Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, United States.
Biochemistry. 2011 Feb 8;50(5):752-62. doi: 10.1021/bi101910n. Epub 2011 Jan 11.
RGS14 is a brain scaffolding protein that integrates G protein and MAP kinase signaling pathways. Like other RGS proteins, RGS14 is a GTPase activating protein (GAP) that terminates Gαi/o signaling. Unlike other RGS proteins, RGS14 also contains a G protein regulatory (also known as GoLoco) domain that binds Gαi1/3-GDP in cells and in vitro. Here we report that Ric-8A, a nonreceptor guanine nucleotide exchange factor (GEF), functionally interacts with the RGS14-Gαi1-GDP signaling complex to regulate its activation state. RGS14 and Ric-8A are recruited from the cytosol to the plasma membrane in the presence of coexpressed Gαi1 in cells, suggesting formation of a functional protein complex with Gαi1. Consistent with this idea, Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex in cells and in vitro using purified proteins. Purified Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex to form a stable Ric-8A-Gαi complex in the absence of GTP. In the presence of an activating nucleotide, Ric-8A interacts with the RGS14-Gαi1-GDP complex to stimulate both the steady-state GTPase activity of Gαi1 and binding of GTP to Gαi1. However, sufficiently high concentrations of RGS14 competitively reverse these stimulatory effects of Ric-8A on Gαi1 nucleotide binding and GTPase activity. This observation correlates with findings that show RGS14 and Ric-8A share an overlapping binding region within the last 11 amino acids of Gαi1. As further evidence that these proteins are functionally linked, native RGS14 and Ric-8A coexist within the same hippocampal neurons. These findings demonstrate that RGS14 is a newly appreciated integrator of unconventional Ric-8A and Gαi1 signaling.
RGS14 是一种脑支架蛋白，可整合 G 蛋白和 MAP 激酶信号通路。与其他 RGS 蛋白一样，RGS14 是一种 G 蛋白激活蛋白 (GAP)，可终止 Gαi/o 信号。与其他 RGS 蛋白不同，RGS14 还含有一个 G 蛋白调节 (也称为 GoLoco) 结构域，该结构域在细胞内和体外与 Gαi1/3-GDP 结合。在这里，我们报告 Ric-8A（一种非受体鸟嘌呤核苷酸交换因子 (GEF)）与 RGS14-Gαi1-GDP 信号复合物在功能上相互作用，以调节其激活状态。在细胞中共表达 Gαi1 的情况下，RGS14 和 Ric-8A 从细胞质招募到质膜，表明与 Gαi1 形成功能性蛋白质复合物。与这一观点一致，Ric-8A 使用纯化蛋白在细胞内和体外刺激 RGS14-Gαi1-GDP 复合物的解离。纯化的 Ric-8A 刺激 RGS14-Gαi1-GDP 复合物的解离，以在没有 GTP 的情况下形成稳定的 Ric-8A-Gαi 复合物。在存在激活核苷酸的情况下，Ric-8A 与 RGS14-Gαi1-GDP 复合物相互作用，刺激 Gαi1 的稳态 GTPase 活性和 GTP 与 Gαi1 的结合。然而，足够高浓度的 RGS14 竞争性地逆转了 Ric-8A 对 Gαi1 核苷酸结合和 GTPase 活性的这些刺激作用。这一观察结果与表明 RGS14 和 Ric-8A 在 Gαi1 的最后 11 个氨基酸内共享重叠结合区域的发现相关。作为这些蛋白质在功能上相互关联的进一步证据，天然 RGS14 和 Ric-8A 共存于同一海马神经元中。这些发现表明 RGS14 是一种新发现的非常规 Ric-8A 和 Gαi1 信号的整合者。
