The bovine brain A1-adenosine receptor was purified 8000-fold by affinity chromatography on xanthine-amine-congener (XAC)-Sepharose. Addition of a 120-fold molar excess of a purified bovine brain G-protein preparation (Go,i a mixture of Go and Gi, containing predominantly Go) decreases the Bmax of the binding of the antagonist radioligand [3H]XAC to the receptor. This decrease is observed not only after insertion into phospholipid vesicles but also in detergent solution, and is reversed by GTP analogues. In the presence of Go,i, about 20 and 40% of the receptors display guanine-nucleotide-sensitive high-affinity binding of the agonist radioligand (-)-N6-3-([125I]iodo-4-hydroxyphenylisopropyl)adenosine after reconstitution into lipid vesicles and in detergent solution, respectively. The ability of Go,i to enhance agonist binding and decrease antagonist binding is concentration-dependent, with a half-maximal effect occurring at approximately 10-fold molar excess of G-proteins over A1-adenosine receptors. In the presence of the receptor, the rate of guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to Go,i is accelerated. This rate is further enhanced if the receptor is activated by the agonist (-)(R)-N6-phenylisopropyladenosine, whereas the antagonist XAC decreases the association rate of GTP[35S] to levels observed in the absence of receptor. These results show (1) that detergent removal is not a prerequisite for the observation of coupling between the A1-adenosine receptor and Go,i, and (2) that the regulatory effect of G-proteins on antagonist binding to the A1-adenosine receptor can be reconstituted by using purified components.
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May 1991
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Research Article|
May 01 1991
Interactions of purified bovine brain A1-adenosine receptors with G-proteins. Reciprocal modulation of agonist and antagonist binding Available to Purchase
M Freissmuth;
M Freissmuth
1Institute of Pharmacology, University of Vienna, Waehringer Strasse 13a, A-1090 Vienna, Austria.
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E Selzer;
E Selzer
1Institute of Pharmacology, University of Vienna, Waehringer Strasse 13a, A-1090 Vienna, Austria.
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W Schütz
W Schütz
1Institute of Pharmacology, University of Vienna, Waehringer Strasse 13a, A-1090 Vienna, Austria.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1991 The Biochemical Society, London
1991
Biochem J (1991) 275 (3): 651–656.
Citation
M Freissmuth, E Selzer, W Schütz; Interactions of purified bovine brain A1-adenosine receptors with G-proteins. Reciprocal modulation of agonist and antagonist binding. Biochem J 1 May 1991; 275 (3): 651–656. doi: https://doi.org/10.1042/bj2750651
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