Signalling complexes associated with adenylyl cyclase II are assembled during their biosynthesis.
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.
Dupré
Denis J
DJ
Department of Pharmacology and Therapeutics, McGill University, Montréal, Canada.
Baragli
Alessandra
A
Rebois
R Victor
RV
Ethier
Nathalie
N
Hébert
Terence E
TE
D000262:Adenylyl Cyclases / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004735:Energy Transfer; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000096:biosynthesis*; D044387:GTP-Binding Protein beta Subunits / Q000096:biosynthesis*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D049449:Luminescence; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions
D044387:GTP-Binding Protein beta Subunits; D018343:Receptors, Adrenergic, beta-2; D049452:Green Fluorescent Proteins; D008156:Luciferases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D000262:Adenylyl Cyclases