Ahmed Syed M, Daulat Avais M, Angers Stéphane
Department of Pharmaceutical Sciences & Biochemistry, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada.
Methods Mol Biol. 2011;756:357-70. doi: 10.1007/978-1-61779-160-4_22.
Heterotrimeric G proteins are the main signal-transducing molecules activated by G protein-coupled receptors. Their GTP-dependent activation leads to the regulation of different effectors such as adenylyl cyclases, phospholipases, and RhoGEFs. To understand the full biological consequences of GPCR signalling and to further understand the cross-talk with other signalling pathways, the complement of proteins associating with heterotrimeric G proteins needs to be identified. Here we describe our mass spectrometry-based proteomic approaches for the study of Gβγ and Gα protein complexes. This approach is predicated on the establishment of mammalian cell lines constitutively or inducibly expressing affinity-tagged versions of Gβγ or wild-type and constitutively active Gα subunits, respectively.
异源三聚体G蛋白是由G蛋白偶联受体激活的主要信号转导分子。它们依赖GTP的激活导致对不同效应器的调节，如腺苷酸环化酶、磷脂酶和RhoGEF。为了了解GPCR信号传导的完整生物学后果，并进一步了解与其他信号通路的相互作用，需要鉴定与异源三聚体G蛋白相关的蛋白质组。在这里，我们描述了基于质谱的蛋白质组学方法，用于研究Gβγ和Gα蛋白复合物。这种方法基于分别建立组成型或诱导型表达Gβγ亲和标签版本或野生型和组成型活性Gα亚基的哺乳动物细胞系。
