Effects of Guanine, Inosine, and Xanthine Nucleotides on β2-Adrenergic Receptor/Gs Interactions: Evidence for Multiple Receptor Conformations
- 1Howard Hughes Medical Institute (R.S., U.K., K.W.-S., B.K.K.) and2Division of Cardiovascular Medicine (B.K.K.), Stanford University Medical School, Stanford, California
Abstract
The aim of our study was to examine the effects of different purine nucleotides [GTP, ITP, and xanthosine 5′-triphosphate (XTP)] on receptor/G protein coupling. As a model system, we used a fusion protein of the β2-adrenergic receptor and the α subunit of the G protein Gs. GTP was more potent and efficient than ITP and XTP at inhibiting ternary complex formation and supporting adenylyl cyclase (AC) activation. We also studied the effects of several β2-adrenergic receptor ligands on nucleotide hydrolysis and on AC activity in the presence of GTP, ITP, and XTP. The efficacy of agonists at promoting GTP hydrolysis correlated well with the efficacy of agonists for stimulating AC in the presence of GTP. This was, however, not the case for ITP hydrolysis and AC activity in the presence of ITP. The efficacy of ligands at stimulating AC in the presence of XTP differed considerably from the efficacies of ligands in the presence of GTP and ITP, and there was no evidence for receptor-regulated XTP hydrolysis. Our findings support the concept of multiple ligand-specific receptor conformations and demonstrate the usefulness of purine nucleotides as tools to study conformational states of receptors.
Footnotes
-
Send reprint requests to: Brian Kobilka, M.D., Howard Hughes Medical Institute, B-157, Beckman Center, Stanford University Medical School, Stanford, CA 94305-5428. E-mail:kobilka{at}cmgm.stanford.edu
-
↵1 Current address: Department of Pharmacology and Toxicology, The University of Kansas, 5001 Malott Hall, Lawrence, KS 66046.
-
↵2 Current address: Department of Cellular Physiology, Institute of Medical Physiology 12.5, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
-
↵3 Current address: Higuchi Biosciences Center, The University of Kansas, 5003 Malott Hall, Lawrence, KS 66045.
-
R.S. and K.W.-S. were supported by a research fellowship of the Deutsche Forschungsgemeinschaft.
- Abbreviations:
- β2-AR
- β2-adrenergic receptor
- GPCR
- G protein-coupled receptor
- AC
- adenylyl cyclase
- XTP
- xanthosine 5′-triphosphate
- XDP
- xanthosine 5′-diphosphate
- GsαL
- long-splice variant of Gsα
- GsαS
- short-splice variant of Gsα
- GTPγS
- guanine 5′-O-(3-thiotriphosphate)
- GppNHp
- guanylyl imidodiphosphate
- DHA
- dihydroalprenolol
- SAL
- salbutamol
- NTP
- nucleoside 5′-triphosphate
- cAMP
- cyclic AMP
- ICI
- [erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol]
- ISO
- isoproterenol
- NDP
- nucleoside 5′-diphosphate
- EPH
- (−)-ephedrine
- DCI
- dichloroisoproterenol
- DOB
- dobutamine
-
- Received January 6, 1999.
- Accepted April 28, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
