Allosteric Control of Beta2-adrenergic Receptor Function.

GPCR;Nanobody;Allosteric modulator;Pharmacy and Pharmacology;Health Sciences;Pharmacology

Mahoney, Jacob PatrickHolinstat, Michael Allan ;

University of Michigan

关键词: GPCR;    Nanobody;    Allosteric modulator;    Pharmacy and Pharmacology;    Health Sciences;    Pharmacology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/133520/jmaho_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins and allow cells to sense their environment and respond accordingly. The transduction of an extracellular stimulus into an intracellular response relies on allosteric communication between two distant binding sites within the receptor. By binding at an extracellular site, GPCR agonists promote interaction of the receptor with a heterotrimeric G protein, thereby initiating an intracellular signaling cascade. The reciprocal interaction has also been observed for decades with many GPCRs - just as agonists promote G protein engagement, G proteins enhance agonist affinity for the receptor. Biophysical and structural studies of the beta2-adrenergic receptor (B2AR), a prototypic GPCR, have illuminated the conformational changes within the receptor that promote agonist-mediated G protein engagement by B2AR. Here we used G protein and a G protein-mimetic camelid antibody fragment (nanobody Nb80) to characterize the allosteric communication between the agonist- and G protein-binding sites on B2AR. We developed a label-free assay to study the B2AR-Nb80 interaction and used this system to demonstrate the selectivity of Nb80 for active-state B2AR, a quality which rendered Nb80 useful to study B2AR activation in living cells. Furthermore, we found that agonists enhance Nb80 affinity primarily by accelerating the association of Nb80 (and presumably G protein). In contrast, using several GPCRs we demonstrate that G protein or G protein-mimetic nanobodies enhance agonist affinity by constricting the agonist-binding site around the bound ligand, thereby slowing its dissociation. We identified key residues within B2AR that form a ;;lid;; over the agonist-binding site in the receptor;;s active state, which form the bottom of a potential binding site in the receptor;;s extracellular vestibule. Targeting this site using virtual screening, we discovered novel allosteric ligands for B2AR. Together, these studies provide a structural mechanism to explain the allosteric communication between the binding of agonists and G protein, as well as highlight the utility of GPCR crystal structures for ligand discovery.