RGS2 interacts with Gs and adenylyl cyclase in living cells.
Regulator of G Protein Signalling (RGS) proteins impede heterotrimeric G protein signalling. RGS2 decreases cAMP production and appears to interact with both adenylyl cyclase (AC) and its stimulatory G protein Gs. We showed previously that Green Fluorescent Protein-tagged RGS2 (GFP-RGS2) localizes to the nucleus in HEK 293 cells and is recruited to the plasma membrane when co-expressed with Gsalpha, or the Gs-coupled beta2-adrenergic receptor (beta2AR). Here, using confocal microscopy we show that co-expression of various AC isoforms (ACI, ACII, ACV, ACVI) also leads to GFP-RGS2 recruitment to the plasma membrane. Bioluminescence Resonance Energy Transfer (BRET) was also used to examine physical interactions between RGS2 and components of the Gs-signalling pathway. A BRET signal was detected between fusion constructs of RGS2-Renilla luciferase (energy donor) and Gsalpha-GFP (energy acceptor) co-expressed in HEK 293 cells. BRET was also observed between GFP-RGS2 and ACII or ACVI fused to Renilla luciferase. Additionally, RGS2 was found to interact with the beta2AR. Purified RGS2 selectively bound to the third intracellular loop of the beta2AR in GST pulldown assays, and a BRET signal was observed between GFP-RGS2 and beta2AR fused to Renilla luciferase when these two proteins were co-expressed together with either ACIV or ACVI. This interaction was below the limit of detection in the absence of co-expressed AC, suggesting that the effector enzyme stabilized or promoted binding between the receptor and the RGS protein inside the cell. Taken together, these results suggest the possibility that RGS2 might bind to a receptor-G protein-effector signalling complex to regulate Gs-dependent cAMP production.
Roy
Anju Anne
AA
Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada, N6A 5C1.
Baragli
Alessandra
A
Bernstein
Leah S
LS
Hepler
John R
JR
Hébert
Terence E
TE
Chidiac
Peter
P
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004735:Energy Transfer; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D049410:Luciferases, Renilla / Q000378:metabolism; D018613:Microscopy, Confocal; D011485:Protein Binding; D020710:RGS Proteins / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction
D007527:Isoenzymes; D020710:RGS Proteins; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; C105345:Rgs2 protein, mouse; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D049410:Luciferases, Renilla; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases