Molecular Mechanism of the Arrestin-Biased Agonism of Neurotensin Receptor 1 by an Intracellular Allosteric Modulator

Chemistry Medicinal Chemistry Biochemistry Life Sciences Pharmacy Medicine Basic Medical Sciences
G protein-coupled receptors allosteric modulators arrestin-biased agonism neurotensin receptor 1 cryo-EM structures signal transduction drug discovery

Academic Background

G protein-coupled receptors (GPCRs) are the most abundant family of cell surface receptors in the human body and are also the most common targets of FDA-approved drugs. GPCRs play a crucial role in the treatment of various diseases, including pain, diabetes, cardiovascular diseases, and cancer. However, drug development targeting GPCRs faces numerous challenges, particularly in achieving receptor subtype selectivity and controlling on-target and off-target side effects. Traditional orthosteric ligands often struggle to address these issues, making the development of allosteric modulators that act outside the orthosteric pocket a promising strategy. Allosteric modulators can selectively enhance or inhibit the signaling of endogenous ligands while providing excellent receptor subtype selectivity.

In recent years, a class of molecules known as biased allosteric modulators (BAMs) has garnered significant attention. BAMs not only possess allosteric modulation capabilities but also can steer receptor signaling toward either the G protein or β-arrestin pathway, thereby avoiding certain side effects. Although structures of GPCRs in complex with G proteins or GPCR kinases (GRKs) in the BAM-bound state have been resolved, the structure of GPCRs in complex with β-arrestin in the presence of BAMs has not yet been determined. This limitation hinders a comprehensive understanding of the pharmacological profile of BAMs.

This study aims to elucidate how BAMs modulate the interaction between GPCRs and β-arrestin, particularly by resolving the high-resolution cryo-EM structures of the neurotensin receptor 1 (NTSR1) in complex with β-arrestin1 and the BAM SBI-553, thereby filling this critical knowledge gap.

Source of the Paper

This paper was jointly completed by researchers including Demeng Sun, Xiang Li, Qingning Yuan, and others from institutions such as the University of Science and Technology of China, Tsinghua University, and the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. It was published online on March 21, 2025, in the journal Cell Research.

Research Process and Results

1. Chemical Synthesis of Phosphorylated NTSR1

The study first developed a chemical protein synthesis strategy to generate NTSR1 with hexa-phosphorylation at its C-terminus. The phosphorylation of the C-terminus of NTSR1 is crucial for the recruitment and activation of β-arrestin1. The research team synthesized NTSR1 C-terminal peptides with different phosphorylation patterns and validated their affinity for β-arrestin1 using fluorescence polarization assays. The results showed that the hexa-phosphorylated peptide (Ser401/Ser403/Ser404 and Thr407/Ser409/Ser410) significantly enhanced the affinity for β-arrestin1, with a dissociation constant (Kd) of 35 nM.

2. Assembly and Structural Determination of the NTSR1-β-arrestin1-SBI-553 Complex

Using chemically synthesized hexa-phosphorylated NTSR1, the research team assembled the NTSR1-β-arrestin1-SBI-553 complex and resolved its high-resolution structure (2.65–2.88 Å) using cryo-EM. The structure revealed that the binding of SBI-553 induced significant remodeling of the intracellular region of NTSR1, particularly in the intracellular loop 3 (ICL3) and transmembrane helix 6 (TM6) regions. This remodeling allowed ICL3 to insert into the cavity of the central crest of β-arrestin1, forming a unique “loop engagement” configuration, which is markedly different from the traditional “core engagement” configuration.

3. Structural Comparison and Functional Analysis

By comparing the structures of the SBI-553-bound and unbound NTSR1-β-arrestin1 complexes, the study found that the binding of SBI-553 significantly altered the intracellular region conformation of NTSR1, especially at the cytoplasmic ends of TM5 and TM6. Additionally, SBI-553 formed extensive hydrophobic interactions with the intracellular region of NTSR1, further stabilizing the complex structure. Functional experiments demonstrated that SBI-553 significantly enhanced the recruitment of β-arrestin1 while inhibiting Gαq protein signaling.

4. Interaction Mechanism Between β-arrestin1 and NTSR1

The study detailed the interaction interface between NTSR1 and β-arrestin1 in the “loop engagement” configuration. ICL3 formed extensive hydrogen bonds and salt bridges with multiple loops in the central crest of β-arrestin1, while ICL1 formed hydrogen bonds with the lariat loop of β-arrestin1. These interactions collectively stabilized the complex structure and promoted the recruitment of β-arrestin1.

5. Binding of the Phosphorylated C-Terminus to β-arrestin1

The study also observed that the phosphorylated C-terminus of NTSR1 bound to the N-lobe of β-arrestin1, forming charge-complementary interactions. The phosphorylated Thr407, Ser409, and Ser410 formed stable hydrogen bonds and salt bridges with multiple positively charged residues in β-arrestin1, further enhancing the stability of the complex.

Research Conclusions and Significance

This study is the first to resolve the structure of a GPCR-β-arrestin complex in the BAM-bound state, revealing how SBI-553 selectively enhances the recruitment of β-arrestin1 by inducing the “loop engagement” configuration of NTSR1. This discovery fills a critical knowledge gap in the regulation of GPCR-β-arrestin interactions by BAMs and provides an important structural basis for the development of safer and more effective GPCR-targeted drugs.

Research Highlights

  1. High-Resolution Structure Determination: This study resolved the high-resolution cryo-EM structure of the NTSR1-β-arrestin1-SBI-553 complex (2.65 Å), which is the highest-resolution structure of a GPCR-β-arrestin complex to date.
  2. Unique “Loop Engagement” Configuration: The study discovered a novel binding mode between β-arrestin1 and GPCR, termed the “loop engagement” configuration, which has never been observed in previous GPCR-β-arrestin complexes.
  3. Chemical Protein Synthesis Strategy: The research team developed a chemical protein synthesis strategy to successfully generate NTSR1 with hexa-phosphorylation at its C-terminus, providing an important tool for studying the effects of phosphorylation on GPCR function.
  4. Biased Signaling Mechanism: The study revealed how SBI-553 selectively enhances the recruitment of β-arrestin1 by modulating the conformation of NTSR1, offering new insights for the development of biased GPCR drugs.

Other Valuable Information

This study also provided detailed molecular mechanisms of SBI-553 binding to NTSR1, including the hydrophobic interactions between SBI-553 and the intracellular region of NTSR1, as well as how SBI-553 hinders G protein binding by altering the conformation of NTSR1. These findings offer new perspectives for understanding the molecular mechanisms of BAMs and provide important references for further optimization of BAM design.

Through this study, scientists have not only filled a critical knowledge gap in GPCR-β-arrestin interactions but also provided an important structural basis for the development of safer and more effective GPCR-targeted drugs. This research achievement is expected to advance the field of GPCR drug development and offer new strategies for the treatment of various diseases.