Inverse Agonist Activity at the α2A-Adrenergic Receptor
- Departments of 1Pharmacology (S.M.W., K-L.L., D.J.M., R.R.N.) and2Internal Medicine/Hypertension (R.R.N.), The University of Michigan, Ann Arbor, Michigan
Abstract
Constitutive activation of G protein-coupled receptors (GPCRs) is now well recognized and many classical GPCR antagonists have been found to be inverse agonists. For the α2A-adrenergic receptor (AR) we determine the relative inverse efficacies of a series of antagonists and utilize the extended ternary complex model to estimate the fraction of constitutively active mutant (CAM) receptors in the active state. Stable Chinese hamster ovary cell lines expressing the porcine α2A-AR in its wild-type (WT) and constitutively activated (CAM-T373K) form were isolated. Activation of both Gi and Gs was enhanced for CAM receptors. cAMP production was suppressed in cells with the CAM α2A-AR and this suppression was reversed by α2-adrenergic antagonists with an order of inverse efficacy of rauwolscine > yohimbine > RX821002 > MK912, whereas phentolamine and idazoxan were essentially neutral antagonists. This striking difference in inverse efficacy between idazoxan and RX821002 may account for in vivo pharmacological differences between these two α2-adrenergic antagonists. Agonist binding affinity to the non-G protein-coupled CAM receptor was 3- to 9-fold higher than to WT, whereas binding of the most efficacious inverse agonists, yohimbine and rauwolscine, was 1.7- and 2.1-fold weaker. Analysis of this difference by the extended ternary complex model indicates that approximately 50% of the CAM α2A-AR is in the active (R*) state although there is no detectable constitutive activity of the WT receptor in the absence of agonist.
Footnotes
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Send reprint requests to: Dr. Richard R. Neubig, Department of Pharmacology, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. E-mail: RNeubig{at}umich.edu
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↵1 In addition to the three experiments with the WT-Tag 19 line (shown in Table 1), Kd values were also determined in paired experiments comparing CAM with WT-Tag L1 (n = 5) and WT-Tag L9 (n = 1).
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↵2 We have left out the G protein component for the calculations involving inverse agonist binding because the receptor density (∼20 pmol/mg of protein) is greater than that of Gi (∼10 pmol/mg for CHO cells) (Gerhardt, 1992), and we have included GppNHp in the binding assays to further reduce any G protein contribution. Chen et al. (2000) recently compared the ETC and cubic ternary complex models but did not address the question of inverse agonist affinities for the different receptor states.
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↵3 In these calculations, we use association equilibrium constants to simplify the calculations. The conclusions would be the same if dissociation constants were used.
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↵4 A less stringent assumption will also lead to the same final conclusions. If K is the same for WT and CAM receptors and L for the WT receptor is very low, then eq. 2 is still valid.
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Supported by National Institutes of Health HL46417. Production and flow cytometry screening of the mutant receptors received assistance from UM-MAC, National Institutes of Health P60-AR20557.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- CAM
- constitutively active mutant
- ETC
- extended ternary complex
- WT
- wild-type
- AR
- adrenergic receptor
- 5-HT
- 5-hydroxytrytamine
- PTX
- pertussis toxin
- IBMX
- isobutylmethylxanthine
- HA
- hemagglutinin
- CHO
- Chinese hamster ovary
- DMEM
- Dulbecco's modified Eagle's medium
- GppNHp
- 5′-guanylyimidodiphosphate
- NLLSQ
- non-linear least squares regression
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- Received June 12, 2000.
- Accepted November 17, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
