From China: This study reveals the mechanism of self-activation of adhesion receptors and the structural basis of mechanical force perception

In the process of biological evolution, human beings have evolved a variety of sensory systems including hearing, vision, smell, touch, taste, pain, and itching in order to better adapt to their living environment. Since the sensory system is the basic response of human beings to perceive the outside world, and it is also the basis for the organism to communicate with the outside world and protect itself, the exploration of the sensory system has long been a research hotspot of scientists.
Xu Huaqiang’s research group from Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Sun Jinpeng’s research group from Shandong University have long been committed to the research of GPCR receptors related to sensation. Xu Huaqiang’s research group analyzed the complex structure of rod pigment and Arrestin, the complex structure of G protein and GRK, and the basis of visual perception. Sun Jinpeng’s research group published the research results of the auditory-related receptor VLGR1 and the itch receptor MGPRX2. Although some achievements have been made in the study of the relationship between adhesion-like GPCRs and mechanical force in recent years, the mechanism by which adhesion-like receptors sense mechanical force is still unclear.
On April 13, Xu Huaqiang’s team from Shanghai Institute of Materia Medica, Sun Jinpeng’s team from Shandong University and Ines Liebscher’s team from Rudolf Schonheimer Institute of Biochemistry in Germany jointly published the latest research titled Structural basis for the tethered peptide activation of GPCRs online in Nature As a result, the complex electron microscope structure of the adhesion receptors GPR133, GPR114 and downstream protein Gs that sense mechanical force was analyzed, and it was proved that the receptor activates the receptor through the Stachel sequence after sensing the mechanical force. ) recognition and the mechanism of receptor self-activation.
“Force” can determine the fate of cells. The perception of force is very important for living organisms. Mechanical force stimulation drives many physiological processes, including touch, pain, hearing and balance regulation. In the development of cell biology, people gradually realize that cells can sense the signals transmitted by mechanical external force and make various physiological changes. Mechanical external force is very important for the dynamics of cells and the normal growth and operation of the body. For example, the ear’s perception of wind, the lung’s perception of gas intrusion, and the skin cell’s perception of pressing force, these physiological processes are inseparable from the perception of mechanical force. Some of these specialized cells have evolved to become specialized “terminals” for sensing mechanical force, such as the tactile receptor Piezo family proteins capable of sensing mechanical force, and the auditory-related receptor TMC mediating auditory conduction through mechanical force in cochlear hair cells. These are common Cognitive ion channel receptors capable of sensing mechanical forces. As another large class of membrane receptors, GPCRs can also sense mechanical force, and most of their force sensing is carried out through the adhesion receptor family, such as GPR114, GPR133, GPR126, GPR56, etc., due to their unique structural and functional characteristics. Adhesion-like receptors (aGPCRs) have a huge extracellular end that contains many domains. The extracellular end is involved in the interaction between receptors and receptors and between receptors and the cell matrix. It is closely related to the perception of mechanical force. However, adhesion How receptor-like receptors are activated and signal transduction after sensing mechanical force is unclear.
Another significant structural feature of adhesion receptors is that they contain a GAIN (GPCR autoproteolysis-inducing) domain and a GPS (GPCR proteolytic site) autohydrolysis site. Most adhesion receptors GPS can undergo autohydrolysis, cutting the receptor. There are two fragments, NTF (N-terminal fragment) and CTF (C-terminal fragment), about a dozen amino acids at the N-terminal of the receptor CTF can act as an agonist to activate the receptor and recruit downstream G proteins, this partial sequence is called Stachel Serial or tethered agonist (TA, Tethered agonist). An important feature that distinguishes aGPCRs from other families of GPCRs is their ability to rely on Stachel sequences for self-activation. Previous studies have demonstrated that small molecule ligands can directly bind to the 7-transmembrane core of adhesion-like receptors and activate the receptors. However, the mechanism of action of adhesion receptors in the self-activated state is still unclear. At least three activation modes have been reported for Stachel sequence-mediated aGPCR activation (Fig. 1): (1) After autohydrolysis of Stachel sequence at GPS, NTF dissociates from CTF, Stachel sequence is exposed, and then interacts with TMD; ( 2) The Stachel sequence has a pre-binding or intermittent interaction with TMD, which is stabilized by ligand stimulation or mechanical force; (3) The contact between the Stachel sequence and TMD is regulated by the internal conformational change of the GAIN domain. Each hypothesis has experimental support, but also contradicts each other. For example, the crystal structure of the GAIN domain shows that the Stachel sequence is hidden as a β-sheet that cannot interact with TMD, which conflicts with the high constitutive activity of some aGPCRs that can be activated by the Stachel sequence. It is therefore unclear how the Stachel sequence activates the receptor.
In response to the above two problems, the researchers used the adhesion receptors GPR133 and GPR114 as models for structural analysis to explore the Stachel sequence-mediated aGPCR activation mechanism. The GPR133 GPS site undergoes hydrolysis and NTF-CTF separation occurs at the plasma membrane. GPR114 does not undergo autohydrolysis and can sense mechanical force.
Stachel sequences can act as agonists to activate GPR133, GPR114 receptors and recruit downstream Gs proteins. Biochemical experiments confirmed that the background activity of GPR133/GPR114 after removing NTF was significantly higher than that of the full-length receptor and the CTF structure lacking the Stachel sequence. By stimulating the CTF fragment of the receptor Stachel sequence to remove the Stachel sequence in vitro, it was also proved that the Stachel sequence can act as an agonist to activate the receptor. In addition, the study found that mechanical force activates the receptor by regulating the binding of Stachel to the receptor. The researchers performed mechanical force detection on wild-type and GPR133 GPS site mutant (loss of self-cleaving function) receptors, and found that both can sense mechanical force. The study also found that mutating important sites on human NTF did not affect the perception effect of its mechanical force, which confirmed that the mechanical force acts by changing the vibrational conformation of NTF, exposing the Stachel sequence, and then binding to the receptor. thereby activating the receptor. Based on this result, the study mutated the interaction site with Stachel in the receptor binding pocket, and found that the effect of mechanical force was reduced to varying degrees. Therefore, it was concluded that mechanical force activates receptors by regulating the binding of Stachel to receptors. body (Figure 2).
Through the structural analysis of the complex cryo-electron microscope, the study found that the HIM structure of the Stachel sequence mediates its interaction with the receptor. In order to explore the relationship between the Stachel sequence and the receptor, the researchers analyzed the cryo-EM structures of the GPR133-CTF-Gs-Nb35 and GPR114-CTF-Gs-scFv16 complexes with a resolution of 3.1 angstroms and 3.3 angstroms, respectively (Fig. 3a). The overall TMD arrangement of GPR133-CTF and GPR114-CTF is quite different from the structure of the adhesion receptor GPR97 recently solved by the team, more similar to the secretin-like GPCR (ClassB1) structure, and there are two kink structures in TM6 and TM7. The Stachel sequences of both GPR133-CTF and GPR114-CTF were folded within an orthosteric binding site pocket surrounded by transmembrane helices and ECL2, and the Stachel sequences formed an α-helical structure inside the pocket (Fig. 3b). The conserved HIM (Fss-03xφφφxφss-09) composed of five hydrophobic amino acids in the Stachel sequence plays a central role in the interaction of the Stachel sequence with the receptor (Fig. 3c). The three amino acid side chains of Fss-03, φss-06, φss-07 face the inside of the ligand binding pocket, the side chain of φss-05 faces the outside of the pocket, and the last φss-09 forms a hydrophobic pocket with the amino acids on TM5 and ECL2 ( Figure 3d-i).

A conserved switch, W6.53, was found in both GPR133-CTF and GPR114-CTF receptors, which senses the binding of Stachel sequences and mediates receptor activation. Q/Y7.49 and the conserved N5.50 form a hydrogen bond network with the P6.47/V6.47 φφG6.50 motif, which is important for stabilizing the kink structures of TM6 and TM7. Below the kink structure, the ends of TM6 and TM7 slope outward to form a larger cytoplasmic cavity that facilitates coupling to G proteins. Unlike other GPCRs, GPR114 has an extended TM7 that inserts into the groove created between the Gas and Gβ1 subunits, and the extended helix may play an important role in its intracellular signaling.
In summary, the researchers first proved that the receptor senses mechanical force and then activates the receptor through the Stachel sequence through functional experiments. Cryo-electron microscopy structure analysis further identified a conserved HIM (Fss-03xφφφxφss-09) composed of five hydrophobic amino acids in Stachel. ) plays a central role in the interaction between Stachel sequences and receptors, elucidating the structural basis for the recognition of Stachel sequences by receptors, revealing the activation mechanism of adhesion receptors under the action of mechanical force relying on Stachel sequence activation, expanding the understanding of Stachel sequences The understanding of the mechanism of aGPCR-mediated coupling with G protein provides a basis for the design of exogenous short peptides with Stachel sequences.
The research work has been supported by the National Key Research and Development Program Fund, the National Science Fund for Distinguished Young Scholars, the National Science Fund for Outstanding Young Scholars, the National Natural Science Foundation of China Key Fund and the Shandong Provincial Outstanding Youth Fund.
Paper link


FIG. 2. Schematic diagram of mechanical force-sensing aGPCR activation mechanism & NBSP;

图3.GPR133 GPR114结构模型

Related Articles

Leave a Reply

Your email address will not be published. Required fields are marked *

Back to top button