Dupré Denis J, Baragli Alessandra, Rebois R Victor, Ethier Nathalie, Hébert Terence E
Department of Pharmacology and Therapeutics, McGill University, Montréal, Canada.
Cell Signal. 2007 Mar;19(3):481-9. doi: 10.1016/j.cellsig.2006.07.021. Epub 2006 Sep 18.
We have previously demonstrated that adenylyl cyclase II (ACII) interacts with beta2-adrenergic receptors and heterotrimeric G proteins as part of a pre-assembled signalling complex. In this study, we further show that AC interacts with these proteins before it is targetted to the cell surface. Using a combination of approaches including bioluminescence resonance energy transfer (BRET) in concert with subcellular fractionation, we show that ACII and beta2AR initially interact in the ER. Further, dominant-negative Rab1 and Sar1 GTPases which block anterograde trafficking out of the ER have no effect on either ACII/receptor or ACII/Gbetagamma protein interactions. However, DN Rab1 and Sar1 constructs (but not DN Rabs 2, 6, 8 or 11) prevent the inclusion of Galpha subunits in ACII signalling complexes suggesting it assembles into the complex at a slightly later stage. Thus, like Kir3.1 inwardly rectifying potassium channels, signalosomes containing ACII are formed during their biosynthesis and not in response to agonist at the cell surface.
我们之前已经证明，作为预组装信号复合物的一部分，腺苷酸环化酶II（ACII）与β2 - 肾上腺素能受体和异源三聚体G蛋白相互作用。在本研究中，我们进一步表明，AC在靶向细胞表面之前就与这些蛋白质相互作用。通过结合生物发光共振能量转移（BRET）与亚细胞分级分离等多种方法，我们表明ACII和β2AR最初在内质网中相互作用。此外，阻断从内质网的顺行转运的显性负性Rab1和Sar1 GTP酶对ACII/受体或ACII/Gβγ蛋白相互作用均无影响。然而，DN Rab1和Sar1构建体（但不是DN Rab 2、6、8或11）会阻止Gα亚基纳入ACII信号复合物，这表明它在稍晚阶段组装到复合物中。因此，与Kir3.1内向整流钾通道一样，含有ACII的信号体在其生物合成过程中形成，而不是在细胞表面对激动剂作出反应时形成。
