Reconstitution of bovine A1 adenosine receptors and G proteins in phospholipid vesicles: betagamma-subunit composition influences guanine nucleotide exchange and agonist binding.
We have studied the interactions of purified A1 adenosine receptors and G proteins reconstituted into phospholipid vesicles to investigate how the betagamma composition of G protein heterotrimers influences coupling. Recombinant hexahistidine-tagged bovine A1 adenosine receptors were expressed in Sf9 cells and purified to homogeneity by sequential chromatography over heparin-sepharose, xanthine amino congener-agarose, and nickel-nitrilotriacetic acid columns. These receptors were reconstituted with pure recombinant G proteins of defined subunit composition. Receptor-G protein complexes containing alphai2 and beta1gamma2 or beta1gamma3 and stimulated with the agonist, (R)-phenylisopropyladenosine, exchange guanine nucleotide 2-3 times more rapidly than do complexes containing beta1gamma1. This difference is not overcome by increasing the concentration of betagamma subunits. Receptor-G protein complexes containing beta1gamma1 also bind less of the agonist, [125I]-iodoaminobenzyladenosine (125I-ABA), than do complexes containing beta1gamma3. Kinetic experiments show that 125I-ABA dissociates 2-fold more rapidly from receptor-G protein complexes containing beta1gamma1 than from complexes containing the other betagamma subunits. The affinity of the interaction between immobilized Galphai2 subunits and beta1gamma1 or beta1gamma2 measured with an optical biosensor in the absence of receptor is similar. Taken together, these data implicate the gamma-subunit in influencing the interaction between the A1 adenosine receptor and G proteins.
Figler
R A
RA
Department of Molecular Physiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Lindorfer
M A
MA
Graber
S G
SG
Garrison
J C
JC
Linden
J
J
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D015374:Biosensing Techniques; D019921:Biotinylation; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism* / Q000494:pharmacology; D007462:Iodobenzenes / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010660:Phenylisopropyladenosine / Q000378:metabolism* / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1 / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism
D001386:Azides; D007462:Iodobenzenes; D008081:Liposomes; D010743:Phospholipids; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D014970:Xanthines; C056217:3-(3-iodo-4-azido)phenethyl-1-propyl-8-cyclopentylxanthine; D010660:Phenylisopropyladenosine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C044323:3-iodo-N(6)-4-aminobenzyladenosine; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019204:GTP-Binding Proteins; D000241:Adenosine