G protein-coupled receptors form stable complexes with inwardly rectifying potassium channels and adenylyl cyclase.
A large number of studies have demonstrated co-purification or co-immunoprecipitation of receptors with G proteins. We have begun to look for the presence of effector molecules in these receptor complexes. Co-expression of different channel and receptor permutations in COS-7 and HEK 293 cells in combination with co-immunoprecipitation experiments established that the dopamine D(2) and D(4), and beta(2)-adrenergic receptors (beta(2)-AR) form stable complexes with Kir3 channels. The D(4)/Kir3 and D(2) receptor/Kir3 interaction does not occur when the channel and receptor are expressed separately and mixed prior to immunoprecipitation, indicating that the interaction is not an artifact of the experimental protocol and reflects a biosynthetic event. The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein alpha subunit function. However, using a peptide that binds Gbetagamma (betaARKct), we show that Gbetagamma is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the beta(2)-adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, beta(2)AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.
Lavine
Natalie
N
Centre for Addiction and Mental Health, Department of Pharmacology, Institute of Medical Science, University of Toronto, Ontario M5T 1R8, Canada.
Ethier
Nathalie
N
Oak
James N
JN
Pei
Lin
L
Liu
Fang
F
Trieu
Phan
P
Rebois
R Victor
RV
Bouvier
Michel
M
Hebert
Terence E
TE
Van Tol
Hubert H M
HH
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D015221:Potassium Channels / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction; D014981:Xenopus
D017931:DNA Primers; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C497066:KCNJ3 protein, human; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011956:Receptors, Cell Surface; D020962:Heterotrimeric GTP-Binding Proteins; D000262:Adenylyl Cyclases