Abstract
G protein-coupled receptors (GPCRs) transduce a diverse variety of extracellular stimuli into intracellular signaling. These receptors are the most clinically productive drug targets at present. Despite decades of research on the signaling consequences of molecule-receptor interactions, conformational components of receptor-effector interactions remain incompletely described. The β2-adrenergic receptor (β2AR) is a prototypical and extensively studied GPCR that can provide insight into this aspect of GPCR signaling thanks to robust structural data and rich pharmacopeia. Using bioluminescence resonance energy transfer -based biosensors, second messenger assays, and biochemical techniques, we characterize the properties of β2AR-F193A. This single point mutation in extracellular loop 2 of the β2AR is sufficient to intrinsically bias the β2AR away from β-arrestin interaction and demonstrates altered regulatory outcomes downstream of this functional selectivity. This study highlights the importance of extracellular control of intracellular response to stimuli and suggests a previously undescribed role for the extracellular loops of the receptor and the extracellular pocket formed by transmembrane domains 2, 3, and 7 in GPCR regulation that may contribute to biased signaling at GPCRs.
SIGNIFICANCE STATEMENT The role of extracellular G protein-coupled receptor (GPCR) domains in mediating intracellular interactions is poorly understood. We characterized the effects of extracellular loop mutations on agonist-promoted interactions of GPCRs with G protein and β-arrestin. Our studies reveal that F193 in extracellular loop 2 in the β2-adrenergic receptor mediates interactions with G protein and β-arrestin with a biased loss of β-arrestin binding. These results provide new insights on the role of the extracellular domain in differentially modulating intracellular interactions with GPCRs.
Footnotes
- Received June 7, 2021.
- Accepted November 21, 2021.
Research reported in this publication was supported by the National Institutes of Health awards R35GM122541 (J.L.B.), R01HL136219 (J.L.B.), P01HL114471 (J.L.B.), T32GM100836 (M.I.), and F31HL139104 (M.I.), and used the MetaOmics Shared Resource at Sidney Kimmel Cancer Center at Jefferson Health supported by National Institutes of Health award P30CA056036. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. A.I. was funded by KAKENHI 21H04791 from The Japan Society for the Promotion of Science (JSPS); the PRIME 20gm5910013, the LEAP JP20gm0010004 and the BINDS JP20am0101095 from the Japan Agency for Medical Research and Development (AMED), and the Uehara Memorial Foundation.
No author has a conflict with the contents of this article.
- Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics
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Extracellular Control of Signaling Bias at the β2AR
