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Reconstitution of bovine A1 adenosine receptors and G proteins in phospholipid vesicles: betagamma-subunit composition influences guanine nucleotide exchange and agonist binding.
- Source :
-
Biochemistry [Biochemistry] 1997 Dec 23; Vol. 36 (51), pp. 16288-99. - Publication Year :
- 1997
-
Abstract
- 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.
- Subjects :
- Adenosine metabolism
Amidohydrolases metabolism
Animals
Azides metabolism
Biosensing Techniques
Biotinylation
Cattle
Chromatography, Affinity
Electrophoresis, Polyacrylamide Gel
GTP-Binding Proteins chemistry
Guanosine 5'-O-(3-Thiotriphosphate) pharmacology
Kinetics
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Phenylisopropyladenosine pharmacology
Phospholipids metabolism
Protein Binding
Protein Conformation
Purinergic P1 Receptor Agonists
Receptors, Purinergic P1 chemistry
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Xanthines metabolism
Adenosine analogs & derivatives
GTP-Binding Proteins metabolism
Guanosine 5'-O-(3-Thiotriphosphate) metabolism
Iodobenzenes metabolism
Liposomes metabolism
Phenylisopropyladenosine metabolism
Receptors, Purinergic P1 metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 36
- Issue :
- 51
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 9405064
- Full Text :
- https://doi.org/10.1021/bi972000q