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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. G-proteins are structurally trimeric proteins that are composed of three subunits: α, β, and γ. 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.

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 359 amino acid proteins, with varying sequences, that both contain a helical domain and a GTPase binding domain. 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 discovery of GPCRs… Nobel prize (insert information about that here: discovery, scientists, method, etc.) nobel prize, methods of visualization, and the history of known diseases and their associated mutations

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

Talk about there a ton of different ligands can bind Highlight the few that we want to talk about: Peptide vs. manufactured/artificial vs. non-peptide Agonists, inverse agonists, and antagonists and their effects on the mechanism

Mechanism

mechanism of binding and the associated chemical interactions signaling cascades associated with the Human Itch GPCRs regulatory processes within the signaling cascade that directly affect the g-proteins

Relevance

Implications for Disease

significant mutations that have been found drug design for GPCR targeting

Clinical Relevance

drug hypersensitivity article

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