Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides

doi:10.1016/j.bbrc.2005.03.088

Biochemical and Biophysical Research Communications

Volume 330, Issue 4, 20 May 2005, Pages 1146-1152

https://doi.org/10.1016/j.bbrc.2005.03.088 Get rights and content

Abstract

Proadrenomedullin N-terminal 20 peptide (PAMP[1–20]/PAMP-20) and its truncated analog, PAMP[9–20]/PAMP-12, are endogenous peptides that elicit hypotension through inhibiting catecholamine secretion from sympathetic nerve endings and adrenal chromaffin cells. Although the binding sites for PAMP are widely distributed, the nature of its receptor has been elusive. In an effort to identify potential PAMP receptor(s), we found that a human G-protein-coupled receptor, MrgX2, was specifically activated by PAMP. Although a previous study revealed that MrgX2 was a receptor for cortistatin, a neuropeptide involved in sleep regulation and locomotor activity, our present data indicated that the rank order of the agonistic effect against MrgX2 was “PAMP-12 ≥ cortistatin > PAMP-20”. These activities were confirmed by the inhibition of the forskolin-elevated cAMP accumulation, Ca²⁺ mobilization, and [³⁵S]guanosine 5′-(γ-thio)triphosphate binding assays. These findings suggest that MrgX2 couples with not only G_αq but also G_αi, consistent with previous reports on the pharmacological profile of PAMP signaling. Furthermore, by immunostaining, we found that MrgX2 was expressed in the adrenal chromaffin cells as well as the dorsal root ganglia. From these results, we concluded that MrgX2 is a potential human PAMP-12 receptor that regulates catecholamine secretion from adrenal glands. The present discovery will eventually lead to a better understanding of the pathophysiological role of proadrenomedullin peptides.

Section snippets

Materials and methods

Materials. Human PAMP-12 and PAMP-20 were purchased from the Peptide Institute (Osaka, Japan). Human cortistatin (CST-14) and BAM-22P were from Phoenix Pharmaceutical (Belmont, CA, USA) and Bachem (Bubendorf, Switzerland), respectively. 3-Isobutyl-1-methylxanthine (IBMX), pertussis toxin (PTX), and guanosine 5′-diphosphate (GDP) were from Sigma (St. Louis, MO, USA). Forskolin and methotrexate were from Wako (Osaka, Japan). [³⁵S]guanosine 5′-(γ-thio)triphosphate ([³⁵S]GTPγS) (1000 Ci/mmol) was

PAMP-12 specifically responds to MrgX2

Previous studies revealed that it is possible for a presumed PAMP receptor(s) to be a G_αi/o-protein-coupled receptor [16], [17], [18]. Therefore, based on the change in intracellular cAMP, we have searched for a PAMP-responsive receptor(s) from each type of orphan GPCR-expressing cell. By exploring these potential sources, we found that CHO cells stably expressing MrgX2 were specifically activated by PAMP-12 (Fig. 1A). MrgX2 has been classified as a member of the GPCR subfamily referred to as

Discussion

The proadrenomedullin gene products, PAMP-12 and PAMP-20, act as circulating hormones with hypotensive activities. Clinically, plasma levels of PAMP have been reported to increase in patients with a variety of diseases such as essential hypertension [28], chronic renal failure [29], congestive heart failure [30], and chronic glomerulonephritis [31]. This suggests that the vasophysiological function of PAMP relates to many diseases. Although clinical evidence indicates the importance of PAMP

Acknowledgments

We thank Dr. Shigekazu Nagata for providing pEF-BOS, and Ms. Mariko Isshiki, Ms. Akiko Wakita, and Mr. Hideki Miyamoto for excellent technical assistance.

References (37)

K. Kitamura et al.
FEBS Lett.
(1994)
K. Kitamura et al.
Biochem. Biophys. Res. Commun.
(1993)
T. Ishimitsu et al.
Biochem. Biophys. Res. Commun.
(1994)
K. Kuwasako et al.
FEBS Lett.
(1997)
H. Kobayashi et al.
Mol. Brain Res.
(2001)
H. Washimine et al.
Biochem. Biophys. Res. Commun.
(1994)
T. Eto
Peptides
(2001)
Y. Ishizaka et al.
Biochem. Biophys. Res. Commun.
(1994)
Y. Shimekake et al.
J. Biol. Chem.
(1995)
N. Robas et al.
J. Biol. Chem.
(2003)

M. Kamohara et al.

J. Biol. Chem.

(2000)

T. Soga et al.

Biochem. Biophys. Res. Commun.

(2003)

J. Takasaki et al.

Biochem. Biophys. Res. Commun.

(2000)

M. Kamohara et al.

Biochem. Biophys. Res. Commun.

(2001)

M. Matsumoto et al.

Gene

(2000)

H. Kinoshita et al.

Am. J. Kidney Dis.

(1999)

X. Dong et al.

Cell

(2001)

T. Shinohara et al.

J. Biol. Chem.

(2004)

Cited by (86)

Targeting active sites of inflammation using inherent properties of tissue-resident mast cells
2023, Acta Biomaterialia
Mast cells play a pivotal role in initiating and directing host's immune response. They reside in tissues that primarily interface with the external environment. Activated mast cells respond to environmental cues throughout acute and chronic inflammation through releasing immune mediators via rapid degranulation, or long-term de novo expression. Mast cell activation results in the rapid release of a variety of unique enzymes and reactive oxygen species. Furthermore, the increased density of mast cell unique receptors like mas related G protein-coupled receptor X2 also characterizes the inflamed tissues. The presence of these molecules (either released mediators or surface receptors) are particular to the sites of active inflammation, and are a result of mast cell activation. Herein, the molecular design principles for capitalizing on these novel mast cell properties is discussed with the goal of manipulating localized inflammation.
Mast cells are immune regulating cells that play a crucial role in both innate and adaptive immune responses. The activation of mast cells causes the release of multiple unique profiles of biomolecules, which are specific to both tissue and disease. These unique characteristics are tightly regulated and afford a localized stimulus for targeting inflammatory diseases. Herein, these important mast cell attributes are discussed in the frame of highlighting strategies for the design of bioresponsive functional materials to target regions of inflammations.
MAS-related G protein-coupled receptors X (MRGPRX): Orphan GPCRs with potential as targets for future drugs
2022, Pharmacology and Therapeutics
Citation Excerpt :
Besides neuropeptides, another endogenous peptide, proadrenomedullin N-terminal peptide-12 (PAMP-12) activated MRGPRX2 in calcium, cyclic adenosine monophosphate (cAMP) inhibition, and β-arrestin assays with EC50 values ranging from 40 nM to 3 μM (X2-24, Table 8) (Kamohara et al., 2005; Lansu et al., 2017). PAMP-20, a PAMP 12-derivative containing nine additional amino acids on the C-terminus, showed lower potency than PAMP-12 in both cAMP and β-arrestin assays using CHO cells recombinantly expressing MRGPRX2 (X2-25, Table 8) (Kamohara et al., 2005). Kallidin, a bradykinin homolog cleaved from kininogen by kallikreins, caused a concentration-dependent calcium signal in HEK293T cells recombinantly expressing MRGPRX2 with an EC50 value of 11.5 μM (X2-26, Table 8) (Lansu et al., 2017) Endogenous secretagogues that bind to MRGPRX2 are typically less than 4000 Da in size and contain cationic amino acid residues like arginine (R) and lysine (K), and hydrophobic, aromatic amino acids such as tryptophan (W), tyrosine (Y) and phenylalanine (F).
MAS-related G protein-coupled receptors (GPCRs) of subfamily X, designated MRGPRX, are primate-specific orphan receptors that belong to the δ-branch of rhodopsin-like, class A GPCRs. Four distinct subtypes exist, MRGPRX1, -2, -3, and -4, MRGPRX2 having the lowest degree of similarity with the others. Due to their expression on sensory neurons and immune cells, and their roles in pain perception and transmission, itch, inflammation, immune defense, pseudo-allergic reactions, wound healing, and possibly cancer, they have recently attracted much attention as novel drug targets. In particular MRGPRX2 was identified as an important mast cell receptor, responsible for anaphylactoid drug reactions and involved in skin and mucosal diseases, e.g. urticaria, atopic dermatitis, rosacea, and allergic rhinitis. A major hurdle has been the lack of animal models for studying these primate-specific receptors. However, recently humanized mice have been created. Moreover, a mouse ortholog of MRGPRX2, MRGPRB2, was identified, both receptors having a certain degree of similarity. MRGPRX1 and -2 can be activated by various peptides and small (partly peptidomimetic) molecules. MRGPRX2 is additionally activated by a very broad range of basic molecules, positively charged at physiologic pH value of 7.4, including many drugs. MRGPRX4 is activated by small acidic molecules including bile acids. For MRGPRX3, no ligands have been reported yet. Antagonists with reasonable potency and selectivity have been described for MRGPRX1, and few antagonists also for MRGPRX2, but not for the other subtypes. The recent elucidation of cryogenic electron microscopy structures of MRGPRX2 and -4 is expected to facilitate and advance drug development for these receptors. Currently, research on MRGPRX is still in its infancy, and exciting discoveries can be awaited. These receptors have great potential as future drug targets.
Atypical opioid receptors: unconventional biology and therapeutic opportunities
2022, Pharmacology and Therapeutics
Endogenous opioid peptides and prescription opioid drugs modulate pain, anxiety and stress by activating four opioid receptors, namely μ (mu, MOP), δ (delta, DOP), κ (kappa, KOP) and the nociceptin/orphanin FQ receptor (NOP). Interestingly, several other receptors are also activated by endogenous opioid peptides and influence opioid-driven signaling and biology. However, they do not meet the criteria to be recognized as classical opioid receptors, as they are phylogenetically distant from them and are insensitive to classical non-selective opioid receptor antagonists (e.g. naloxone). Nevertheless, accumulating reports suggest that these receptors may be interesting alternative targets, especially for the development of safer analgesics. Five of these opioid peptide-binding receptors belong to the family of G protein-coupled receptors (GPCRs)—two are members of the Mas-related G protein-coupled receptor X family (MrgX1, MrgX2), two of the bradykinin receptor family (B_1, B₂), and one is an atypical chemokine receptor (ACKR3). Additionally, the ion channel N-methyl-d-aspartate receptors (NMDARs) are also activated by opioid peptides. In this review, we recapitulate the implication of these alternative receptors in opioid-related disorders and discuss their unconventional biology, with members displaying signaling to scavenging properties. We provide an overview of their established and emerging roles and pharmacology in the context of pain management, as well as their clinical relevance as alternative targets to overcome the hurdles of chronic opioid use. Given the involvement of these receptors in a wide variety of functions, including inflammation, chemotaxis, anaphylaxis or synaptic transmission and plasticity, we also discuss the challenges associated with the modulation of both their canonical and opioid-driven signaling.
Action of substance P and PAMP(9-20) on different excitation sites of MRGPRX2 induces differences in mast cell activation
2021, International Immunopharmacology
MRGPRX2 on mast cells (MCs) is the target that directly mediates MC activation through the activity of small molecular substances. Previous work has attempted to prove that substance P (SP) and PAMP(9-20) induce an MRGPRX2-mediated MC degranulation reaction. However, SP activates MRGPRX2-induced histamine release, which may lead to allergic airway inflammation, while PAMP(9-20)-induced MrgprB2 activation releases more tryptase and fewer monoamines. Due to the lack of direct available comparisons, the different types of sensitizing mediators released by the action of SP and PAMP(9-20) inducing pseudo-allergic reactions via MRGPRX2 are unclear. To investigate whether the action sites of excited MRGPRX2 are different for SP and PAMP(9-20), leading to different effects, the release of inflammatory mediators was measured using MC degranulation reactions and RNA-seq assay in vitro. Mice were treated to observe local inflammation and MC degranulation in vivo. Moreover, site-directed mutagenesis was used to verify the excited sites of SP and PAMP(9-20). SP and PAMP(9-20) both activated MRGPRX2 and led MCs to release inflammatory mediators. Significantly different levels of histamine, tryptase, TNF-α, MCP-1, and other cytokines were released in vivo and in vitro. G165E, D184N, W243R, and H259Y were necessary for SP to activate MRGPRX2, while only D184N and W243R were important for PAMP(9-20). The downstream signaling pathways activated by SP and PAMP(9-20) also differed in the phosphorylation level of PKC. There were differences in the sites via which SP and PAMP(9-20) activate MRGPRX2 and also in the activated downstream signaling pathways, which led to the differences the activation of the pathways and effects of SP- and PAMP(9-20)-induced MRGPRX2 activation.
Identification of short peptide sequences that activate human mast cells via Mas-related G-protein coupled receptor member X2
2021, Acta Biomaterialia
Citation Excerpt :
We therefore were interested in studying the crucial role of the physiochemical properties of amino acids within a peptide molecule in MRGPRX2 activation. To elucidate the molecular basis for peptide-MRGPRX2 interactions, a peptide library based on Proadrenomedullin N-terminal 12 (PAMP-12) was used to identify amino acid residues responsible for MRGPRX2 induced activation of mast cells [4,9]. PAMP-12 based peptide library was developed using common screening techniques: N and C-terminal truncations, and alanine scanning.
Peptide based therapeutics are desirable owing to their high biological specificity. However, a number of these fail in clinical testing due to an adverse inflammatory response. Mast cells play a key role in directing the host response to drugs and related products. Although the role of FcεRI receptor is well known, Mas-related G-protein coupled receptor X2 (MRGPRX2) binding of endogenous peptides, and drugs will activate mast cells independent of FcεRI. Identifying peptides that activate mast cells through MRGPRX2, and their respective activation potency, can be used to reduce the failure rate of peptide therapeutics at clinical trial. Moreover, it will allow for peptide design where mast cell activation is actually desired. It was found that FRKKW and WNKWAL are two motifs that activate human LAD2 cells similar to PAMP-12 controls. Peptide activators of MRGPRX2 could be reduced to X_a-(Y)_{(n ≥ 3)}-X_b where: X_a is an aromatic residue; X_b is a hydrophobic residue; and Y is a minimum 3 residue long sequence, containing a minimum of one positively charged residue with the remainder being uncharged residues. Artificial peptides WKKKW and FKKKF were constructed to test this structural functionality and were similar to PAMP-12 controls. Peptides with different activation potentials were found where FRKKW = WKKKW = FKKKF > PAMP-12 = WNKWAL > YKKKY > FRKKANKWALSR = FRKKWNKAALSR > KWKWK > FRKK = WNKWA > KYKYK > NKWALSR = YKKY = WNK. These sequences should be considered when designing peptide-based therapeutics.
Mast cells release immune regulating molecules upon activation that direct host's immune response. MRGPRX2 receptor provides an alternate pathway for mast cell activation that is independent of FcεRI receptor. It is thought that mast cell activation through MRGPRX2 plays a critical role in high failure rates of drugs in clinical trials. Identifying peptide sequences that activate mast cells through MRGPRX2 can serve two important purposes, namely, sequences to avoid when designing peptide therapeutics, and artificial peptides with different activation potentials for mast cells. Herein, we have identified a general amino acid sequence that induces mast cell activation through MRGPRX2. Furthermore, by modulating the identified sequence, artificial peptides have been designed which activate mast cells by varying degrees for therapeutic applications.
Innate Immune Regulation of Dermatitis
2021, Immunology and Allergy Clinics of North America

View all citing articles on Scopus

^☆: Abbreviations: GPCR, G-protein-coupled receptor; PAMP, proadrenomedullin N-terminal peptide; AM, adrenomedullin; CST, cortistatin; Mrg, Mas-related gene; PTX, pertussis toxin; GTPγS, guanosine 5′-(γ-thio)triphosphate; DRG, dorsal root ganglia; EC₅₀, median effective concentration.

¹: These authors contributed equally to this work.

View full text

Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides☆

Abstract

Section snippets

Materials and methods

PAMP-12 specifically responds to MrgX2

Discussion

Acknowledgments

FEBS Lett.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Mol. Brain Res.

Biochem. Biophys. Res. Commun.

Peptides

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Gene

Am. J. Kidney Dis.

Cell

J. Biol. Chem.