Activation of mas-related G-protein-coupled receptors by the house dust mite cysteine protease Der p1 provides a new mechanism linking allergy and inflammation.

Cysteine and serine proteases function via protease-activated and mas-related G-protein-coupled receptors (Mrgprs) to contribute to allergy and inflammation. Der p1 is a cysteine protease and major allergen from the house dust mite and is associated with allergic rhinitis and allergic asthma. Der p1 activates protease-activated receptor 2 and induces the release of the pro-inflammatory cytokine IL-6 from cells. However, the possibility that Der p1 acts on Mrgprs has not been considered. We report here that ratiometric calcium imaging reveals that Der p1 activates the human receptor MRGPRX1 and the mouse homolog MrgprC11, implicated previously in itch. Der p1 cleavage of N-terminal receptor peptides followed by site-directed mutagenesis of the cleavage sites links receptor activation to specific amino acid residues. Der p1 also induced the release of IL-6 from heterologous cells expressing MRGPRX1. In summary, activation of Mrgprs by the allergen Der p1 may contribute to inflammation.

Brief communication: MRGPRX2, atopic dermatitis and red man syndrome.

Vancoymycin causes red man syndrome, an itchy erythematous eruption involving the face, neck and upper torso. Atopic dermatitis also manifests itch and erythema, and staphylococcus δ-toxin contributes to this process. The antibiotic and toxin each provoke mast cell degranulation but the mechanism had not been understood. We have determined that these compounds evoke degranulation via interaction with the same receptor, MRGPRX2, on mast cells. A receptor antagonist inhibits this process. Antagonists of this receptor may have therapeutic potential.

Substance P activates Mas-related G protein-coupled receptors to induce itch.

BACKGROUND: Substance P (SP) is linked to itch and inflammation through activation of receptors on mast cells and sensory neurons. There is increasing evidence that SP functions through Mas-related G protein-coupled receptors (Mrgprs) in addition to its conventional receptor, neurokinin-1. OBJECTIVE: Because Mrgprs mediate some aspects of inflammation that had been considered mediated by neurokinin-1 receptor (NK-1R), we sought to determine whether itch induced by SP can also be mediated by Mrgprs. METHODS: Genetic and pharmacologic approaches were used to evaluate the contribution of Mrgprs to SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons from mice. RESULTS: SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons was dependent on Mrgprs rather than NK-1R. CONCLUSION: We deduce that SP activates MrgprA1 on sensory neurons rather than NK-1R to induce itch.

Dual action of neurokinin-1 antagonists on Mas-related GPCRs.

The challenge of translating findings from animal models to the clinic is well known. An example of this challenge is the striking effectiveness of neurokinin-1 receptor (NK-1R) antagonists in mouse models of inflammation coupled with their equally striking failure in clinical investigations in humans. Here, we provide an explanation for this dichotomy: Mas-related GPCRs (Mrgprs) mediate some aspects of inflammation that had been considered mediated by NK-1R. In support of this explanation, we show that conventional NK-1R antagonists have off-target activity on the mouse receptor MrgprB2 but not on the homologous human receptor MRGPRX2. An unrelated tripeptide NK-1R antagonist has dual activity on MRGPRX2. This tripeptide both suppresses itch in mice and inhibits degranulation from the LAD-2 human mast cell line elicited by basic secretagogue activation of MRGPRX2. Antagonists of Mrgprs may fill the void left by the failure of NK-1R antagonists.

GRPR/PI3Kγ: Partners in Central Transmission of Itch.

The gastrin-releasing peptide (GRP) and its receptor (GRPR) are important components of itch transmission. Upstream, but not downstream, aspects of GRPR signaling have been investigated extensively. We hypothesize that GRPR signals in part through the PI3Kγ/Akt pathway. We used pharmacological, electrophysiological, and behavioral approaches to further evaluate GRPR downstream signaling pathways. Our data show that GRP directly activates small-size capsaicin-sensitive DRG neurons, an effect that translates into transient calcium flux and membrane depolarization (∼ 20 mV). GRPR activation also induces Akt phosphorylation, a proxy for PI3Kγ activity, in ex vivo naive mouse spinal cords and in GRPR transiently expressing HEK293 cells. The intrathecal injection of GRP led to intense scratching, an effect largely reduced by either GRPR antagonists or PI3Kγ inhibitor. Scratching behavior was also induced by the intrathecal injection of an Akt activator. In a dry skin model of itch, we show that GRPR blockade or PI3Kγ inhibition reversed the scratching behavior. Altogether, these findings are highly suggestive that GRPR is expressed by the central terminals of DRG nociceptive afferents, which transmit itch via the PI3Kγ/Akt pathway. SIGNIFICANCE STATEMENT: Itch is the most common symptom of the skin and is related to noncutaneous diseases. It severely impairs patients' quality of life when it becomes chronic and there is no specific or effective available therapy, mainly because itch pathophysiology is not completely elucidated. Our findings indicate that the enzyme PI3Kγ is a key central mediator of itch transmission. Therefore, we suggest PI3Kγ as an attractive target for the development of new anti-pruritic drugs. With this study, we take a step forward in our understanding of the mechanisms underlying the central transmission of itch sensation.

Redefining the concept of protease-activated receptors: cathepsin S evokes itch via activation of Mrgprs.

Sensory neurons expressing Mas-related G-protein-coupled receptors (Mrgprs) mediate histamine-independent itch. We show that the cysteine protease cathepsin S activates MrgprC11 and evokes receptor-dependent scratching in mice. In contrast to its activation of conventional protease-activated receptors, cathepsin S-mediated activation of MrgprC11 did not involve the generation of a tethered ligand. We demonstrate further that different cysteine proteases selectively activate specific mouse and human Mrgpr family members. This expansion of our understanding by which proteases interact with G-protein-coupled receptors (GPCRs) redefines the concept of what constitutes a protease-activated receptor. The findings also implicate proteases as ligands to members of this orphan receptor family while providing new insights into how cysteine proteases contribute to itch.

Cathepsin S signals via PAR2 and generates a novel tethered ligand receptor agonist.

Protease-activated receptor-2 is widely expressed in mammalian epithelial, immune and neural tissues. Cleavage of PAR2 by serine proteases leads to self-activation of the receptor by the tethered ligand SLIGRL. The contribution of other classes of proteases to PAR activation has not been studied in detail. Cathepsin S is a widely expressed cysteine protease that is upregulated in inflammatory conditions. It has been suggested that cathepsin S activates PAR2. However, cathepsin S activation of PAR2 has not been demonstrated directly nor has the potential mechanism of activation been identified. We show that cathepsin S cleaves near the N-terminus of PAR2 to expose a novel tethered ligand, KVDGTS. The hexapeptide KVDGTS generates downstream signaling events specific to PAR2 but is weaker than SLIGRL. Mutation of the cathepsin S cleavage site prevents receptor activation by the protease while KVDGTS retains activity. In conclusion, the range of actions previously ascribed to cysteine cathepsins in general, and cathepsin S in particular, should be expanded to include molecular signaling. Such signaling may link together observations that had been attributed previously to PAR2 or cathepsin S individually. These interactions may contribute to inflammation.

Maxadilan, the PAC1 receptor, and leishmaniasis.

Maxadilan is a vasodilator peptide isolated from sand fly salivary glands. The vasodilator effects of maxadilan are mediated by the PAC1 receptor, although maxadilan and PACAP do not share sequence homology. Sand flies are the vector of the parasitic disease leishmaniasis. The peptide aids the sand fly in obtaining a blood meal while enhancing the infectivity of leishmania parasites transmitted by this arthropod vector. Aspects of maxadilan, PAC1, and leishmaniasis are discussed.

Cowhage-evoked itch is mediated by a novel cysteine protease: a ligand of protease-activated receptors.

Cowhage spicules provide an important model for histamine-independent itch. We determined that the active component of cowhage, termed mucunain, is a novel cysteine protease. We isolated mucunain and demonstrate that both native and recombinant mucunain evoke the same quality of itch in humans. We also show that mucunain is a ligand for protease-activated receptors two and four. These results support and expand the relationship between proteases, protease-activated receptors, and itch.

Maxadilan, a PAC1 receptor agonist from sand flies.

In 1991, a potent 61 amino acid vasodilator peptide, named maxadilan, was isolated from the salivary glands of the sand fly. Subsequently, it was shown that this peptide specifically and potently activated the mammalian PAC1 receptor, one of the three receptors for PACAP. These studies and the link between maxadilan and leishmaniasis are discussed.

Functional analysis of recombinant mutants of maxadilan with a PAC1 receptor-expressing melanophore cell line.

Maxadilan, a 61-amino-acid vasodilatory peptide, was initially isolated from the salivary glands of the sand fly Lutzomyia longipalpis. Although its primary sequence has no homology to that of pituitary adenylate cyclase-activating peptide, maxadilan is an agonist for the PAC1 receptor. A total of 58 substitution and deletion mutants was engineered in an effort to determine which residues were important for receptor activation. The mutants were characterized functionally using an assay based on pigment granule translocation in PAC1-expressing Xenopus laevis melanophores. Substitution of charged residues and proline 43 could alter (but not eliminate) the agonist activity of the mutants. In contrast, we found that several multiple substitution mutants of the predicted beta-strand threonine residues became antagonists at the PAC1 receptor. The results suggest that these threonine residues are cooperatively involved in PAC1 activation.

Identification of novel hexapeptide agonists at the Xenopus laevis melanophore melanocortin receptor.

We used a combinatorial chemical approach to identify novel agonists for the endogenous melanocortin receptor expressed in Xenopus laevis melanophores. A random one-bead one-compound hexapeptide library was screened to detect new molecules able to induce pigment dispersion in melanophores. Our approach led to the discovery of seven related novel peptides able to stimulate pigment dispersion with EC50 in the range of 0.1-10 microM. Their action was inhibited by the amphibian melanocortin receptor antagonist dWRL. These novel peptides share no significant sequence homology with known melanocortins. This study may aid in the understanding of the chemical interaction between the melanocortin receptors and their ligands.