevant to osteoblast (OB) biology because overexpression of GRK2 attenuated Wnt/beta-catenin signaling in calvarial OBs. Coimmunoprecipitation studies found that: 1) GRK2 bound APC; 2) The GRK2-APC interaction was promoted by GRK2 enzymatic activity; and 3) Deletion of the RGS domain in GRK2 prevented both the GRK2-APC interaction and GRK2-dependent inhibition of canonical signaling. These data suggest that: 1) GRK2 negatively regulates Wnt signaling; 2) GRK2-dependent inhibition of canonical signaling requires a protein-protein interaction between the RGS domain in GRK2 and APC; and 3) Enzymatic activity promotes the GRK2-APC interaction and is required for the negative regulatory effect on canonical signaling. We speculate that inhibiting GRK2 activity in bone-forming OBs might be a useful therapeutic strategy for increasing bone mass.