Analysis of the Molecular Recognition and Signal Transduction Mechanism of PRRP and PRRPR

Research Background

Neuropeptides, as the most abundant signaling molecules in the nervous system, have over 100 identified types and play critical roles in processes like metabolism, pain sensation, and reproduction. Among them, RF-amide neuropeptides are characterized by their C-terminal Arg-Phe-NH₂ (RF-amide) motif, including prolactin-releasing peptide (PRRP), neuropeptide FF (NPFF), kisspeptin, etc. RF-amide neuropeptides regulate a wide range of physiological functions by binding to specific G protein-coupled receptors (GPCRs). However, despite the importance of PRRP and its receptor PRRPR in regulating stress, appetite, pain, and cardiovascular functions, the molecular mechanisms by which it binds and activates the receptor remain not fully understood.

PRRP is a highly conserved RF-amide neuropeptide identified as the endogenous ligand for PRRPR through reverse pharmacology. PRRP and PRRPR primarily signal through the Gq/11 pathway and may also activate the Gi/o pathway. However, current structural studies on PRRP and PRRPR are limited, hindering a deeper understanding of their functional mechanisms. Therefore, this study utilized cryo-electron microscopy (Cryo-EM) to reveal the structure of PRRP bound to PRRPR in complex with both Gq and Gi heterotrimers, providing a structural foundation for developing selective drugs.

Source of Research

This article was published in Cell Discovery by researchers from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Nanjing University of Chinese Medicine, and Shanghai Jiao Tong University, among others. The primary authors include Yang Li, Qingning Yuan, and others, with H. Eric Xu and Li-Hua Zhao serving as the corresponding authors. This study provides fundamental structural insights into the key interactions between PRRP and PRRPR, laying a foundation for further mechanistic research and drug development for neuropeptide-receptor systems.

Research Methods

1. Research Process

The researchers constructed and expressed the full-length wild-type receptor of PRRPR, introducing tags and small probes to stabilize the GPCR-G protein complex. Then, Cryo-EM was used to analyze the structure of PRRP20 (a 20-amino acid active isoform of PRRP) bound to PRRPR, in complex with Gq and Gi, with resolutions of 2.96 Å and 2.97 Å, respectively. Additionally, molecular dynamics (MD) simulations and functional experiments were conducted to analyze binding energy, flexibility, and activation characteristics of different complexes.

2. Key Experimental Techniques

• Complex Preparation: An insect cell expression system was used to co-express PRRPR and its ligand, and purification techniques such as affinity purification and size-exclusion chromatography were used to obtain high-purity complex samples.
• Cryo-EM Analysis: Samples were frozen in a liquid nitrogen environment, and Cryo-EM data were collected using a Titan Krios G4 electron microscope. The data were enhanced by deep learning and molecular modeling to reconstruct the molecular structures.
• Molecular Dynamics Simulation: PRRPR-Gq/Gi complex models were constructed, followed by 200 ns simulations to study the dynamic behavior of ligand binding and receptor activation.
• Functional Validation: BRET2 assays were used to analyze the Gq and Gi dissociation efficiency induced by PRRP20. Mutational experiments were also conducted to verify the roles of key amino acids in signal transduction.

Research Results

1. Key Structures of PRRP20 Binding to PRRPR

PRRP20 adopts an L-shaped conformation, with its C-terminal RF-amide motif inserted into the ligand-binding pocket of PRRPR. Structural analysis indicated that the C-terminal of PRRP20 forms multiple polar and hydrophobic interaction networks with PRRPR, involving key amino acids such as C113².⁵⁷, T117².⁶¹, Q141³.³², and H321⁷.³⁹. These interactions are crucial for the high affinity of PRRP20. Additionally, the conserved residues R19 and F20 underscore their universal importance in various RF-amide ligand receptors.

2. Activation Mechanism of PRRPR

Activation of PRRPR involves an outward movement of the transmembrane region TM6 and an inward movement of TM7. These conformational changes transmit signals to the intracellular regions, leading to G protein coupling. F20, through polar interaction with Q141³.³², triggers conformational changes in TM3, further activating PRRPR. The study also revealed a unique activation pathway where Y146³.³⁷ interacts with TM5 to facilitate TM6 movement, a mechanism distinct from other GPCRs.

3. Selective Coupling of PRRPR with Gq/Gi

Significant conformational differences were observed between the coupling of PRRPR with Gq and Gi, especially in the relative positions of TM6 and the α5 helix of the Gα subunit. PRRPR prefers coupling with Gq, which may be attributed to the unique polar network in the “wavy hook” region of the α5 helix. Mutation experiments further confirmed the importance of these interactions in signal selectivity.

Significance of the Study

This study, through high-resolution Cryo-EM structures, elucidated the molecular mechanism of PRRP binding to and signaling through PRRPR, providing a structural basis for the selective drug development targeting neuropeptide-receptor systems. These findings not only deepen the understanding of the RF-amide neuropeptide recognition mechanisms but also offer new perspectives for designing treatment strategies for conditions related to appetite regulation, stress, and pain. Moreover, the innovative methodologies employed in this study hold significant value for future GPCR research.

Highlights of the Study

1. Revealed molecular details of PRRP20 binding to PRRPR, providing a universal mechanism for RF-amide motif recognition.
2. Clarified the unique activation pathway of PRRPR and its differences from other GPCRs.
3. Provided a structural basis for the selective coupling of PRRPR with Gq/Gi, aiding in the development of selective drugs.

This research not only fills the gap in PRRPR structural studies but also provides a valuable resource for future GPCR mechanistic studies and drug design.