Using cryo-electron microscopy, researchers of Xi'an Jiaotong University (XJTU) have made significant progress in the general mechanism of Adhesion G Protein-coupled Receptor or aGPCR.

This research explained in detail the molecular mechanism of aGPCR self-activation, and proposed a finger model of aGPCR activation based on the observed Stachel motif activation mechanism. 

On the basis of this model, a general method for the design of aGPCR polypeptide agonists/antagonists has been developed, which has important reference value and guiding significance for the development of molecule drugs targeting aGPCR.

In this research, the polarity of Fss03 was modified, and a general modification method of aGPCR family antagonistic short peptide ligands was developed, which will greatly promote the development of aGPCR family polypeptide antagonists.

On April 13, the research results were published in Nature under the title of Tethered peptide activation mechanism of the adhesion GPCR ADGRG2 and ADGRG4. (Link to the paper: https://www.nature.com/articles/s41586-022-04590-8)

Gou Lu, a doctoral student of Professor Zhang Lei of XJTU's State Key Laboratory for Strength and Vibration of Mechanical Structures, is the co-first author of the paper. Professor Zhang, Professor Sun Jinpeng of the Second Hospital of Shandong University, Professor Yu Xiao of the School of Basic Medical Science, Shandong University, and Professor Kong Liangliang of the National Center for Protein Science are the co-corresponding authors.

Compared with other members of the GPCR family, aGPCR has remarkable structural features, including a complex multi-domain N-terminal extracellular region and GPCR autoproteolysis-inducing, GAIN domain that can undergo autohydrolysis, which contains a conserved GPCR proteolysis site, known as GPS.

The vast majority of aGPCR members can spontaneously undergo autohydrolysis at the GPS position to generate α and β sub-units, of which the α sub-unit is an extracellular domain (NTF), and the β sub-unit is a region containing seven transmembrane helices (CTF). The hydrolyzed NTF and CTF remain non-covalently linked on the cell surface.

After autohydrolysis of aGPCR, the most N-terminal region of its β sub-unit is called the Stachel sequence. Studies have shown that aGPCR can sense a variety of external signal stimuli and have distinct activation modes, such as being activated by mechanical forces such as intracellular fluid flow or vibration, and activated by soluble small molecules or protein ligands.

Activation patterns of aGPCRs associated with Stachel sequences

In addition to the direct activation by small molecule ligands, it is currently believed that the activation of aGPCR and Stachel is mainly divided into three modes.

In the first mode, binding of a soluble ligand (small molecule or extracellular protein) induces a conformational change in the extracellular domain, promoting the interaction between the Stachel sequence and the 7-transmembrane region (7TM) of aGPCR.

In the second mode, binding or mechanical action of matrix proteins may lead to the separation of α and β sub-units following autohydrolysis of GPCR autohydrolysis sites (GPS, HLT), which exposes the Stachel sequence. Stachel binds to 7-transmembrane (7TM) bundles that ultimately activate downstream signaling in a Stachel-independent manner.

Interaction mode of Stachel sequence with ADGRG2

a. Structural model of the ADGRG2-β-Gs complex activated by the Stachel sequence.

b. The Stachel sequence binds to the pocket formed by the seven transmembrane helices of ADGRG2 in a "U" configuration, and is divided into a polar hydrophilic upper half and a hydrophobic lower half along the direction of the bottom opening.

c. The five hydrophobic amino acid fingers and positions of the hydrophobic part of the Stachel sequence interact directly with the five hydrophobic pockets of the receptor.

The activation of adhesion-like GPCRs caused by Stachel sequences has always been the core content of adhesion-like GPCR signals and functions. Although it has been reported that Stachel sequences are closely related to the function of adhesion-like receptors, but how they interact with receptors and the general mechanism that regulates the activation state of receptors remain unclear.

Based on this, the joint team analyzed the complex structure of ADGRG2-β-Gs and ADGRG4-β-Gs activated by Stachel sequence by cryo-electron microscopy, revealing the direct mechanism of Stachel sequence and aGPCR.

The results show that the Stachel sequence of ADGRG2 and ADGRG4 was bound in the pocket formed by the seven transmembrane helices of the receptor in a "U" shape, and are divided along the midline of the "U" divide into a solvent-exposed hydrophilic part (upper rim) and a hydrophobic moiety (lower rim) that intercalates and forms a strong hydrophobic interaction with the receptor 7TM.

The five hydrophobic amino acids Fss03, I/Vss05, Lss06, L/Mss07 and Lss09 in the hydrophobic part of the Stachel sequence are in the form of fingers and are in contact with the five hydrophobic pockets of the receptor. Combined with biochemical experiments, it is indicated that this hydrophobic interaction mode is the Stachel sequence required for activation of ADGRG2-β and ADGRG4-β.

Further sequence comparison analysis found that there is a highly conserved F/Y/LXφφφXφ hydrophobic structural motif on the Stachel sequence of the aGPCR family (X represents any amino acid, and φ represents a hydrophobic amino acid).

Structural modeling analysis revealed that this hydrophobic motif forms strong hydrophobic interactions with receptors in other aGPCRs in a finger-like mode of action similar to ADGRG2-β and ADGRG4-β. Biochemical experiments also demonstrated that amino acid mutations that affect hydrophobic interactions can significantly reduce the activation ability of aGPCRs.

The study explained in detail the general mechanism of Stachel sequence activation of aGPCRs, and found that five hydrophobic amino acids were distributed in a finger-like manner, which played a major role in the Stachel sequence-mediated activation process, and proposed a "finger" model of aGPCR activation.

General peptide antagonist engineering strategy for aGPCRs development

At present, there are few reports on the antagonists of the aGPCR family, which restricts the research on the function of the aGPCR family and the development of related targeted drugs.

Using the newly developed agonist VPM-IP15, a series of mutations were made in the three "finger" residues near W6.53. In the biochemical verification, it was found that the negative electrical modification of Fss03 in IP15 by replacing D/E could eliminate the Gs of ADGRG2 While maintaining high affinity for the receptor, these two derived peptides can significantly inhibit the activation activity of ADGRG2, and also inhibit the ADGRG2-mediated CFTR calcium flux response.

This strategy of obtaining polypeptide antagonists by negative electric engineering of polypeptide agonists is like putting a ring on the thumb of an activated finger model, which decisively changes the function of adhesion receptor-like polypeptides. This method is also applicable to ADGRG4, ADGRG5 and ADGRG6.