Fong C W, Milligan G
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Biochem J. 1999 Sep 1;342 ( Pt 2)(Pt 2):457-63.
Direct measures of G-protein activation based on guanine nucleotide exchange and hydrolysis are frequently impossible to monitor for receptors which interact predominantly with G(s)alpha. An isolated FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-epitope-tagged human IP prostanoid receptor and fusion proteins generated between this form of the receptor and the alpha subunits of its cognate G-protein G(s), G(i1), a G-protein which it fails to activate in co-expression studies, and a chimaeric G(i1)-G(s)6 (a form of G(i1) in which the C-terminal six amino acids were replaced with the equivalent sequence of G(s)) were stably expressed in HEK293 cells. These were detected by [(3)H]ligand-binding studies and by immunoblotting with both an anti-FLAG antibody and with appropriate antisera to the G-proteins. Each construct displayed similar affinity to bind the agonist iloprost. Iloprost stimulated adenylate cyclase activity in clones expressing both IP prostanoid receptor and the IP prostanoid receptor-G(s)alpha fusion protein, and both constructs were shown to interact with and activate endogenously expressed G(s)alpha. Addition of iloprost to membranes of cells expressing the isolated receptor resulted in a small stimulation of high-affinity GTPase activity. Iloprost produced no stimulation of GTPase activity which could be attributed to the IP prostanoid receptor-G(i1)alpha fusion. However, the fusion proteins containing either G(s)alpha or G(i1)-G(s)6alpha produced substantially greater stimulation of GTPase activity than the isolated IP prostanoid receptor. Treatment of cells expressing the IP prostanoid receptor-G(i1)-G(s)6alpha fusion protein with a combination of cholera and pertussis toxins allowed direct measurement of agonist activation of the receptor-linked G-protein. Normalization of such results for levels of expression of the IP prostanoid receptor constructs demonstrated a 5-fold higher stimulation of GTPase activity when using the G(s)alpha-containing fusion protein and a 9-fold improvement when using the fusion protein containing G(i1)-G(s)6alpha to detect G-protein activation compared with expression of the isolated receptor.
对于主要与G(s)α相互作用的受体，基于鸟嘌呤核苷酸交换和水解的G蛋白激活的直接测量通常是无法监测的。一种分离的带有FLAG（天冬氨酸-酪氨酸-赖氨酸-天冬氨酸-天冬氨酸-天冬氨酸-天冬氨酸-赖氨酸）表位标签的人IP前列腺素受体，以及这种形式的受体与其同源G蛋白G(s)、G(i1)（一种在共表达研究中它无法激活的G蛋白）的α亚基，和一种嵌合的G(i1)-G(s)6（一种G(i1)形式，其中C末端的六个氨基酸被G(s)的等效序列取代）之间产生的融合蛋白，在HEK293细胞中稳定表达。通过[³H]配体结合研究以及用抗FLAG抗体和针对G蛋白的适当抗血清进行免疫印迹来检测这些。每个构建体对激动剂伊洛前列素的结合显示出相似的亲和力。伊洛前列素刺激表达IP前列腺素受体和IP前列腺素受体-G(s)α融合蛋白的克隆中的腺苷酸环化酶活性，并且两种构建体都显示与内源性表达的G(s)α相互作用并激活它。将伊洛前列素添加到表达分离受体的细胞膜中导致高亲和力GTP酶活性有小的刺激。伊洛前列素对可归因于IP前列腺素受体-G(i1)α融合的GTP酶活性没有刺激。然而，含有G(s)α或G(i1)-G(s)6α的融合蛋白对GTP酶活性的刺激比分离的IP前列腺素受体大得多。用霍乱毒素和百日咳毒素组合处理表达IP前列腺素受体-G(i1)-G(s)6α融合蛋白的细胞，可以直接测量受体连接的G蛋白的激动剂激活。将这些结果针对IP前列腺素受体构建体的表达水平进行归一化，结果表明，与分离受体的表达相比，使用含G(s)α的融合蛋白检测G蛋白激活时，GTP酶活性的刺激高5倍，使用含G(i1)-G(s)6α的融合蛋白时提高9倍。
