The Third Extracellular Loop of the β2-Adrenergic Receptor Can Modulate Receptor/G Protein Affinity
- Department of Molecular Cardiology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
Abstract
Chimeric receptors of the β2-adrenergic receptor in which the extracellular loops were replaced with the corresponding amino acids of the α1a-adrenergic receptor were generated to measure changes in α1-antagonist affinity. Although no changes in α1-antagonist affinity were measured in the β2/α1a chimeras, a decreased IC50 (10-fold) for agonists as compared with wild type β2 control was found because of the replacement of the third extracellular loop (EX3). These agonist high affinity changes were because of a greater proportion of high affinity sites (2-fold) that were convertible to low affinity sites with guanosine 5′-3-O-(thio)triphosphate. Adenylate cyclase activity evoked by the EX3 chimera showed commensurate increases in the basal signal transduction as well as the isoproterenol-stimulated potency, suggesting constitutive activity. However, unlike other constitutively active adrenergic receptor mutants in which the mutation causes G protein-independent changes, the mechanism of the EX3 chimera seems to be attributable to a greater ease with which the active ternary complex is formed because of a higher affinity/coupling of the G protein. Although the changes because of EX3 are indirect and most likely affect helical packing, they support an emerging hypothesis that G protein-coupled receptors have evolved their structure-function relationships to constrain the receptor in an inactive state.
Footnotes
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Send reprint requests to: Dr. Dianne M. Perez, Department of Molecular Cardiology, FF3-01, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, Ohio 44195. E-mail:perezd{at}cesmtp.ccf.org
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↵1 Current affiliation: Division of Hematology, Vanderbilt University, Nashville, TN 37232.
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This work was supported in part by National Institutes of Health Grant RO1-HL52544 (D.M.P.) and an unrestricted research grant from Glaxo Wellcome. This work was done under the tenure of an Established Investigator Award from the American Heart Association (D.M.P.).
- Abbreviations:
- AR
- adrenergic receptor
- CAM
- constitutively active mutation
- EX
- extracellular loop
- [125I]CYP
- iodocyanopindolol
- TCM
- ternary complex model
- TM
- transmembrane
- WT
- wild-type
- GTPγS
- guanosine 5′-3-O-(thio)triphosphate
- ANOVA
- analysis of variance
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
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- Received August 19, 1997.
- Accepted November 21, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
