MRGPRX2 is a certain type of GPCR that is located in the cellular membranes of mast cells.

Figure 1. MRGPRX2 as it sits within the cellular membrane. Phospholipid bilayer is represented by grey dots, with labeled cellular locations

Background

GCPR’s or G-Protein Coupled Receptors are a large family of protein receptors that promote cellular signaling and signal transduction ^[1]. GPCRs transmit extracellular signals to intracellular messages. Many essential pathways utilize GPCRs, including human vision by the GPCR Rhodopsin, and the adrenaline fight-or-flight response by the β2-adrenoceptor GPCR. Understanding GPCR’s and how they produce their desired intracellular signal is essential to studying essential cellular pathways, especially in their diseased states. GCPRs are common drug targets, with 475 drugs acting on over 100 GPCRs. An additional 300 drugs are in clinical trial stages, and 20% of those drugs are targeting novel GPCRs ^[2]. Because of the clinical relevance of GPCRs, new structures provide new avenues for drug development to both treat disease or modulate the harmful side effects.

Some cells in the human body that express the MRGPRX2 receptor include mast cells in the skin, intestines, and trachea ^[3][4]. Mast cells are immune cells responsible for triggering inflammatory responses and are densely packed with granules containing inflammatory chemicals, such as histamine^[4] and heparin. Mast cells can be activated by either antibodies from the immune response or upon ligands binding to MRGPRX2 receptors on their surface^[5]. Upon activation, mast cells will release histamine-containing granules which can trigger a larger inflammatory response ^[4][5]. These responses induce common allergic reaction or [1] symptoms, such as cutaneous itching sensations or airway constriction^[6][7][5].

Ligands that bind to MRGPRX2 in the natural environment to produce an allergic response include exogenous molecules such as contents of insect venom, molecules like Compound 48/80 (C48/80), or other polycationic molecules^[4][3]. They can also respond to endogenous signaling molecules involved in inflammation pathways such as cytokines, anaphylatoxins, or neuropeptides^[3]. Many pseudo-allergic drug reactions have been tied to overactivity of MRGPRX2 receptors on mast cells^[5], so research into receptor-ligand interactions of the MRGPRX2 receptor has the potential to mediate many adverse itching and allergic reaction side effects seen in drugs today^[5].

GPCRs are categorized into 6 different classes based on shared sequences and functions. MRGPRX2 is categorized into the Class A receptor family. However, itch receptors like MGPRX2 have unique structural features from most class A receptors ^[6][7]. These unique structural features, as seen in Class A Family Differences, cause conformational changes throughout the protein that impact what ligands bind to the receptor^[7].

GPCR Structure

The MRGPRX2 receptor structure was determined by cryo-electron microscopy (cryo-EM) ^{[6] [7]}. Despite MRGPRX2’s novel characteristics, these structures still confirmed MRGPRX2 classification as an A-family GPCR. MRGPRX2 therefore shares the same general structural domains of all GPCR’s. This includes a that interacts with a heterotrimeric domain, consisting of , , and subunits. The G-protein serves as the intracellular relay for ligand binding to the receptor. In preparing the protein sample, MRGPRX2 was prepared with an in order to stabilize the transmembrane domain for proper imaging. For simplicity and to focus on the MRGPRX2 receptor, the antibody has been removed in structural scenes.

Transmembrane Domain

The transmembrane domain spans the cell membrane (Figure 1) and it consists of and (three extracellular loops, and three intracellular loops). The transmembrane helices are numbered 1-7 and contain special conserved motifs that are shared across other A family receptors. These motifs are expanded upon later, as they heavily contribute to the structure and therefore function of the transmembrane domain as a whole. The extracellular region of the 7 transmembrane domain forms a single binding pocket with . Sub-pocket 1 is negatively charged due to negatively charged residues (Asp-184 and Glu-164), while sub-pocket 2 contains hydrophobic amino acids which contribute to hydrophobic interactions between the ligand and protein. The intracellular region (Figure 1) is what connects the transmembrane helices with the G-protein.

G-Protein

GTP-binding proteins, also known as G-proteins, are heterotrimeric complexes consisting of , , and subunits that interact with the intracellular transmembrane region at an ( Figure 2b). G-proteins are responsible for transmitting extracellular signals into the cell upon activation. Activation leads to a substitution of GDP with GTP within the alpha subunit, causing the alpha subunit to disassociate from the beta and gamma subunits to initiate an intracellular signaling cascade. There are different families of G-alpha subunits, Gαi, Gαs, Gα12/13, and Gαq ^[8]. MRGPRX2 binds to both Gαi and Gαq subunits with nearly identical structures despite slightly different amino acids present ( Figure 2a) ^{[6] [7]}. Throughout this page, MGPRX2 is always shown with Gq. The major difference between the Gq and Gi bound structures comes from one amino acid difference (valine on Gq versus phenylalanine on Gi) that pushes the Gi subunit 2Å away from the arginine residue on helix 6 of the transmembrane protein.

Figure 2a. Overlay of MGPRX2-Gq (red-dark blue) and MGPRX2-Gi (cyan-yellow). Figure 2b. Important residues involved in the interface between MGPRX2 and Gq/ Gi subunits. Arrow pointing to the major difference between the interfaces, which comes from the final C-terminus residue on the G-alpha subunit. In Gq, there is a valine while in Gi, there is a phenylalanine. This pushes the Gi subunit 2Å away from the arginine residue on helix 6 of the transmembrane protein. All other interactions are nearly identical.

Class A Family Differences

The MRGPRX2 receptor shows surprising differences between it and all other previously characterized class A GPCRs including many conserved class A structural motifs which are absent on MRGPRX2. These structural motif differences contribute to a ligand binding site closer to the membrane surface for MRGPRX2 rather than a ligand binding site deep within the helices (Figure 3). To demonstrate this difference and other structural differences, an of MRGPRX2 and 5-HT2AR, another class A GPCR with more conserved structural motifs is provided.

Figure 3. Comparison of ligand Cortistatin-14 binding in MRGPRX2 (left) and binding in 5HT2AR (right)

Toggle Switch

Key toggle switch residues in the ligand binding pocket can act as molecular switches to turn the GPCR “on” or “off". Toggle switches initiate the transmission of the molecular signal through the 7 TMD helices to the intracellular G protein. Trp-336 is the "iconic" toggle switch in class A GPCR’s ^[9], and is a part of another motif, known as the CWxP motif. However in MRGPRX2, this tryptophan has been replaced with a ^{[6] [7]}. This shift leads to a significant modification to the receptor structure. By replacing the large tryptophan residue with a small glycine, the membrane helices, especially helix 7 on which the toggle switch is found, pack more tightly. The ligands that interact with MRGPRX2 are able to bind much of the receptor, as opposed to deeper within the helices (Figure 3). This shallower binding pocket expands the types of ligands that are able to interact with X2 and therefore what types of molecules can activate the Human Itch GPCR. More details about what kinds of ligands bind to this receptor are discussed later.

Sodium Site

The allosteric sodium site in class A GPCRs has been characterized as important in inactive state GPCR stabilization ^[10]. Katritch et al ^[10] describe that class A GPCRs lacking conserved D2.50 and other polar residues within the sodium pocket are typically inactive. The MRGPRX2 consists of conserved D2.50, or ASP-75, and GLY-116 compared to the previously conserved polar residues in this binding pocket such as S3.39. Other class A GPCRs demonstrate a larger sodium binding pocket with a higher negative character allowing for a suitable environment for sodium ions to bind. In MRGPRX2, this sodium binding pocket lacks the same amount of with the shift to a glycine residue rather than serine. However, evidence suggests that sodium is still able to bind in X2's sodium binding site even with fewer conserved residues.

PIF/LLF Motif

Figure 3. Conserved PIF motif in 5HT2AR (teal) compared to the LLF motif found in MRGPRX2 (red). Transmembrane helices and residues are numbered and labeled to show how this structural change shifts the orientation of the helices.

Another motif found in most, but not all, A family GPCR’s is the PIF motif. The PIF residues () are found on transmembrane helices 5, 3, and 6, respectively. In MRGPRX2, the PIF motif is changed to LLF residues. Figure 3 shows the conserved PIF motif on 5HT2AR, to the LLF motif on MRGPRX2. This change to LLF shifts helix 6 towards helix 3, and contributes to the tighter packing of helices ^{[6] [7]} and therefore a more surface-level ligand binding site.

DRY/ ERC Motif

Figure 4. ERC Motif

The E/DRY motif in most class A GPCRs is responsible for forming salt bridges with surrounding residues and TM6^[11]. These salt bridges maintain the inactive conformation of the receptor until ligand binding breaks the ionic "lock" from these interactions. MRGPRX2 has an ERC motif rather than the typically conserved E/DRY Motif. The amino acid residue shift from TYR-174 to CYS-128 allows compaction of the helices in MRGPRX2 where the standard TYR physically pushes the TMD helices apart(Figure 4). The conserved residues E and R still form salt bridges with nearby residues. This and the closer packing of the helices contribute to a less significant TMD conformational change upon ligand binding (Figure 10).

Disulfide Bonds

Figure 5. Overlay of the 5HT2AR and MRGPRX2 TMP for comparison of disulfide bond location.

In a large majority of class A GPCRs, there is a conserved disulfide bond between extracellular loop 2 (ECL2) and transmembrane helix 3 (TM3). This bond has been proposed to have a role in structural stability, expression, and function of GPCRs^[12]. The MRGPRX2 disulfide bond is between on TM helices 5 and 4, respectively. For example, the shows this disulfide bond between the ECL2 and TM3. Although this bond is in a different location than other class A GPCRs, there is evidence to suggest its location is essential for the signaling of the X2 receptor as the ECL2 instead located at the top of TM4 and TM5 allowing for the large, extracellular binding pocket observed in X2^[6].

Further Information

The MRGPRX2 GPCR also contains semi-conserved or fully-conserved motifs that are seen in other class A GPCRs.

NPxxY Motif

The residues in the are pivotal for receptor activation in all Class A GPCRs. This motif is conserved in the MRGPRX2 receptor with residues VAL-231, ASP-75, ASN-275, and TYR-279.

CWxP Motif

The is almost fully conserved except for TRP-236, or the toggle switch, which is replaced with GLY-236. CYS-235, LEU-237, and PRO-238 are all conserved. CYS6.47 may play a fundamental role in GPCR activity by participating in the rearrangement of TM6 and TM7 upon receptor activation^[13]. This residue's role is supported by its conservation in MRGPRX2 as it is a functional GPCR.

Ligands

MRGPRX2 binds a wide range of small molecule and peptide ligands. These ligands have positively charged regions which can interact with the negative binding pocket, sub-pocket 1. Some of these larger ligands also have a hydrophobic region which can interact with sub-pocket 2. MRGPRX2 has been known to interact with PAMP-12, Cortistatin-14, C48/80, and Zinc-3573^[7],^[6].

Common MRGPRX2 ligands with positive regions in blue, negative regions in red, and hydrophobic regions in green

Ligands' charge or hydrophilicity are shown to demonstrate where they may interact with sub-pockets 1 and 2. , specifically, interacts with sub-pocket 1 through LYS-3 and sub-pocket 2 through LYS-8.

Function

GPCRs undergo a conformational change in their 7TMD region upon ligand binding. This signal is then transduced to the G-protein allowing for downstream responses due to between the alpha subunit of the G-protein and the transmembrane protein which activates g-protein by GTP exchange.

Before Activation

The culmination of different motifs observed in MRGPRX2 compared to other class A GPCRs leads to external membrane ligand binding. The MRGPRX2 GPCR undergoes a much smaller conformational change upon ligand binding compared to other Class A GPCRs due to surface level binding versus deep helix binding (Figure 10).

Figure 10. Overlay of unbound (transparent) and bound (opaque) transmembrane proteins of both MRGRPX2 (left) and 5-HT2AR (right).

After Activation

After ligand binding and transmembrane protein activation, this signal is transmitted to the alpha subunit of the G-protein which undergoes chemical and conformational changes. The alpha subunit is initially bound with GDP which is then physically replaced by GTP leading to conformational changes. These changes can be seen in this video of a G-protein alpha subunit activation derived from common rats.

Clinical Relevance

Figure 11.Schematic representation of cellular response

Some cells in the human body that express the MRGPRX2 receptor include mast cells in the skin, intestines, and trachea ^[3]. Mast cells are involved in allergic responses by their release of histamine or other inflammatory chemicals in the body. ^[5] These responses induce common allergic reaction and anaphylaxis symptoms, such as cutaneous itching sensations or airway constriction ^[6][7][5]. Mast cells can be activated by either antibodies in the human immune response or upon ligands binding to their MRGPRX2 receptors ^[5]. Ligands that bind to MRGPRX2 in the natural environment to produce an allergic response include some contents of insect venom, molecules like C48/80 or other polycationic molecules. They can also respond to endogenous signaling molecules involved in inflammation pathways and immune responses such as cytokines, anaphylatoxins, or neuropeptides ^[3]. Binding to MRGPRX2 triggers an intracellular signaling pathway that eventually leads to mast cells releasing their contents containing histamine, cytokines, or other inflammatory molecules that may go on to trigger the itching sensation ^{[6] [7]}.

Many medications commonly list chronic skin itching or inflammatory responses as side effects, such as nateglinide, an anti-diabetic drug ^[6]. Upon analysis of some medications that list itching as side effects such as nateglinide, they structurally share many similarities to known MRGPRX2 ligands, and could therefore initiate an unwanted itch response in patients. Other drugs such as morphine or codeine also share structural features to known MRGPRX2 ligands, which can contribute to itching side effects seen in users of these drugs ^[6].

Because of how some drugs initiate an unwanted itch response,

MRGPRX2 has been identified as a potential target for drugs either involved in the inflammatory response or to mediate drugs from causing an unwanted reaction such as Atracurium, Rocuronium, Ciprofloxacin, and Levofloxacin^[14]. These drugs have similarly charged regions which may interact with sub-pockets 1 and/or 2. By binding these target drugs, MRGPRX2 can elicit or prevent a response to aid with itch-related side effects.

3D Structures

7s8l, MRGPRX2 Gq
7s8m, MRGPRX2 Gi
6wha, 5HT2AR