Abstract
G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the β2-adrenergic receptor, we developed a new agonist radioligand for binding assays. [3H](R,R′)-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total β2-adrenoceptor (AR) population as determined with the antagonist [3H]CGP-12177. The β2-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and Ki values determined for fenoterol analogs model much better the cAMP activity of the β2-AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [3H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel β2-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the β2-adrenoceptor.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This research was supported in part by the Intramural Research Program of the National Institutes of Health National Institute on Aging; by the National Institutes of Health National Institute on Aging [Contract N01-AG31009]; and by the Foundation for Polish Science (TEAM Programme). The article was developed using the equipment purchased within the Project “The equipment of innovative laboratories doing research on new medicines used in the therapy of civilization and neoplastic diseases” within the Operational Program Development of Eastern Poland 2007–2013, Priority Axis I Modern Economy, Operations I.3 Innovation Promotion.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- β2-AR
- β2-adrenoceptor
- CGP-12177
- 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one
- GPCR
- G protein-coupled receptor
- Fen
- fenoterol
- HEK
- human embryonic kidney
- HEK-β2-AR
- HEK293 cells stably transfected with β2-AR
- MFen
- (R,R′)-4-methoxyfenoterol
- ICI 118-551
- (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol
- PDB
- Protein Data Bank
- MVD
- Molegro Virtual Docker
- GTPγS
- guanosine 5′-O-(3-thio)triphosphate
- BI-167,107
- 5-hydroxy-8-{2-[2-(2-methylphenyl)-1,1-dimethyl-ethylamino]-1-hydroxyethyl}-4H-benzo[1,4]oxazin-3-one
- TM
- transmembrane.
- Received December 20, 2011.
- Accepted March 15, 2012.
- U.S. Government work not protected by U.S. copyright
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|