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Introduction

Function and Biological Role

Human Itch G-coupled protein receptors (GPCRs), or Mast cell-related GPCRs (MRGPRX), have been identified as pruritogenic receptors and are found in human sensory neurons, specifically in the connective tissue mast cells and the dorsal root ganglia in humans. Some MRGPRXs have even been found in the dorsal root ganglia of primates. They are a group of receptors that respond to a diverse number of agonists, antagonists, and inverse agonists. They are involved in host defense, pseudo-allergic reactions, non-histaminergic itch, periodontitis, neurogenic inflammation, and inflammatory pain. Non-histaminergic itch receptors are GPRCs that are commonly seen in dorsal root ganglia and their peripheral fibers, as well as in keratinocytes and immune cells located in the dermis. The novelty of non-histaminergic itch responses, in contrast to the histaminergic itch responses, is that histamine is not binding to the N-terminal domain of the receptor and activating the itch response. Instead, it was found that there is a distinct separation between the two pathways which is maintained in the spinal cord and ascending pathways to the brain.^[1]

G proteins and Signaling

G-proteins when paired with a receptor assist in signal transduction, which is the conversion of information collected by the receptors by a chemical process to induce a cellular response.^{[2] [3]} G-proteins are structurally trimeric proteins that are composed of three subunits: , , and . The α subunit acts as the main signal mediator and contains a binding site for GDP or GTP, which acts as a biological “switch” to regulate the transmission of a signal from the activated receptor. Specifically, when the α subunit is bound to GDP, the signal transmission is terminated and no cellular response is generated. However, when the alpha subunit is bound to GTP, the g-protein is activated which initiates dissociation of the β and γ subunits, as a dimer, from the α subunit. The α subunit can then move in the plane of the membrane from the receptor to bind to downstream effectors to continue signal transmission and ultimately produce a cellular response.^{[4] [5]}

G_q and G_i family alpha subunits

The G-protein actions induced can be classified based on the sequence homology of α subunit (G_α) present in the structure. The most well-known are referred to as G_i, G_s, and G_q. The G_s and G_q proteins are stimulatory, while the G_i protein is inhibitory. In addition, the g-proteins can be classified based on the signaling pathway that they regulate. For example, G_q proteins are seen in a signaling pathway that relies on phospholipase C enzymes, while G_s and G_i proteins are regulators of adenylate cyclase. In this case, we will be focusing solely on the structures of the G_q and G_i proteins and their interactions with mast-cell receptors.

G_αq and G_αi are proteins comprised of 359 amino acid residues, with varying sequences, that both contain a helical domain and a GTPase binding domain.^[6] The GTPase binding domain is responsible for the hydrolysis of GTP as well as the binding of the β and γ subunits which form the trimeric protein structure. The helical domain contains six alpha-helices which are responsible for the binding of the g-protein to the coupled receptor.

The conformations of the g-proteins are variant based on their association with a particular membrane receptor due to the interactions of the amino acids in the N-terminal of the α subunit and the C-terminal of the receptor.

Significance of Human Itch GPCR

The determination of the first structures of a ligand-activated G protein-coupled receptor was achieved by Robert J. Lefkowitz and Brian K. Kobilka which won them the 2012 Nobel Prize in Chemistry. They also successfully captured images of the first activated G protein-coupled receptor in complex with a G protein. Nobel Prizes for 3D Molecular Structure

Related Enzymes

Human Itch GPCRs are a specific class of receptor called MRGPRX2 of the family of receptors mentioned above. MRGPRX2 is a class A GPCR that specifically regulates mast cell degranulation and itch-related hypersensitivity reactions. In pharmaceuticals, it has been found to be a target of morphinan alkaloids, like morphine, codeine, and dextromethorphan. MRGPRX2 has also been found to couple effectively to nearly all G-protein families and subtypes through interactions with the alpha subunit with robust coupling to G_q and G_i family α subunits.

In addition, MRGPRX4 is another sub-group of the MRGPRX family which mediates cholestatic itch. Pharmaceutically it has been shown to be a target of nateglinide drugs. MRGPRX4 also couples to G_q and G_i family α subunits in similar ways to the MRGPRX2 receptor.

Structure

The MRGPRX2 receptor consists of 7 transmembrane helices, 3 intracellular loops, and 3 extracellular loops. The N-terminus, which is in the extracellular domain, is involved in ligand binding to the receptor. The intracellular domain consists of helix VII and a specific c-terminal sequence which binds the g-protein and promotes downstream signaling. The MRGPRX4 receptor consists of the same key structures and interacts with ligands and g-proteins in the same structural regions.

When comparing the structures of MRGPRX2 and MRGPRX4, they are very similar in their intracellular domains. However, they differ in their extracellular domains which highlight key differences in the functions of the two receptors. First, the extracellular tip of transmembrane helix 3 (TM3) is displaced 5.2 Å inward in the MRGPRX4 structure. This results in a more compact binding site. In addition, because of the displacement of TM3, extracellular loop 2 (ECL2) shifts into the orthosteric binding pocket in between TM3 and TM6 affecting the binding pocket of MRGPRX4, which will be discussed later.

Active Site

The active site of MRGPRX2 is comprised of two binding sub pockets. Sub pocket 1 is formed by TM3, TM6, and ECL2. This sub pocket is both small and deep which results in the binding of only a single amino acid residue, namely arginine or sometimes lysine. The binding is mediated by two key residues on the MRGPRX2 protein within the binding site, glutamic acid 164 (E164) and aspartic acid (D184). The strong charge interactions of these two residues create a highly negatively charged electrostatic interaction within this sub-pocket.

Sub pocket 2 is formed by TM1, TM2, TM6, and TM7. This binding sub pocket is much more shallow and allows for the binding of larger structures. The key residues involved are tryptophan 243 (W243) and phenylalanine 170 (F170) which allow for binding through hydrophobic interactions. The hydrophobicity of this binding pocket accounts for the large electrostatic difference observed between the two sub pockets.

In addition, a conserved disulfide bond between TM3 and ECL2 found in class A GPCRs is absent in MRGPRX2. Instead, a disulfide bond between cysteine 168 (C168) of TM4 and cysteine 180 (C180) of TM5 is observed which structurally flips ECL2 to the top of TM4 and TM5. This creates the wide ligand-binding surface described above and allows for the binding of a diverse number of ligands.

As described above the binding site of MRGPRX4 is more compact due to structural rearrangements of TM3. The location of TM3 in this protein shifts the conformation of ECL2 which blocks the key residues that formed sub pocket 1 of MRGPRX2. Specifically, W168 on ECL2 covers E164 and D184 superiorly which makes them solvent inaccessible. This results in a single binding pocket for MRGPRX4 formed by interactions with W168 and two arginine residues (R82 and R95) which forms an electrostatically positive binding pocket.

The differences in the active sites of the MRGPRX2 and MRGPRX4 receptors help to explain their differing functions in the body. Since MRGPRX2 is found in more diverse regions of the body, it has a binding site that can bind to ligands with more diverse structures due to the lack of specificity in sub pocket 2 and the different electrostatic environments observed in sub pocket 1 and 2. In contrast, MRPGRX4 is found largely in the digestive tract, and the positive nature of the binding pocket helps to increase affinity to the negatively charge bile acids that are present.

Substrates/Ligands

Some ligands that bind to MRGPRX2 and X4 are discussed below. There are many more ligands that can bind to these receptors, especially MRGPRX2 because of the electrostatic surface of its binding pocket.

(R)-zinc-3573

(R)-zinc-3573 ** is an agonist that binds to MRGPRX2 when it is associated with a G_i or G_q protein with no change in molecular interactions. This agonist is a small cationic molecule that forms largely ionic interactions with the negatively-charged sub-pocket 1 and has no interactions with sub-pocket 2. (R)-zinc-3573 forms hydrogen bonds and hydrophobic interactions with D184 and E164 of sub-pocket 1. In experimental research, this agonist was manufactured to further research the characteristics of mast cells including mast cell proliferation, receptor expression, mediator release and inhibition, and signaling. This agonist was created to stimulate LAD2 mast cell degranulation.

C48/80

C48/80 is a peptide agonist that can bind to MRGPRX2 when it is associated with a G_i or G_q protein. The binding interactions of C48/80 with G_i and G_q do not change, but their binding efficacies are different. The structure of the peptide itself consists of three phenethylamine groups that are arranged in a Y shape with a semicircular arrangement. Upon its binding, the D184 and E164 within sub-pocket 1 interact only with the central phenethylamine ring, forming hydrogen bonds and charge-charge interactions. The central phenethylamine ring is inserted into the binding pocket at a depth of 5.6 A. The binding of this ligand causes the separation of all of the ECLs from the receptor's N-terminus.

Cortistatin-14

Cortistatin-14 is an endogenous peptide agonist which interacts with MRGPRX2 the same way whether it is coupled to G_i or G_q proteins. This is a fairly large ligand and it binds near the ECLs in the shallow binding pocket of the receptor, which reduces its local resolution. Specifically, a basic lysine residue (L3) on Cortistatin-14 will bind in the negatively charged sub-pocket 1 and forms strong charge interactions with D184 and E164. This structure will bind to both binding pockets, so the remaining residues of Cortistatin-14 will extend over to sub-pocket 2 and bind through hydrophobic interactions.

Mechanism

As mentioned above, the ligands for this g-coupled receptor can vary, but they often interact with the binding pockets through charge interactions, hydrogen bonding, or hydrophobic interactions. When considering agonistic interactions, once bound to the receptor, the ligand triggers a conformational change in the C-terminus of the receptor. This conformational change results in the activation of the coupled G protein. This alpha subunit of this G protein will then exchange a GDP for GTP which will allow the α subunit to dissociate from the βγ dimer and bind to downstream target proteins to allow the signaling pathway to continue. However, when considering the activity of the G proteins, it is important to remember that G_q proteins are stimulatory and will drive forward the signaling pathway, while G_i proteins are inhibitory and will stop signaling pathways. G_q proteins are seen in signaling pathways that rely on phospholipase C enzymes, while G_i proteins regulate pathways that involve adenylate cyclase. GPCR pathways can be regulated by a variety of modulators. For example, there are many known agonists, inverse agonists, and antagonists that are capable of binding to the human itch GPCRs.

Relevance

Implications for Disease

significant mutations that have been found drug design for GPCR targeting

Clinical Relevance

Most often MRGPRs, specifically MRGPRX2, have been studied in response to anaphylactic reactions. In an article written by Porebski et al., they focused on IgE-mediated and MRGPRX2-mediated anaphylactic reactions. In which they looked at the characteristics of the two pathways and how mutations to the receptors affected the responses generated. They found that MRGPRX2-mediated responses occur more quickly that IgE-mediated responses, but the responses tended to be more transient. They also demonstrated that common commercial drugs, like icatibant and cetrorelix, activate mast cells through the MRGPRX2 pathway and that neuromuscular blocking agents also activated responses through MRGPRX2.

Porebski et al. also determined many mutations that affect the actions of MRGPRX2. For example, a single residue mutation in sub-pocket 1 (Glu164Arg) prevented interactions between the receptor and ligands like C48/80. In addition, single nucleotide polymorphisms (SNPs) have been linked to many variations of MRGPRX2 which predispose patients to hyperactivation of the receptors. Two of the most common SNPs are Asn62Thr which affects the cytoplasmic domain and Asn16His which affects the extracellular domain. Ultimately, there have only been 5 SNPs detected that can potentially affect the extracellular domains and binding pockets of the receptors: Asn16His, Pro6Thr, Gly165Glu, Asp252Tyr, and His259Tyr. These mutations can potentially protect patients from drug-induced mast cell degranulation and hypersensitivity reactions.

However, even within the MRGPRX2-mediated pathway reactions can still differ in frequency which leads to many theoretical possibilities which need further exploration. Notably, in the same patients, it has been shown that reactions can occur through both pathways and that both mechanisms may be responsible for cross-reactivity between drugs. If the findings of Porebski et al. are accurate it could point to drugs interacting with MRGPRX2 on different active sites within the receptor or that the intracellular signaling pathways triggered by the binding of varying drugs have different responses thus producing different overall reactions.

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