Resonating with the signaling bias of CXCR7

Abstract

Kleist et al. combine NMR spectroscopy and residue contact network analysis to identify a potential allosteric network in CXCR7, a β-arrestin-biased chemokine receptor, which links the extracellular ligand-binding pocket and the intracellular transducer-coupling region through the receptor transmembrane core.

G protein-coupled receptors (GPCRs), also referred to as seven transmembrane receptors (7TMRs), are intricately involved in nearly every aspect of human physiology and pathophysiology. The major hallmarks of prototypical GPCRs include their 7TM architecture and down-stream signaling via heterotrimeric G proteins and β-arrestins (βarrs). While most natural agonists typically activate both G proteins and βarrs, synthetic ligands referred to as biased agonists capable of preferentially engaging and activating one of these pathways over the other, have been described for multiple GPCRs (Kolb et al., 2022). Interestingly, there are several 7TMRs, including multiple chemokine receptors, which couple selectively to βarrs but not to G proteins, even when stimulated by their natural agonists. Such receptors have been referred to as non-canonical GPCRs, atypical chemokine receptors (ACKRs), or arrestin-coupled receptors (ACRs), and they essentially represent natural examples of intrinsically biased 7TMRs (Figure 1A) (Nibbs and Graham, 2013; Pandey et al., 2021). Our current understanding of these receptors, especially in terms of their functional divergence vis-à-vis prototypical GPCRs, is still in its infancy. Now, Kleist et al. utilize nuclear magnetic resonance (NMR) spectroscopy to monitor agonist-induced conformational changes at specific residues in CXCR7, a βarr-biased chemokine receptor, and employ residue contact network analysis to propose a potential allosteric network that links agonist binding to transducer coupling (Kleist et al., 2022) (Figure 1B).

Figure 1. An allosteric connection for CXCR7 activation and βarr coupling.

(A) Schematic representation showing the intrinsically biased nature of CXCR7 compared to prototypical GPCRs.

(B) A proposed allosteric pipeline that links the ligand-binding pocket on the extracellular side of the receptor to βarr-coupling interface on the cytoplasmic side through Asn¹²⁷ localized in the transmembrane core of the receptor upon agonist binding (right panel) but not for antagonist (left panel).

Kleist et al. set out to explore the conformational landscape of CXCR7 upon its activation by a set of different ligands having distinct pharmacological efficacies in order to connect ligand binding and receptor activation with βarr coupling. In addition to CXCL12, the natural chemokine agonist of CXCR7, Kleist et al. also used two strong partial agonists, namely LIH383 and CCX777, and a nanobody-based competitive antagonist, VUN701, to induce and stabilize active and inactive conformations in the receptor, respectively. They selectively labeled native methionine residues scattered along the different regions of the receptor as sensitive local probes of conformational change monitored using NMR spectroscopy (Kleist et al., 2019). In particular, the authors focused their efforts on monitoring the chemical shift changes of two distinct methionine residues, namely Met²¹² located toward the extracellular end of TM5 and Met¹³⁸ positioned toward the cytoplasmic end of TM3. The chemical shift signatures of Met²¹² cluster together in response to the two peptide agonists, i.e., CXCL12 and LIH383, while being different for the small molecule agonist, CCX777. Similarly, although the rotameric state of Met²¹² is distinct between the peptide and small-molecule agonist, it suggests overall conformational stabilization in this region. In contrast, antagonist binding to the receptor results in a chemical shift pattern that is intermediate between the peptide and non-peptide agonists and exhibits sustained conformational heterogeneity at and around Met²¹² based on rotamer conformation. On the other hand, all three agonists elicit nearly identical chemical shift signatures at Met¹³⁸ with conformational stabilization while the antagonist binding not only results in a distinct chemical shift signature but also reflects persistent conformational heterogeneity.

How did the authors rationalize these NMR chemical shift signatures observed here to link agonist-binding with βarr coupling at CXCR7? Kleist et al. employed residue contact network analysis to compare the overall ligand contacts between the βarr-binding states of selected GPCRs and the antagonist-bound state of the same receptors using previously determined structural snapshots. Interestingly, agonists in the βarrbound states appear to engage TM5 as an anchor to make a relatively higher number of contacts with the residues in ECL2 and TM7 compared to the antagonists. Interestingly, a similar contact analysis of the residues on the cytoplasmic side of the receptors combined with experimental validation helped the authors identify Tyr³¹⁵ in TM7 of CXCR7 as a potential determinant for βarr recruitment, which is also corroborated by conformational stabilization around Met¹³⁸ as observed in NMR measurement potentially through a direct interaction with Tyr³¹⁵ (Figure 1B). Furthermore, the extracellular ligand-binding pocket and the cytoplasmic region guiding βarr recruitment appear to be linked through a conserved Asn¹²⁷ in TM3, as Asn¹²⁷Lys mutation results in diminished βarr recruitment while Asn¹²⁷Ser substitution yields an enhanced level of basal βarr binding, suggesting constitutive activity. Interestingly, chemical shift signatures of Met¹³⁸ and Met²¹² are consistent with a change in conformational stabilization and de-stabilization upon Asn¹²⁷Lys and Asn¹²⁷Ser mutations, respectively. Collectively, these observations suggest an allosteric connection between the ligand-binding pocket and transducer-coupling interface via Asn¹²⁷ in the receptor transmembrane core.

While this study offers a potential framework to link agonist binding and receptor activation with βarr interaction, there are several key questions that remain unanswered. For example, how much of the effects observed on βarr recruitment upon Tyr³¹⁵ or Asn¹²⁷ mutations reflect a direct impact on βarr binding versus receptor phosphorylation? Considering that receptor phosphorylation is a key driver of βarr binding to GPCRs and ACRs, including CXCR7 (Maharana et al., 2022), it is tempting to speculate that some of the proposed allosteric connections may be exerting differential GPCR kinase (GRK) engagement and receptor phosphorylation than direct measure of βarr recruitment. Considering that previous studies have linked distinct βarr conformations with different functional outcomes in the context of GPCRs versus ACRs, including CXCR7 (Sarma et al., 2022), it also remains to be explored if the proposed mechanism is likely to be operative for other ACRs and potentially for GPCRs in response to stimulation of biased agonists. Such studies may shed light on distinct modalities of receptor-βarr engagement, conformational outcomes, and diversity in this versatile class of receptors. Finally, how the receptors, including CXCR7, select against G protein coupling is not explored in the current study. However, considering the powerful approach of contact network analysis (Hauser et al., 2021) with ever-increasing coverage of structural snapshots of GPCR G proteins (Congreve et al., 2020), it would be tempting to apply this approach to understand the driving mechanism(s), which dissuade ACRs from coupling to heterotrimeric G proteins. Although a recent study describing cryo-EM structure of CXCL12-bound CXCR7 has offered some interesting insights into the lack of G protein coupling to CXCR7 (Yen et al., 2022), additional studies are still required to further delineate the precise molecular mechanism of intrinsic bias at CXCR7 and other ACRs.

In summary, Kleist et al. identify an interesting allosteric pipeline in CXCR7, linking the ligand binding event on the extracellular side with transducer-coupling on the intracellular side. However, the central question to decipher the molecular basis of the functional divergence exhibited by ACRs, including CXCR7 compared to prototypical GPCRs, remains very much open.