Abstract
G protein coupled receptors (GPCRs) are integral membrane proteins that change conformation subsequent to 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 GPCR 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 about 25% of the total β2-AR receptor 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.
- Adrenergic
- Gs family
- Structure-activity relationships and modeling
- Thermodynamic and kinetic processes and modeling
- Receptor binding studies
- Received December 20, 2011.
- Revision received March 14, 2012.
- Accepted March 15, 2012.
- The American Society for Pharmacology and Experimental Therapeutics