Location bias contributes to functionally selective responses of biased CXCR3 agonists

Abstract

Some G protein-coupled receptor (GPCR) ligands act as “biased agonists” that preferentially activate specific signaling transducers over others. Although GPCRs are primarily found at the plasma membrane, GPCRs can traffic to and signal from many subcellular compartments. Here, we determine that differential subcellular signaling contributes to the biased signaling generated by three endogenous ligands of the GPCR CXC chemokine receptor 3 (CXCR3). The signaling profile of CXCR3 changes as it traffics from the plasma membrane to endosomes in a ligand-specific manner. Endosomal signaling is critical for biased activation of G proteins, β-arrestins, and extracellular-signal-regulated kinase (ERK). In CD8 + T cells, the chemokines promote unique transcriptional responses predicted to regulate inflammatory pathways. In a mouse model of contact hypersensitivity, β-arrestin-biased CXCR3-mediated inflammation is dependent on receptor internalization. Our work demonstrates that differential subcellular signaling is critical to the overall biased response observed at CXCR3, which has important implications for drugs targeting chemokine receptors and other GPCRs.

Location bias: A “Hidden Variable” in GPCR pharmacology

“…However, at the endosome, only CXCL11 was able to recruit β-arrestin, while all three ligands still maintained G protein activation. [12] Similar findings were obtained in the study on the AT1R, where the authors found that inhibiting internalization had a significant impact on a ligand's ability to recruit β-arrestin, but did not impact G protein activation. [79] It is plausible that these observations occur due to differential localization and availability of G proteins and β-arrestins in subcellular compartments.…”

“…[41][42][43] Subsequent work demonstrated endosomal activation of Gαs, Gαi/o, and Gαq at a diverse array of GPCRs. [12,[44][45][46] GPCRs which activate the Gαs subunit and exhibit receptor signaling from endosomes have demonstrated sustained and consequential cAMP signaling upon receptor internalization. [43,[47][48][49][50] Additionally, endosomal cAMP production has been shown to promote nuclear cAMP and PKA activation in ways that cAMP generated at the plasma membrane cannot.…”

“…Unfortunately, some debate in the literature has focused on a paradigm that G proteins and β-arrestins act in purely an antagonistic fashion, [57,58] while it appears that βarrestins and G proteins contribute to intracellular signaling cascades in complex patterns, [59] with β-arrestin promoting some signaling cascades while antagonizing others in a location-biased fashion. [12] Other sites of subcellular signaling Endosomes are only one of the various subcellular locations at which GPCR signaling has been detected. GPCRs have been extensively studied at the primary cilium, an organelle specialized to act as the "cell's antenna," with specific subcellular group of receptors demonstrates prominent roles in sensing mechanical and chemical signals, and integrating these signals into physiologic processes important during development.…”

“…A recent publication by our group demonstrated that G protein activation by three ligands at the chemokine receptor CXCR3 is location-dependent. [12] By modifying a recently published biosensor F I G U R E 3 Subcellular location can impact G protein-coupled receptor (GPCR) pharmacology. (A) Hypothetical dose-response curve (left) and saturation-binding curve (right).…”

“…[ 11 ] “Biased agonists” can exist endogenously, such as in the chemokine system, where multiple ligands target the same receptor and differentially activate G proteins and β‐arrestins. [ 12 ] A variety of synthetic small molecules and peptide analogs that similarly demonstrate biased GPCR signaling also exist. These biased agonists have important clinical and pharmaceutical implications and have been actively pursued as a potential avenue to develop specific drugs that activate beneficial pathways while inhibiting those that lead to adverse effects.…”