Epitope-tagged Gq alpha subunits: expression of GTPase-deficient alpha subunits persistently stimulates phosphatidylinositol-specific phospholipase C but not mitogen-activated protein kinase activity regulated by the M1 muscarinic acetylcholine receptor.
Epitope-tagged Gq alpha subunits: expression of GTPase-deficient alpha subunits persistently stimulates phosphatidylinositol-specific phospholipase C but not mitogen-activated protein kinase activity regulated by the M1 muscarinic acetylcholine receptor.
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表位标记的 Gq α 亚基：GTPase 缺陷的 α 亚基的表达持续刺激磷脂酰肌醇特异性磷脂酶 C，但不刺激由 M1 毒蕈碱乙酰胆碱受体调节的丝裂原激活蛋白激酶活性。
DOI：
10.1073/pnas.90.9.4077
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发表时间：
1993
影响因子：
11.1
通讯作者：
Johnson,GL
中科院分区：
文献类型：
--
作者：
Qian,NX;Winitz,S;Johnson,GL
Gq是异源三聚体鸟嘌呤核苷酸结合蛋白，可激活磷脂酰肌醇特异性磷脂酶C（PI-PLC）的β亚型。通过添加9-aa序列YPYDVPDYA对Gq α-亚基多肽（α qa）进行N-末端修饰。将9-aa表位标签放置在N末端允许表达功能性α q多肽并选择性鉴定质粒表达的野生型和突变型G蛋白α亚基。谷氨酰胺-209突变为亮氨酸的N-末端表位标记的α q（N（epi）α qQ 209 L）抑制GTdR活性和持续激活PI-PLC，导致高稳态水平的肌醇磷酸。由N（epi）alpha qQ 209 L表达引起的肌醇磷酸水平升高与卡巴胆碱激活M1毒蕈碱乙酰胆碱受体获得的水平相似。刺激PI-PLC活性的Gq偶联M1受体和通过蛋白激酶C作用的佛波酯激活COS细胞中的细胞质有丝分裂原激活蛋白激酶。然而，由GTP酶缺陷型α q表达引起的PI-PLC酶活性的组成性激活不能持续激活该激酶。结果表明，持续的PI-PLC激活是不足以维持刺激的细胞质丝氨酸/苏氨酸蛋白激酶调节Gq偶联受体信号转导途径。
Gq is the heterotrimeric guanine nucleotide-binding protein that activates the beta isoforms of phosphatidyl-inositol-specific phospholipase C (PI-PLC). The Gq alpha-subunit polypeptide (alpha qa) was N-terminally modified by addition of a 9-aa sequence, YPYDVPDYA. Placement of the 9-aa epitope tag at the N terminus allowed expression of functional alpha q polypeptides and selective identification of plasmid-expressed wild-type and mutant G-protein alpha subunits. Mutation of glutamine-209 to leucine in the N-terminally epitope-tagged alpha q (N(epi) alpha qQ209L) inhibited GTPase activity and persistently activated PI-PLC, resulting in high steady-state levels of inositol phosphates. The elevated levels of inositol phosphates resulting from N(epi) alpha qQ209L expression were similar to those obtained with carbachol activation of the M1 muscarinic acetylcholine receptor. The Gq-coupled M1 receptor, which stimulates PI-PLC activity, and phorbol esters, acting via protein kinase C, activate the cytoplasmic mitogen-activated protein kinase in COS cells. However, the constitutive activation of PI-PLC enzymatic activity resulting from expression of GTPase-deficient alpha q was unable to persistently activate this kinase. The results indicate that persistent PI-PLC activation is insufficient to sustain the stimulation of a cytoplasmic serine/threonine protein kinase regulated by Gq-coupled receptor signal-transduction pathways.