Signalling complexes associated with adenylyl cyclase II are assembled during their biosynthesis

We have previously demonstrated that adenylyl cyclase II (ACII) interacts with beta(2)-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 beta(2)AR 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/G beta gamma protein interactions. However, DN Rab1 and Sar1 constructs (but not DN Rabs 2, 6, 8 or 11) prevent the inclusion of G alpha 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. (c) 2006 Elsevier Inc. All rights reserved.