Cell research reveals the structural basis of frogus family receptor recognition of itch polypeptides

On November 3, 2022, the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, published the research results
entitled "Molecular recognition of itch-associated neuropeptides by bombesin receptors" online on Cell Research 。 This achievement is the first to report that the frogeetin family receptor Neuromedin B receptor (NMBR) and Gastrin-releasing peptide receptor (GRPR) bind endogenous polypeptide ligands - neuromedin B (NMB) and gastrin-releasing peptide (GRP), respectively.
and the near-atomic resolution structure of the Gq protein signaling complex, revealing the unique ligand binding characteristics, receptor selective recognition, and molecular mechanisms
mediating itch signaling for members of this family.
Throughout human evolution, itching is part of the scratching reflex that helps avoid potential extraneous damage; However, persistent itching or itching can lead to skin infections, mental anxiety, or sleep disturbances
due to repeated scratching.

As a skin disease, pruritus is mainly treated
clinically with certain antihistamines, immunomodulator drugs, and drugs that target the nervous system.
However, considering that antihistamines have a central inhibitory effect, patients who have been engaged in dangerous work such as driving and working at height for a long time are generally prohibited from using such drugs
.
If hormonal immunomodulator drugs are used for a long time, the body will produce many adverse reactions, such as dry skin, atrophy, inflammation, and weight gain
.
Chronic itching, on the other hand, causes uncontrolled scratching and can cause serious infections
.

Therefore, it is particularly important
to develop new drugs that effectively treat itching.
The biological mechanism behind pruritus is still unclear, which greatly hinders the development
of pruritus-related drugs.

From itch perception to signal transmission, central processing and scratching feedback, it is a complex multi-step, multi-factor regulation physiological process, the first step is the perception and recognition of itch-causing substances, involving a variety of membrane proteins, among which GPCR members MRGPRX2 and MRGPRX4 mediate the production
of a variety of itch signals.
However, after the itch-causing substance is perceived, how does the itch signal generated transmit to the brain center?

Previous studies have found that histamine and non-histamine skin itching signals caused by external stimulation are conducted through the dorsal root ganglion, and the soma at the end of the ganglion releases NMB and GRP polypeptide ligands, respectively, which activate the corresponding neuromodulatory peptide receptor NMBR and gastrin-releasing peptide receptor GRPR, respectively, while NMBR and GRPR are activated, the signal is processed at the dorsal corner of the polio gray and continues to be transmitted to the brain, resulting in itching in the brain, making people or animals scratch unconsciously to get rid of itching (Figure
。

Both NMBR and GRPR are at the heart of pruritus transmission and play a key role in pruritus biology, and as such, they are gaining traction and are considered to be potentially powerful targets for antipruritus interventions
.
In order to understand the molecular mechanism by which NMBR and GRPR mediate itch signaling to the central nervous system, the research team used single-particle cryo-EM technology to analyze the cryo-EM structures of NMMB and GRPR and the corresponding endogenous ligands NMB and GRP and downstream G protein complexes, respectively, with resolutions of 3.
15 angstroms and 3.
30 angstroms (Figure b-g),
respectively.
Cryo-EM structure
of froggetin receptor NMBR and GRPR complexes with NMB and GRP, respectively.
NMBR and GRPR mediate itch signaling; b-g.
cryo-EM structures of NMBR and GRPR complexes with NMB and GRP, respectively; h-k.
Comparison
of recognition patterns of NMB and GRP in NMBR and GRPR ligand-binding pockets, respectively.

In the structure of NMBR and GRPR, the researchers found that both the itch polypeptide ligands, NMB and GRP, exhibit dumbbell-shaped surface features and bind to the positive binding pocket of the receptor in such a way that the C-terminus of the peptide is facing the inside of the pocket of the frogestin receptor (Figure d, g).

。 The dumbbell-shaped tails of the itch-sensing peptide ligands NMB and GRP mediate most of the receptor interactions, among which the conserved HF(L)M motif at the C-terminal of the polypeptide is particularly important for receptor activation; The three conserved amino acids of NMBR and GRPR, P3.
29, Q3.
32 and P45.
52, are critical
for ligand recognition.

It has been reported that NMBR and GRPR are activated by two different endogenous ligands, NMB and GRP, respectively, with a difference of more than
600 times.
Although the ligand binding methods in the two receptor structures are similar and the differences between the structures are small, combined with literature reports and functional experimental verification, the researchers analyzed the interaction details of froggetin receptor NMBR and GRPR to identify their endogenous ligands, respectively, and elucidated the specific mechanism of activation differences caused by subtle differences in the structure of NMBR and GRPR, including conformational differences on ECL2 of NMBR and GRPR and differences in hydrophobic environment at the bottom of ligand structure
pockets 。 Similar to most peptide receptors, the activation of froggetin receptors NMBR and GRPR is also affected by the level of C-terminal amidation of its endogenous polypeptide ligand, and the researchers detected through functional experiments that the C-terminal amidation forms of NMB and GRP have more than 100 times the activation capacity of their respective receptors than unamidated polypeptides, and the interaction characteristics of ligand C-terminal residues in their receptor structures are combined by contrasting amidation NMB and unamidated GRP, respectively.
The researchers found that the amidophores can form additional polar interactions
.

In conclusion, these structural and functional studies of NMBR and GRPR elaborate the recognition characteristics and selective structural basis of the two itch polypeptide ligands NMB and GRP, which is helpful for the development
of anti-pruritus and other diseases based on the structure of NMBR and GRPR.

Changyao Li, a 2019 master's student in Xu Huaqiang's research group of Shanghai Institute of Materia Medica, Dr.
Youwei Xu, an associate researcher, and Heng Liu, a postdoctoral fellow, are the co-first authors
of the paper.
Researcher Yin Wanchao of Shanghai Institute of Materia Medica/Zhongshan Institute of Drug Innovation of Chinese Science and Technology and researcher Xu Huaqiang of Shanghai Institute of Materia Medica, are co-corresponding authors, and Shanghai Institute of
Materia Medica.
The work has been funded
by the Shanghai Municipal Science and Technology Major Project, the National Natural Science Foundation of China, the National Major Science and Technology Project, the National Key Basic Research Program, and the Guangdong High-level New R&D Institution/Guangdong High-level Innovation Research Institute.