Abstract
In this study, we investigated the hypothesis of agonist-directed trafficking of receptor signaling for the α2A-adrenergic receptor (α2A-AR). α2A-ARs couple to both Gs and Gi to stimulate or inhibit adenylyl cyclase activity. Chinese hamster ovary-K1 cell lines expressing the porcine α2A-AR at high (α2A-H) and low (α2A-L) levels were used to estimate the relative efficacies (R.e.s) of a series of agonists for the Gs and Gi pathways. Gs-mediated responses were measured after pertussis toxin treatment to inactivate Gi in α2A-H, whereas Gi responses were measured in α2A-L, where Gs responses were absent. The full agonist UK-14,304 showed a large receptor reserve for Gi responses in α2A-H but little receptor reserve for Gs responses in α2A-H or for Gi responses in α2A-L. With the exception ofl-isoproterenol (ISO), all agonists showed similar R.e.s at the α2A-AR for Gs and Gi responses, with rank orders of R.e.s as follows: l-epinephrine =l-norepinephrine = UK-14,304 >p-aminoclonidine ≥ BHT-920 ≥ BHT-933 > clonidine = p-iodoclonidine ≥ xylazine ≥ guanabenz. Interestingly, ISO had the highest efficacy at the α2A-AR for activating Gs versus Gi (9-fold higher); however, it had low potency for both. By several criteria, the ISO response was mediated by the α2A-AR, supporting the hypothesis of agonist-directed trafficking of receptor signaling or agonist-specific G protein selectivity. In contrast, the apparent Gi pathway selectivity of oxymetazoline appears to be mediated by an endogenous serotonergic receptor. It is intriguing that a classic β-AR agonist that activates Gs through β2-ARs also appears to produce a Gs-selective conformation of the Gi-coupled α2A-AR.
Footnotes
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Send reprint requests to: Richard R. Neubig, M.D., Ph.D., 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 This work was supported by National Institutes of Health Grant HL46417 and funds from Eli Lilly (Indianapolis, IN). The development of the R3 and B2 mutants was also supported by the University of Michigan Multipurpose Arthritis Center (AR20557).
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↵2 Present address: Division of Pharmacology, School of Pharmacy, Potchefstroom University for CHE, Potchefstroom, 2520 South Africa.
- Abbreviations:
- ADTRS
- agonist-directed trafficking of receptor signaling
- AR
- adrenergic receptor
- α2A-H
- high α2A-adrenergic receptor expressing Chinese hamster ovary-K1 cell line
- α2A-L
- low α2A-adrenergic receptor expressing Chinese hamster ovary-K1 cell line
- α2A-B2
- B2 mutant α2A-adrenergic receptor expressing Chinese hamster ovary-K1 cell line
- α2A-R3
- R3 mutant α2A-adrenergic receptor expressing Chinese hamster ovary-K1 cell line
- 5-HT
- 5-hydroxytryptamine
- CHO
- Chinese hamster ovary
- ISO
- l-isoproterenol
- KA(app.)
- apparent dissociation constant of the agonist-receptor complex
- HEK
- human embryonic kidney
- TCA
- trichloroacetic acid
- DMEM
- Dulbecco's modified Eagle's medium
- PTX
- pertussis toxin
- R.e.
- relative efficacy
- IBMX
- 3-isobutyl-1-methyl-xanthine
- Emax
- maximal response
- RIA
- relative intrinsic activity
- q
- fraction of receptors still functional after partial receptor alkylation
- Smax
- maximal stimulus
- Received December 2, 1999.
- Accepted April 19, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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