Cell research Shanghai Drug revealed the structural basis of frogetin family receptors to recognize itch polypeptides

Throughout the long evolutionary process of humans, itching was part of the scratching reflex and helped avoid potential external 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 have 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 itching is still unclear, which greatly hinders the development
of itching-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 itching, by a variety of membrane proteins involved, of 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 are transmitted to the brain center? Previous studies have found that the histamine-type and non-histamine skin itching signals caused by external stimuli are transmitted through the dorsal root ganglion, and the cell body at the end of the ganglion releases NMB and GRP polypeptide ligands, respectively, which activate the corresponding neuromodulatory peptide receptors NMBR and gastrin-releasing peptide receptors GRPR, respectively, and after NMBR and GRPR are activated, the signals are processed at the dorsal corner of the polio of the spinal cord and continue to be transmitted to the brain, resulting in itching sensations in the brain, making people or animals scratch unconsciously to get rid of itching (Figure A).

。 Both NMBR and GRPR are at the heart of pruritus transmission and play a key role in pruritus biology, so they are gaining traction as potentially potent targets for antipruritus interventions
.
In order to understand the molecular mechanism of NMBR and GRPR mediating 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 (Fig.
b-g),
respectively.

In the structure of NMBR and GRPR, the researchers found that the two itchy polypeptide ligands, NMB and GRP, both exhibit dumbbell-shaped surface features and bind to the positive binding pocket of the receptor in a way that the C-end of the peptide faces the inside of the girdleetin receptor (Figure d, g).

。 The dumbbell-like tails at both ends of the itchy polypeptide ligands NMB and GRP mediate most 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 P3.
29, Q3.
32 and P45.
52 of NMBR and GRPR are critical
for ligand recognition.
It has been reported that NMBR and GRPR are affected by the activation of two different endogenous ligands, NMB and GRP, respectively, by 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 the frogetin receptors NMBR and GRPR to identify their endogenous ligands, respectively, and elaborated the specific mechanism of activation differences caused by subtle differences in the structure of NMBR and GRPR, including the conformational differences on ECL2 of NMBR and GRPR and the hydrophobic environment differences
at the bottom of the ligand structure pocket 。 Similar to most peptide receptors, the activation of frogetin receptors NMBR and GRPR is also affected by the level of C-terminal amidation of its endogenous polypeptide ligands, and the researchers detected through functional experiments that the activation ability of the C-terminal amidated forms of NMB and GRP to their respective receptors is more than 100 times that of non-amidated polypeptides, and by contrasting the interaction characteristics of amidated NMB and non-amidated GRP binding to the C-terminal residues of ligands in their receptor structures, respectively.
The researchers found that the amidation group can form additional polar interactions
.
In conclusion, these structural and functional studies on NMBR and GRPR elaborate the recognition characteristics and selective structural basis of NMB and GRP, two pruritic polypeptide ligands, which are helpful for the development
of anti-pruritus and other diseases based on NMBR and GRPR structures.

Li Changyao, a 2019 master student in Xu Huaqiang's research group of Shanghai Institute of Materia Medica, Dr.
Xu Youwei, associate researcher, and Liu Heng, a postdoctoral fellow, are the co-first authors
of the paper.
Yin Wanchao, researcher of Shanghai Institute of Materia Medica/Zhongshan Institute of Drug Innovation of Zhongke, and Xu Huaqiang, researcher of Shanghai Institute of Materia Medica, are the co-corresponding authors, and Shanghai Institute of Materia Medica, is the first to complete
.
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.

Fremotin receptors NMBR and GRPR with cryo-EM structures
of NMB and GRP complexes, respectively.
a.
NMBR and GRPR mediate itch signaling; b-g.
Cryo-EM structure of NMBR and GRPR with NMB and GRP complexes, respectively; h-k.
Comparison
of recognition patterns of NMB and GRP in NMBR and GRPR ligand binding pockets, respectively.

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(Contributing department: Xu Huaqiang research group; Contributor: Lee Chang-yiu)