1521-0081/66/3/570–597$25.00 http://dx.doi.org/10.1124/pr.113.

008425
PHARMACOLOGICAL REVIEWS Pharmacol Rev 66:570–597, July 2014
Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: FINN OLAV LEVY

Pharmacology and Signaling of MAS-Related

G Protein–Coupled Receptors
Hans Jürgen Solinski, Thomas Gudermann, and Andreas Breit
Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, Munich, Germany

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
II. Genes Encoding MAS-Related G Protein–Coupled Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
A. Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
B. Phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
C. Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
D. Evolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
III. Pharmacology and Physiology of MAS-Related G Protein–Coupled Receptors . . . . . . . . . . . . . . . . . 577
A. Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
B. MAS-Related G Protein–Coupled Receptors A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577

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1. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
2. Signaling Cascades and Physiologic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
C. MAS-Related G Protein–Coupled Receptors B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
D. MAS-Related G Protein–Coupled Receptors C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
1. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
2. Signaling Cascades and Physiologic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
a. Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
b. Pain-enhancing effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
c. Analgesic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583
d. Pruritogenic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583
E. MAS-Related G Protein–Coupled Receptors D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
1. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
2. Signaling Cascades and Physiologic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
F. MAS-Related G Protein–Coupled Receptors E to -H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
G. MAS-Related G Protein–Coupled Receptors X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
1. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
a. MAS-related G protein–coupled receptors X1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
b. MAS-related G protein–coupled receptors X2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
c. MAS-related G protein–coupled receptors X3 and -4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
2. Signaling Cascades and Physiologic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
a. Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
b. Somatosensory functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
c. Primary sensory neuron plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
d. Mast cell biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
e. Putative role of MAS-related G protein–coupled receptors X2 in sleep and
blood pressure regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
IV. Agonist-Promoted Internalization and Desensitization of MAS-Related
G Protein–Coupled Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591

This work was supported by a grant from the “Deutsche Forschungsgemeinschaft” [Grant BR 3346/3-1].
Address correspondence to: Dr. Andreas Breit, Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-
Universität München, Goethestrasse 33, 80336 Munich, Germany. E-mail: Andreas.Breit@lrz.uni-muenchen.de
T.G. and A.B. are co-senior authors.
dx.doi.org/10.1124/pr.113.008425.

570
 MAS-Related G Protein–Coupled Receptors 571

V. Functional Interactions of MAS-Related G Protein–Coupled Receptors with

Other Receptor Families. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
A. Heteromultimerization among MAS-Related G Protein–Coupled
Receptors and Receptor Subtypes of Other GPCR Families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
B. Inhibition of Tolerance to Morphine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
VI. Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594

Abstract——Signaling by heptahelical G protein– angiotensin-(1–7), alamandine, GABA, cortistatin-14,

coupled receptors (GPCR) regulates many vital body and cleavage products of proenkephalin, pro-
functions. Consequently, dysfunction of GPCR sig- opiomelanocortin, prodynorphin, or proneuropeptide-
naling leads to pathologic states, and approximately FF-A. The full spectrum of biologic roles of all MRGPR
30% of all modern clinical drugs target GPCR. One is still ill-defined, but there is evidence pointing to a role
decade ago, an entire new GPCR family was discov- of distinct MRGPR subtypes in nociception, pruritus,
ered, which was recently named MAS-related G sleep, cell proliferation, circulation, and mast cell
protein–coupled receptors (MRGPR) by the HUGO degranulation. This review article summarizes
Gene Nomenclature Committee. The MRGPR family findings published in the last 10 years on the
consists of ∼40 members that are grouped into nine phylogenetic relationships, pharmacology, sig-
distinct subfamilies (MRGPRA to -H and -X) and are naling, physiology, and agonist-promoted regula-
predominantly expressed in primary sensory neurons tion of all MRGPR subfamilies. Furthermore, we
and mast cells. All members are formally still con- highlight interactions between MRGPR and other
sidered "orphan" by the Committee on Receptor No- hormonal systems, paying particular attention
menclature and Drug Classification of the International to receptor multimerization and morphine toler-
Union of Basic and Clinical Pharmacology. However, ance. Finally, we discuss the challenges the field
several distinct peptides and amino acids are dis- faces presently and emphasize future directions of
cussed as potential ligands, including b-alanine, research.

I. Introduction identified for ;100 of the GPCR identified so far,

G protein–coupled receptors (GPCR), also known
as seven-transmembrane domain receptors, constitute
a large protein family that senses a plethora of distinct
physical and chemical stimuli (biogenic amines, amino
acids, ions, lipids, nucleotides, peptides, proteins, light,
odorants, and pheromones) outside the cell. Upon
GPCR activation, a host of intracellular signaling pro-
teins (enzymes, transcription factors, ion channels) are
engaged, affecting vital cellular and organismal func-
tions, such as cell proliferation, differentiation, de-
velopment, survival, circulation, metabolism, neuronal
signal transmission, and sensory perception. Approxi-
mately 800 putative GPCR have been identified in
humans and more than 300 of them are nonodorant
receptors (Fredriksson et al., 2003). Interestingly, en-
dogenous or natural exogenous ligands have not been
suggesting that many biologic functions of GPCR may
not have been discovered yet (Davenport et al., 2013).
It is reasonable to assume that these "orphan" GPCR
have an immense therapeutic potential, because ;30%
of all clinically relevant drugs already target GPCR
directly or modulate their cognate signaling pathways
(Overington et al., 2006). Hence, in recent years, con-
siderable efforts have been undertaken to identify
endogenous ligands of "orphan" GPCR by high-
throughput screening strategies. As a result of one of
these efforts, Lembo and coworkers (2002) reported a
group of human "orphan" receptors that are activated
by proenkephalin (PENK) cleavage products of the
bovine adrenal medulla (BAM) peptide family. The
authors called these receptors sensory neuron–specific
G protein–coupled receptors (SNSR) because of their
restricted expression pattern in primary sensory

ABBREVIATIONS: BAM, bovine adrenal medulla; CAP, capsaicin; CCR2, chemokine receptors 2; CFA, complete Freund’s adjuvant; CGRP,
calcitonin gene-related peptide; CHO, Chinese hamster ovary; D-APV, D-(2)-2-amino-5-phosphonopentanoic acid; DOP, d-OR; DRG, dorsal
root ganglia; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; GPCR, G protein–coupled receptors; HC-030031,
1,2,3,6-tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7H-purine-7-acetamide, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-
7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; HEK, human embryonic kidney; MK-801, (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo
[a,d]cyclohepten-5,10-imine; MOP, m-OR; Mrg, MAS-related gene; MRGPR, MAS-related G protein–coupled receptors; MSH, melanocyte
stimulating hormone; NC-IUPHAR, Committee on Receptor Nomenclature and Drug Classification of the International Union of Basic and
Clinical Pharmacology; NFAT, nuclear factor of activated T cells; NMDA, N-methyl-D-aspartate; nNOS, neuronal nitric oxide synthetase;
NPAF, neuropeptide AF; NPFF, neuropeptide FF; OR, opioid receptors; PAR, protease-activated receptors; PENK, proenkephalin; PIP2,
phosphatidylinositol-3,4-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; PTX, pertussis toxin; SNSR, sensory neuron–specific
receptors; TrkA, neurotrophic tyrosin kinase receptor 1; TRP, transient receptor potential cation channel; TRPA, TRP ankyrin; TRPM, TRP
melastatin 8; TRPV, TRP vanilloid 1; YM58483, N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-
carboxamide.
572 Solinski et al.
neurons and postulated a role for SNSR in pain
perception. Shortly before, Dong and colleagues
(2001) compared cDNA libraries from wild-type mice
and from mice lacking the transcription factor neuro-
genin 1. Mice without functional neurogenin 1 fail to
develop the neurotrophic tyrosine kinase receptor 1
(TrkA)–positive subclass of dorsal root ganglia (DRG)
and trigeminal ganglia neurons that detect painful
stimuli, so called TrkA+ nociceptors (Ma et al., 1999).
Thus, the authors postulated that genes found in the
library of wild-type mice, but not in neurogenin 1–
deficient mice, should be specific for TrkA+ nociceptors.
By this approach, the authors discovered an entire new
family of selectively expressed GPCR and named these
proteins MAS-related genes (Mrg), because of their
homology to the oncogenic GPCR MAS1. The Mrg
family in rodents and humans comprises ;40 members
that can be divided into several subfamilies. It soon
turned out that one of these subfamilies, the MrgX
family, is identical to the SNSR proteins (Lembo et al.,
2002), such that the same receptor proteins were given
two different names. Even more confusing, the rat
MrgC/SNSR was named after its proposed cognate
ligand BAM peptide-activated receptor with nonopioid
activity, and MRG is also used as an abbreviation for
mortality factor on chromosome 4–related genes
[mortality factor on chromosome 4/MRG functions in
aging are reviewed elsewhere (Chen et al., 2010a)],
a group of proteins distinct from the GPCR superfam-
ily. Thus, inconsistencies in the nomenclature de-
veloped over the last decade may have contributed to
confusions in the field. To overcome this problem, the
HUGO Gene Nomenclature Committee now refers to
Mrg/SNSR proteins as MAS-related G protein–coupled
receptors (MRGPR) (www.genename.org). So far, a to-
tal of 38 genes encoding MRGPR proteins has been
listed in National Center for Biotechnology Informa-
tion databases (orthologous proteins from distinct
mammalian species are counted as one member, see
Fig. 1 and Table 1). Therefore, to the best of our
knowledge, MRGPR represent the nonodorant GPCR
family with the largest number of members known so
far. At present, MRGPR are arranged in nine sub-
families, designated by capital letters, whereas individ-
ual subtypes are indicated by numbers. For instance,
the MRGPR subtype 1 of subfamily X, originally termed
MrgX1 (according to Dong et al., 2001) or SNSR4
(according to Lembo et al., 2002), is now officially called
MRGPRX1. This nomenclature is also recognized by
Committee on Receptor Nomenclature and Drug Clas-
sification of the International Union of Basic and Clin-
ical Pharmacology (NC-IUPHAR; Davenport et al.,
2013) and is recommended for use to improve scientific
communication.
Since their first description, considerable efforts
have been undertaken to characterize MRGPR in
terms of ligand binding profile, regulation, signaling
cascades, and physiologic effects. Significant progress
has been made recently and, thus, several MRGPR
members emerged as most appealing pharmacological
targets for analgesic, antipruritogenic, and antihyper-
tensive therapies. However, at the same time, advan-
ces in our understanding of the physiology and
pathophysiology of MRGPR are hampered by a complex
phylogeny, redundancy in ligand binding, and multi-
faceted functions. This review is an attempt to summa-
rize our present knowledge about the MRGPR family
and to point out current caveats. We present an over-
view of MRGPR genetics, including phylogeny and evo-
lution, and of pharmacologic and physiologic details of
each MRGPR subfamily. Finally, we will highlight
agonist-induced MRGPR regulation and interactions
with other GPCR families identified so far.
II. Genes Encoding MAS-Related G
Protein–Coupled Receptors
A. Preface
MRGPR-encoding genes have been detected in
mammals, including rodents, Canis lupus, Bos taurus,
and primates. No MRGPR genes have been identified
in lower vertebrates so far, probably because of less
advanced gene sequencing and annotation processes of
lower vertebrate genomes. Only one MRGPR member
was annotated in the genome of birds: Solely the
MRGPRH was found in Gallus gallus. The GPCR
superfamily is divided based on sequence homology
into class A to F. According to the existence of several
conserved motifs, for instance the NPxxY motif in
transmembrane domain seven, the MRGPR family is
assigned to the rhodopsin-like class A (Dong et al.,
2001; Lembo et al., 2002; Fredriksson et al., 2003;
Katritch et al., 2013). The rhodopsin-like GPCR class A
can be further subdivided into four groups designated
a to d. MRGPR belong to the d-group of class A, which
also comprises glycohormone, purinergic and the very
large family of olfactory receptors, with glycohormone
receptors being the receptor family most closely related
to the MRGPR family (Dong et al., 2001; Lembo et al.,
2002; Fredriksson et al., 2003; Katritch et al., 2013).
B. Phylogeny
The mammalian family of MRGPR can be subdivided
into nine separate subfamilies (A–H and X) because of
sequence similarities (see Fig. 1 and Table 1). Sub-
families A, B, C, and H exist only in rodents, whereas
subfamily X is specific to primates including humans,
macaque, and rhesus monkey (Dong et al., 2001;
Lembo et al., 2002; Zylka et al., 2003; Zhang et al.,
2005; Burstein et al., 2006). In contrast, subfamilies D
to G are conserved in different mammalian species,
including rodents and primates (Dong et al., 2001;
Lembo et al., 2002; Zylka et al., 2003).
 MAS-Related G Protein–Coupled Receptors 573

Fig. 1. Phylogeny of MAS-related G protein–coupled receptors. A phylogenetic tree of all 38 MRGPR members from the nine MRGPR subfamilies (A–
H, X) of mice (m), rat (r), human (h), and rhesus monkey (Rh) was computed using Geneious 7 (Biomatters, Auckland, New Zealand; Blosum62 cost
matrix, Jukes-Cantor genetic distance model, Neighbor-Joining tree build method). GeneID of MRGPR genes and their aliases are given in Table 1.
The scale bar indicates amino acid substitutions per site. Deeper insights into the phylogenetic relationship of the human MRGPR and other GPCR
families are provided within the excellent articles of Fredriksson or Katritch (Fredriksson et al., 2003; Katritch et al., 2013).

The MRGPRA subfamily composition differs consid-
erably among rodents. Although only one gene is found
in rats, mice harbor 18 protein-encoding MRGPRA
genes as well as several pseudogenes (Dong et al.,
2001; Zylka et al., 2003). The MRGPRB subfamily
comprises seven protein-encoding genes in rats, nine in
mice, and several pseudogenes in both species (Dong
et al., 2001; Zylka et al., 2003). According to Zylka et al.
(2003), a further subdivision of MRGPRB genes into
the subgroups B2, B4, and B8 is procurable and reflects
the basic set of MRGPRB in other rodent species, such
as gerbil. The MRGPRC subfamily consists of only one
gene in rats and one protein-encoding gene in addition
to 13 pseudogenes in mice (Han et al., 2002; Zylka et al.,
2003). The MRGPRD subfamily comprises only one
protein-encoding gene per species (Dong et al., 2001;
Lembo et al., 2002; Zylka et al., 2003). Just like
MRGPRD, MRGPRE to -H subtypes exhibit only one
protein-encoding gene per species (Zylka et al., 2003). The
MRGPRX subfamily was originally reported to consist of
four distinct genes that are all located on chromosome
11p15.1 in humans (Dong et al., 2001). Three of the four
receptors discovered earlier were identified independently
by other researchers, and three additional receptors were
isolated from the same laboratory (Lembo et al., 2002),
resulting in a total of seven distinct MRGPRX receptors.
However, three of the original putative MRGPRX
members (SNSR2, -3, and -5) have not yet been listed
by NC-IUPHAR, most likely because these receptors are
up to 98% identical to the listed MRGPRX and, thus,
probably represent polymorphisms but not distinct sub-
types. Hence, four distinct MRGPRX proteins (MRGPRX1
to -4) are currently listed by NC-IUPHAR and will be
reviewed herein. Table 2 provides an overview of the
identities and relationships of all MRGPRX members
published and officially listed so far.
574 Solinski et al.

TABLE 1
Phylogeny of MAS-related G protein–coupled receptors
All 38 MRGPR from 4 different species are listed by their designated names, aliases, and GeneID. The ascribed RefSeq
records have been reviewed by National Center for Biotechnology Information (NCBI) staff and are annotated to the
reference genome (mouse: annotation release 103, rat: build 5.1, human: annotation release 105, rhesus monkey: build
1.2). A straight line below the GeneID indicates that the RefSeq record has not yet been subject to individual review by
NCBI staff. A GeneID in parentheses indicates that the underlying information was provided by an NCBI collaborator, an
NCBI RefSeq record is yet missing, and accordingly no annotation to the reference genome exists. Notice that if MRGPRA
and -B subfamily members are not numbered consecutively, numbers in between designate pseudogenes.

Subfamily Number Aliases GeneID

MRGPRA 1 MrgA1 (M) 233221*

— MrgA10, AdeninR, MRGPRX3 (R) 252960†
2a - (M) 668727*
2b MrgA2 (M) 235712*
3 MrgA3 (M) 233222*
4 MrgA4 (M) 235854*
5 MrgA5 (M) 404235*
6 MrgA6 (M) 381886*
7 MrgA7 (M) 404236*
8 MrgA8 (M) (404237)*
9 MrgA9 (M) 668725*
10 MrgA10 (M) (404243)*
11 MrgA11 (M) (404244)*
12 MrgA12 (M) (404245)*
13 MrgA13 (M) (404246)*
14 MrgA14 (M) (404247)*
15 MrgA15 (M) (404248)*
16 MrgA16, Gm486 (M) (404249)*
19 MrgA19 (M) (404250)*
MRGPRB 1 MrgB1 (M, R), MRGPRX2 (R) 233231*/404640†
2 MrgB2 (M, R), MRGPRX2-like (R) 243979*/404645†
3 MrgB3 (M, R) 404238*/(404656)†
4 MrgB4 (M, R) 233230*/404658†
5 MrgB5 (M, R) 404239*/404644†
6 MrgB6 (R) 502338†
8 MrgB8, MrgB13 (M, R) 404240*/404643†
10 MrgB10, MRGPRX2 (M) 243978*
11 MrgB11 (M) (404251)*
13 MrgB13 (M) 620137*
MRGPRC — Mrgprc11, MRGPRX1 (M, R), 404242*/282547†
MrgC11 (M), SNSR1 (R)
MRGPRD — MrgD, TGR7 (M, R, H, Rh) 211578*/293648†/
116512‡/709555x
MRGPRE — MrgE (M, R, H, Rh), GPR167 (H) 244238*/404660†/
116534‡/706822x
MRGPRF — RTA, MrgF, GPR140, GPR168 (M, R, H, Rh) 211577*/266762†/
116535‡/721546x
MRGPRG — MrgG (M, R, H, Rh), Gm1098 (M), GPR169 (H) 381974*/49929†
386746‡/721253x
MRGPRH — GPR90, MrgH (M, R) 80978*/404641†
MRGPRX 1 MrgX1 (H, Rh), SNSR4 (H) 259249‡/692101x
2 MrgX2 (H, Rh), MrgX2-2 (Rh) 117194‡/692078x
3 MrgX3 (H, Rh), SNSR1 (H) 117195‡/692077x
4 MrgX4 (H, Rh), SNSR6 (H) 117196‡/692123x
M, mouse; R, rat; H, human; Rh, rhesus monkey.
GeneIDs: *mouse; †rat; ‡human; xrhesus monkey.

C. Expression and McMahon, 1998). All MRGPRA, MRGPRC to -H,

The majority of MRGPR is expressed in isolectin B4- and MRGPRX members, as well as the MRGPRB4 and
positive small-diameter somatosensory afferents, which -5 subtype, can be subsumed to be expressed in isolectin
represent about half of the nociceptors per DRG (Snider B4-positive DRG neurons (Dong et al., 2001; Lembo

TABLE 2
The MAS-related G protein–coupled receptors subfamily X
An overview of MRGPRX subfamily members, their identity, and their interrelation according to different authors is
given.

Dong et al., 2001 Lembo et al., 2002 Burstein et al., 2006 NC-IUPHAR GeneID or Accession

MrgX1 SNSR4 MrgX1-1 (-2 exists additionally) MRGPRX1 259249

MrgX2 — MrgX2 MRGPRX2 117194
MrgX3 SNSR1 MrgX3-1 (-2 exists additionally) MRGPRX3 117195
MrgX4 SNSR6 MrgX4-1 MRGPRX4 117196
— SNSR5 MrgX4-2 — AF474991
— SNSR2 MrgX6 — AF474988
— SNSR3 MrgX7 — AF474989
 MAS-Related G Protein–Coupled Receptors
et al., 2002; Robas et al., 2003; Zylka et al., 2003; Zhang
et al., 2005; Tatemoto et al., 2006; Cox et al., 2008;
Liu et al., 2009). None of the remaining MRGPRB
members have been detected in primary sensory
neurons so far. However, high amounts of MRGPRB3
and -8 transcripts, as well as lower copy numbers of the
remaining three rat MRGPRB members, were mea-
sured in rat peritoneal mast cells, whereas transcripts
of MRGPRA or -C were not present in these cells
(Tatemoto et al., 2006). Recently, MRGPRX1 and -2
expression was also shown in human mast cells
(Tatemoto et al., 2006; Subramanian et al., 2011b;
Solinski et al., 2013). Thus, human MRGPRX mirror
the expression pattern of rodent MRGPRB and -A/C,
lending support to the current theory of MRGPR evo-
lution, implying that MRGPRX1 and -2 genes inherited
promoter elements from ancestral MRGPRB and -A/C
genes (for details see section II.D).
The initial discovery of the MRGPR family pointed to
a selective expression of MRGPR in primary sensory
neurons derived from the TrkA+ population (Dong
et al., 2001). Perinatally, this results in a largely
overlapping MRGPRA to -D expression in the same
population of neurons (Dong et al., 2001; Zylka et al.,
2003; Liu et al., 2008). After birth, TrkA expression
ceases in roughly half of the formerly TrkA+ neurons
(Molliver et al., 1997). Instead of TrkA, these cells start
to express c-ret, the receptor of the glial-derived
neurotrophic factor (Molliver et al., 1997). In adult-
hood, expression of all MRGPR is maintained in c-ret+,
but not in the remaining TrkA+ neurons (Dong et al.,
2001; Zylka et al., 2003; Liu et al., 2008). Interestingly,
despite the largely overlapping expression pattern of
all MRGPR in perinatal primary sensory neurons, in
adulthood most MRGPR subtypes are not coexpressed
in the same c-ret+ population and exhibit a compart-
mentalized expression pattern in distinct subpopula-
tions (Dong et al., 2001; Zylka et al., 2003; Liu et al.,
2008). Notably, these expression patterns are not
identical in mice and rats, suggesting variations in
the physiologic roles of distinct MRGPR in rodents.
The analysis of transcriptional pathways responsible
for expression of different MRGPR in distinct DRG
populations in mice revealed that expression of
MRGPRA to -D initially depends on the activity of
the runt-related transcription factor 1, explaining the
overlapping expression pattern in embryonic neurons
(Liu et al., 2008). During postnatal development, runt-
related transcription factor 1 suppresses MRGPRA to –C,
but not MRGPRD expression, through its inhibitory
C-terminal domain, leading to the segregation of
murine MRGPRD and MRGPRA to -C expression in
adolescence. Furthermore, the authors found that
the transcription factor Smad4 is indispensable for
MRGPRB4 expression, but does not affect expression
of other MRGPR subtypes (Liu et al., 2008). The
distinct expression pattern of MRGPR in adulthood led
575
to the assumption that neuronal subpopulations
characterized by their MRGPR expression pattern
might have distinct functions. In line with this hypoth-
esis, MRGPRB4- or MRGPRD-positive primary sensory
neurons in mice obtain distinct somatosensory input
from different skin areas (Zylka et al., 2005; Liu et al.,
2007), whereas MRGPRA3 expression specifies neurons
that induce itch without transducing nociceptive cues
(Han et al., 2013).
In addition to the highly selective expression of
several MRGPR in small-diameter primary sensory
neurons and the aforementioned expression of some
MRGPR in mast cells, MRGPRD to -H and MRGPRX2
expression was also found in other tissues. Significant
mRNA levels of MRGPRD were detected in urinary
bladder, testis, uterus, and arteries (Shinohara et al.,
2004). MRGPRE transcripts were also monitored in
medium- and large-diameter neurons of human DRG
sections (Zhang et al., 2005) and in other areas of the
central nervous system, including cerebral cortex,
hippocampus, spinal cord, and cerebellum, as well as
in human, mouse, and rat placenta (Zhang et al., 2005;
Milasta et al., 2006). MRGPRE and MRGPRF are also
expressed in enteric neurons (Avula et al., 2011),
belonging to one of the main divisions of the autonomic
nervous system that regulates gastrointestinal func-
tions. In accordance with these findings, transcripts of
MRGPRF, formally known as rat thoracic aorta re-
ceptor, were detected in small and large intestine (Ross
et al., 1990). More precisely, MRGPRE and MRGPRF
proteins were detected in both myenteric and sub-
mucosal neurons, the latter showing coexpression of
both receptors in 30–50% of neurons analyzed (Avula
et al., 2011). The same study reported that intestinal
schistosomiasis and trinitrobenzene sulfonic acid–
induced ileitis resulted in decreased expression of both
receptors, whereas other neuronal marker proteins,
e.g., calretinin or neuronal nitric oxide synthetase
(nNOS), were not affected by either inflammatory
insult. MRGPRH transcripts were also detectable in
the heart (Wittenberger et al., 2001), and expression of
human MRGPRX2 was described in the adrenal glands
and in several brain areas (Robas et al., 2003; Kamohara
et al., 2005).
D. Evolution
The MRGPR subfamilies D to G constitute evolu-
tionarily old genes that are conserved between rodents
and primates. Accordingly, although all MRGPR are
encoded on only one chromosome in each species
analyzed, the subfamilies D to G lie clustered together
a considerable distance apart from the chromosomal
region encoding subfamilies A to C in rodents or
subfamily X in primates (Zylka et al., 2003). The
phylogenetic relationship between the latter MRGPR
subfamilies poses the question of how and why the
entire MRGPR family developed in such a complex
576 Solinski et al.

manner during mammalian evolution. A current model
(see Fig. 2) rests on the assumption that an ancestral
progenitor gene cluster of one MRGPRA/C/X gene and
one MRGPRB gene existed before rodent speciation
(Zylka et al., 2003). An unequal crossing over event led
to local MRGPRB gene duplication in the rodent
lineage, whereas MRGRPB genes were completely lost
in the primate lineage. In rodents, the MRGPRA/C/X
progenitor developed into two separate gene subfami-
lies because of a further round of local gene duplica-
tion. Specifically in mice, the de novo insertion of the
L1 retrotransposon initiated several additional local
gene duplication events of MRGPRA and -C genes.
However, additional MRGPRC genes lost their tran-
scriptional regulatory elements and are now pseudo-
genes. This assumption is supported by the observation
that all MRGPR genes are found on only one chro-
mosome, e.g., chromosome 1q in rats or chromosome
7B in mice. Notably, such an evolutionary model would
also explain the highly specific expression of rodent
MRGPRA and -C and of human MRGPRX in primary
sensory neurons, assuming that those promoter ele-
ments crucial for the restrictive expression pattern
already existed in the progenitor gene and were pre-
served during gene duplication events (Dong et al.,
2001; Lembo et al., 2002; Zylka et al., 2003; Zhang
et al., 2005). Likewise, additional expression of
MRGPRX1 and -2 in human mast cells could be the result
of conserved ancestral promoter elements responsible for
MRGPRB expression in rat mast cells (Tatemoto et al.,
2006). Thus, MRGPRX1 and -2 inherited their re-
strictive expression in primary sensory neurons and mast
cells by a combination of gene expression regulatory
elements of rodent MRGPRA/C and certain rodent
MRGPRB genes.
The development of single MRGPRX genes in
primate evolution was also initiated by local gene
duplication (note that all human MRGPRX genes are

Fig. 2. Schematic model displaying the putative divergent evolution of the MRGPRA/B/C/X gene cluster in primates and rodents. Based on the data
provided by Zylka et al. (2003), a theoretical model explaining the evolutionary events leading form a putative ancestral MRGPR gene cluster to the
primate and rodent lineage and further on to species-specific distinctions in the rodent lineage is given.
 MAS-Related G Protein–Coupled Receptors
located on chromosome 11p15.1) (Dong et al., 2001;
Choi and Lahn, 2003; Zylka et al., 2003). Diversion of
distinct MRGPRX genes was driven by significant
positive selection pressure (Choi and Lahn, 2003;
Fatakia et al., 2011), resulting in amino acid sub-
stitutions in those parts of transmembrane domains
forming the ligand binding cavity, in extracellular
domains, and in the very C-terminal tail (Choi and
Lahn, 2003; Fatakia et al., 2011). From a structural
point of view, the latter changes may have resulted in
diverging receptor proteins with regard to ligand
specificity or affinity (extracellular domains, trans-
membrane domains) and the regulation of signaling
(C-terminal tail). From an evolutionary point of view,
the strong positive selection pressure indicates that
novel MRGPRX proteins are crucial for primate-
specific physiology and represent important dif-
ferences between primates and rodents. Indeed,
primate-specific genes are clearly over represented in
the fraction of human disease-causing genes (Hao et al.,
2010). Interestingly, a continuing positive selection
pressure on MRGPRX genes may account for frequent
polymorphisms of MRGPRX genes in the human pop-
ulation and, thus, account for differences in published
MRGPRX/SNSR subtypes.
III. Pharmacology and Physiology of
MAS-Related G Protein–Coupled Receptors
A. Preface
To date, no MRGPR subtype has officially been
declared "deorphanized" by NC-IUPHAR (Davenport
et al., 2013). This recommendation would require at
least two independent demonstrations of receptor-
ligand pairing published in refereed papers. To
"deorphanize" a receptor, NC-IUPHAR suggests using
radioligand binding and functional assays in in vitro
systems and native tissues in conjunction with ana-
tomic data indicating that the proposed ligand is
present to activate the receptor in a given tissue.
Additionally, genetic approaches that alter expression
levels of the receptor or the ligand might be beneficial.
So far, at least one of the criteria mentioned is lacking
for any given MRGPR. These criteria are particularly
hard to meet for human MRGPRX subtypes: because of
their specific expression in primates and their re-
stricted expression pattern in primary sensory neurons
and mast cells, it appears difficult to obtain data from
native tissues and to implement genetic approaches
that would alter protein expression levels. However,
knowledge of MRGPR biology and pharmacology is
expanding, a fact that is being appreciated by
NC-IUPHAR (Davenport et al., 2013). Interestingly, a
certain degree of pharmacologic overlap of nonorthologous
MRGPR members exists, although MRGPR phylogeny
is complex, including subfamilies that are specific to
rodents and others that are primate specific. Thus, we
577
will summarize the available pharmacologic data of
MRGPR members ordered by subfamily and review
the current knowledge about signaling cascades and
physiologic effects elicited by a given receptor-ligand
pair.
B. MAS-Related G Protein–Coupled Receptors A
1. Pharmacology. Endogenous or exogenous ligands
have been assigned to only 4 of 19 MRGPRA members,
i.e., rat MRGPRA and three murine MRGPRA (see
Table 3). The murine MRGPRA members 1 and 4 were
the first MRGPR to be analyzed in a functional assay.
After recombinant expression in human embryonic
kidney (HEK)293 cells, both receptors responded to
several peptides of the RFamide family (Dong et al.,
2001) first discovered in mollusks and soon shown
to modulate nociception in vertebrates (Price and
Greenberg, 1977; Yang et al., 1985). The mammalian
neuropeptide FF (NPFF) increased intracellular
calcium concentrations via MRGPRA1 with a potency
of ;200 nM, whereas the mammalian neuropeptide
AF (NPAF) likewise activated the MRGPRA4 sub-
type with a potency of ;60 nM. These findings were
subsequently confirmed by others (Han et al., 2002;
Liu et al., 2009). Both peptides are also known to
activate NPFF receptor 1 or 2 with potencies and
affinities in the nanomolar range (Mollereau et al.,
2002). Thus, if these neuropeptides represent the
endogenous agonists of murine MRGPRA1 or -4,
they would not be selective for the latter receptors.
In addition to endogenously occurring neuropep-
tides, murine MRGPRA1 was found to be a target of
the human version of the b-salusin peptide, employ-
ing fluorescence imaging plate reader technology
and MRGPRA1 overexpressing HEK293 or Chinese
hamster ovary (CHO) cells (Wang et al., 2006b). As
the orthologous murine peptide failed to activate
MRGPRA1, it was concluded that b-salusin is only
a surrogate ligand of MRGPRA1.
The antimalaria drug chloroquine was recently
shown to induce intracellular calcium transients in
cells expressing MRGPRA3 (Liu et al., 2009). This
response was characterized by an EC50 value of ;27 mM
after heterologous expression in HEK293 cells and
was also noted in dissociated primary sensory neurons.
A cluster knockout mouse deficient of several MRGPR
(including MRGPRA1-4, -A10, -A12, -A14, -A16, -A19,
-B4, -B5, and -C) failed to respond to chloroquine as
did wild-type neurons after small interfering RNA–
mediated MRGPRA3 knockdown, indicating that
MRGPRA3 are indeed targeted by chloroquine (Liu
et al., 2009). Notably, chloroquine also induced calcium
signals after recombinant expression of rat MRGPRA in
primary sensory neurons derived from the MRGPR
cluster knockout mouse (Liu et al., 2009).
The purine adenine might serve as an endogenous
agonist of rat MRGPRA, because in silico modeling
578
TABLE 3
Pharmacology of MAS-related G protein–coupled receptors A
MRGPRA subfamily members that have been assigned to a ligand are listed. All data were generated after
recombinant MRGPR expression.
Receptor Ligand EC50
nM
mMRGPRA1 FMRFamide 20
NPFF 200
hb-Salusin 300
chloroquine 300,000
mMRGPRA4 NPAF 60
mMRGPRA3 chloroquine 27,000
rMRGPRA chloroquine N.D.
adenine 3 cAMP inhibition
60
GTPgS, guanosine 59-3-O-(thio)triphosphate; N.D., not determined; r, rat; m, mouse.
revealed a putative adenine binding pocket in the rat
MRGPRA protein (Heo et al., 2007b). This assumption
was corroborated by the observation that adenine
bound to rat MRGPRA with a KD value of 24 nM,
when the receptor was overexpressed in CHO cells
(Bender et al., 2002). In this cellular model, adenine
inhibited forskolin-induced adenylyl cyclase activity
and induced guanosine 59-O-(3-[35S]thio)triphosphate
binding to the plasma membrane with potencies of ;3
and ;60 nM, respectively. However, inhibition of
cAMP production by rat MRGPRA points to Gi/o and
not to Gq/11 as downstream effectors in CHO cells. This
observation contrasts with the functional properties of
murine MRGPRA1 that was found to exclusively
engage classic Gq/11 signaling cascades (Han et al.,
2002; Wang et al., 2006b).
2. Signaling Cascades and Physiologic Effects.
Physiologic effects and detailed signaling cascades of
MRGPRA members have so far been described for
the murine chloroquine-sensitive subfamily member
MRGPRA3 (see Fig. 3). Intradermal application of
chloroquine induced a profound itching behavior at the
injection site (Liu et al., 2009). This procedure did not
lead to alloknesis, an aberrant sensation of itch when
nonitching skin near an itchy skin patch is lightly
touched (Akiyama et al., 2012). Chloroquine-induced
calcium signals in primary sensory neurons were
completely inhibited by removal of extracellular cal-
cium by EGTA and by ;90% after application of
ruthenium red, a blocker of transient receptor poten-
tial (TRP) cation channels (Liu et al., 2009). By use of
calcium imaging of cultured primary sensory neurons
and MRGPRA3 transfected NG108 cells, intracellular
signaling cascades were analyzed that might be re-
sponsible for the sensation of itch (Wilson et al., 2011).
Most interestingly, chloroquine induced an increase in
intracellular calcium through TRP ankyrin 1 (TRPA1)
channels, because chloroquine-induced itching was not
observed in TRPA1-deficient mice (Wilson et al., 2011).
The TRP vanilloid 1 (TRPV1) channel, an established
sensor of different painful stimuli such as heat and
protons (Caterina et al., 1997), is also expressed in
Solinski et al.
Readout System Reference
calcium HEK293-Ga15 Dong et al., 2001
calcium HEK293-Ga15 Dong et al., 2001
calcium HEK293 Wang et al., 2006b
calcium HEK293 Liu et al., 2009
calcium HEK293-Ga15 Dong et al., 2001
calcium HEK293 Liu et al., 2009
calcium mDRG neurons Liu et al., 2009
CHO Bender et al., 2002
GTPgS CHO Bender et al., 2002
chloroquine-responsive neurons (Liu et al., 2009;
Wilson et al., 2011). In contrast to TRPA1, TRPV1 is
not required for MRGPRA3-dependent neuronal exci-
tation, and chloroquine-induced calcium signals were
not detected in cells coexpressing MRGPRA3 and
TRPV1 (Wilson et al., 2011). Functional coupling
between MRGPRA3 and TRPA1 was assigned to Gbg
protein complexes because of the lack of calcium
signals after incubation with the Gbg inhibitor gallein.
The authors further showed that functional interac-
tions between TRPA1 and MRGPRA3 are required for
the physiologic effects of the latter. Than and co-
workers (2013) confirmed that direct activation of
MRGPRA3 by chloroquine excites murine primary
sensory neurons because of TRPA1 activation, because
the response of these neurons was abolished by the
TRPA1 inhibitor HC-030031 [1,2,3,6-tetrahydro-1,3-
dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7H-
purine-7-acetamide, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,
6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)
acetamide]. However, the authors revealed that
MRGPRA3 was also able to induce calcium signals
in TRPA1-negative neurons. By use of YM58483 (N-
[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-
4-methyl-1,2,3-thiadiazole-5-carboxamide) as a nonsubtype
selective canonical TRP and Pyr3 as a TRPC3 blocker,
TRPCs and, in particular, the TRPC3 subtype were
identified as the downstream effectors mediating
MRGPRA3-induced neuronal excitation in the ab-
sence of TRPA1 (Than et al., 2013). In addition, this
work showed profound sensitization of TRPV1 chan-
nels via chloroquine-induced protein kinase C (PKC)
activation and inhibition of the cold sensor TRP
melastatin 8 (TRPM8) via direct binding of the Gaq
subunit to the ion channel, because a Gaq chimera
unable to activate phospholipase C (PLC)b still
blocked TRPM8 activity in cells lacking functional
Gaq proteins (McKemy et al., 2002; Peier et al.,
2002). Thus, although acute signaling of MRGPRA3
via TRPA1 solely leads to itch (Liu et al., 2009;
Wilson et al., 2011), chloroquine also activates cel-
lular signaling cascades that might affect other
 MAS-Related G Protein–Coupled Receptors 579

Fig. 3. Schematic presentation of signaling cascades, downstream targets, and physiologic effects proposed for murine MRGPRA3. Upon chloroquine-
induced activation of MRGPRA3 in primary sensory DRG/trigeminal ganglia neurons, the activity of several TRP channels is modulated via distinct
mechanisms. TRPC3 is activated via an unknown mechanism, TRPA1 activation is mediated by Gbg subunits, TRPV1 is sensitized via PKC-mediated
phosphorylation, and TRPM8 is inhibited via Gaq signaling independently from PLCb activation. MRGPRA3-mediated TRPA1 activation leads to itch,
whereas physiologic effects of TRPA1-independent signaling cascades have not yet been determined. Used with permission.

physiologic processes, such as nociception or tem-
perature sensation.
C. MAS-Related G Protein–Coupled Receptors B
None of the rodent MRGPRB members has been
assigned to any ligand so far. However, in a reporter
mouse that carries a gene for placental alkaline
phosphatase under the control of the MRGPRB4
promoter, a rare subset of primary sensory neurons
that only innervates the hairy skin showed high
promoter activity (Liu et al., 2007). These neurons
were nonpeptidergic, TRPV1 negative, displayed
a broad arborization of their cutaneous termini, and
are often associated with hair follicles. Most interest-
ingly, MRGPRB4 positive neurons project to the spinal
lamina IIo, a spinal pain processing center that is part
of the substantia gelatinosa (Rexed, 1952). These
findings suggested that MRGPRB4 mark C-fiber tactile
afferents that are supposed to detect gentle touch
and stroking. Indeed, activation of MRGPRB4-positive
primary sensory neurons, but not activation of the
MRGPRB4 itself, by gentle stroking was demonstrated by
an elegant in vivo calcium imaging approach. Pharmaco-
genetic activation of MRGPRB4 expressing neurons
in freely moving mice resulted in conditioned place
preference, suggesting that activation of these neurons
is positively reinforcing and might be induced by
interindividual social interactions such as allogroom-
ing (Vrontou et al., 2013).
D. MAS-Related G Protein–Coupled Receptors C
1. Pharmacology. The rodent MRGPRC subfamily
consists of only one protein-encoding gene in rats and
mice, and both receptor subtypes have been suggested
to bind several ligands (see Table 4). After heterologous
expression in different cell systems, MRGPRC from rat
and mouse were activated to elicit calcium signals by
a set of different but structurally related peptides (see
Fig. 4A) with potencies in the low to medium nano-
molar range (Grazzini et al., 2004; Han et al., 2005;
580 Solinski et al.

TABLE 4
Pharmacology of MAS-related G protein–coupled receptors C
MRGPRC subfamily members are listed by the ligands to which they have been assigned. Data were generated after
recombinant MRGPRC expression, despite when endogenous expression is indicated by “(e)”.

Receptor Ligand EC50 System Reference

nM
mMRGPRC 11 HEK293 (GFP-tag) Han et al., 2002
g 2-MSH 340 HEK293 Heo et al., 2007a
290 HEK293 Solinski et al., 2013
53 HEK293 (GFP-tag) Han et al., 2002
BAM8–22 292 HEK293 Heo et al., 2007a
330 HEK293 Solinski et al., 2013
Dynorphin-14 22 HEK293 (GFP-tag) Han et al., 2002
310 HEK293 Solinski et al., 2013
NPFF 54 HEK293 (GFP-tag) Han et al., 2002
358 HEK293 Heo et al., 2007a
NPAF 282 HEK293 (GFP-tag) Han et al., 2002
SLIGRLamide 10,000 CHO Liu et al., 2011
50,000 DRG neurons (e) Liu et al., 2011
rMRGPRC g 2-MSH 44 HEK293-Gaqi5 Grazzini et al., 2004
440 HEK293 Solinski et al., 2013
Tyr6-g 2-MSH6–12 15 HEK293-Gaqi5 Grazzini et al., 2004
BAM8–22 120 HEK293-Gaqi5 Grazzini et al., 2004
2600 HEK293 Solinski et al., 2013
m, mouse; r, rat.

Heo et al., 2007a; Solinski et al., 2010, 2013). Among
these peptides are g 2-melanocyte stimulating hor-
mone (g2-MSH), BAM peptides, dynorphin-14, and
proneuropeptide-FF-A peptides. Notably, these pep-
tides originate from PENK (BAM), pro-opiomelanocortin
(g 2-MSH), prodynorphin (dynorphin-14), and NPPFA
(NPFF or NPAF) (see Fig. 4A). Such a wide range in
the binding profile of a distinct GPCR is quite unique
and suggests that many different physiologic pathways
rely on MRGPRC signaling. Activation of MRGPRC by
BAM peptides was validated in primary sensory
neurons of mice (Liu et al., 2009; Wilson et al., 2011)
and rats (Honan and McNaughton, 2007). However,
their similarities notwithstanding, several pharmaco-
logical differences between murine and rat MRGPRC
have also been observed (see Fig. 4B): NPFF peptides
displayed lower potency to activate the rat MRGPRC
compared with the murine counterpart, and dynorphin-
14 did not activate rat MRGPRC at all (Han et al.,
2002; Grazzini et al., 2004; Solinski et al., 2013).
Regarding efficacy, g2-MSH and BAM peptides are
the most efficacious activators of both MRGPRC, with
g2-MSH clearly surmounting BAM peptides (Solinski
et al., 2013). Using in silico modeling and site-directed
mutagenesis, Heo and coworkers (2007a) revealed
that Tyr110, Asp161, and Asp179 of murine MRGPRC
are crucially involved in agonist binding by interact-
ing with a common C-terminal end of all peptides,
which harbors the consensus sequence RF(Y)amide or
RF(Y)G. Notably, all three ligand-binding amino acids
are conserved in rat MRGPRC.
Several peptides that are now recognized as
MRGPRC agonists were previously shown to activate
distinct receptors from other GPCR families, e.g.,
g2-MSH also binds to melanocortin 3 receptors and
some BAM peptides to opioid receptors (OR). Interest-
ingly, ligand sharing with receptor subtypes of other
GPCR families is not restricted to MRGPRC, but
appears to be a common feature of many MRGPR as
summarized in Table 9 (Bowery et al., 1980; Quirion
and Weiss, 1983; Roselli-Rehfuss et al., 1993; de Lecea
et al., 1996; Fukusumi et al., 1997; Kaupmann et al.,
1997; Oosterom et al., 1999; Han et al., 2002; Lembo
et al., 2002; Mollereau et al., 2002; Robas et al., 2003;
Santos et al., 2003; Grazzini et al., 2004; Shinohara
et al., 2004; Gembardt et al., 2008; Lautner et al.,
2013). Although, ligand sharing between MRGPRC
and other GPCR subtypes is of physiologic interest, it
is also a major concern when considering the specificity
of distinct MRGPRC-activating peptides in vivo. In-
terestingly, after N-terminal truncation of g 2-MSH
(g 2-MSH6–12) or of BAM1–22 (BAM8–22) activity toward
MRGPRC but not toward melanocortin 3 receptor or
OR is preserved (Lembo et al., 2002; Grazzini et al.,
2004). Accordingly, g2-MSH6–12 or BAM8–22 are
regarded as specific MRGPRC ligands and can be used
in in vivo studies to analyze the physiologic effects of
MRGPRC in mice or rats. When considering g2-MSH or
BAM derivatives as putative endogenous MRGPRC
ligands, it is also important to investigate whether
these peptides are actually released from precursors
under physiologic or pathophysiological conditions.
g2-MSH and BAM1–22 have both been shown to be
present in several tissues, including spinal cord and
brain (Mizuno et al., 1980; Pelletier et al., 1981; Hollt
et al., 1982; DeBold et al., 1988a,b; Cai et al., 2007a).
Thus, peptides that bind other GPCRs and MRGPRC
could serve as endogenous ligands of the latter. The
specific MRGPRC agonist BAM8–22 was also identi-
fied in vivo after microdialysis of exogenous BAM1–25
 MAS-Related G Protein–Coupled Receptors
Fig. 4. Schematic presentation and comparison of the pharmacology of
BAM8–22-sensitive MRGPR. (A) Amino acid sequences and precursors
of the classic peptidergic MRGPRC agonists are given. Dashed lines
indicate that the synthesis of the peptide in vivo has not been detected
yet. The C-terminal consensus, which is proposed to be crucial for
MRGPRC binding, is highlighted in red. (B) The most potent agonists of
murine (m) or rat (r) MRGRPC and human (h) MRGPRX1 are provided.
The ligands are drawn to scale by linearly modifying the font size in
accord with their published potencies (Han et al., 2002; Lembo et al.,
2002; Grazzini et al., 2004; Liu et al., 2009, 2011; Solinski et al., 2013).
For simplicity, ligands that exhibit potencies more than 10 times lower
than g 2-MSH (in the case of MRGPRC) or BAM8–22 (in the case of
MRGPRX1) are not shown.
into the striatum of anesthetized rats, using a combi-
nation of solid-phase preconcentration capillary elec-
trophoresis and imaging matrix-assisted laser desorption/
ionization mass spectrometry (Zhang et al., 1999).
However, although the proteolytic machinery to generate
BAM8–22 in vivo is present in the brain, it has not yet
been shown that BAM8–22 or g2-MSH6–12 are produced
endogenously in tissues adjacent to primary sensory
neurons. Thus, it remains unclear whether and under
which circumstances specific MRGPRC ligands exist in
vivo.
Very recently, it was proposed that the "SLIGRL"-
peptide activates murine MRGPRC (Liu et al., 2011).
"SLIGRL" is not a classic neuropeptide released from
common precursor proteins but part of the tethered
ligand domain of murine protease-activated receptors 2
(PAR2) [for further reading see these comprehensive
reviews (Steinhoff et al., 2005; Ramachandran and
Hollenberg, 2008)]. PAR are GPCR that are activated
by serine proteases such as thrombin or trypsin. To
activate PAR, proteases cleave N-terminal peptides
from the PAR protein, thereby exposing a “cryptic”
PAR-activating sequence at the N terminus. Short
peptides that are derived from theses cryptic sequences
have also been shown to activate PAR without previous
cleavage, e.g., the "SLIGRL"-amide was established as
a PAR2-specific ligand. However, it may also be possible
581
that peptides such as the "SLIGRL"-peptide are re-
leased from PAR through consecutive cleavage by two
or more proteases and in turn serve as ligand of
distinct GPCR. By use of CHO cells recombinantly
expressing MRGPRC, small interfering RNA–
mediated MRGPRC knockdown in wild-type primary
sensory neurons, and primary sensory neurons from
MRGPRC-deficient mice, "SLIGRL"-induced calcium
signals in the presence, but not in the absence of the
MRGPRC were identified, indicating that the "SLIGRL"-
peptide is indeed a ligand of this receptor. Analysis of
structure-activity relationships revealed that amida-
tion of the "SLIGRL" C terminus is important for its
function as a ligand, which fits well to the proposed
RF(Y)G/amide consensus sequence of other MRGPRC
agonists. Notably, "SLIGRL"-induced itch, which was
believed to be mediated by PAR2 (Shimada et al.,
2006), was not changed in PAR2-deficient mice (Liu
et al., 2011). In MRGPRC overexpressing CHO cells,
a high EC50 value of ;10 mM for "SLIGRL"-induced
calcium signals was measured (Liu et al., 2011).
However, because PAR2 and MRGPRC are both ex-
pressed in primary sensory neurons, it is appealing to
speculate that the "SLIGRL" peptide might physiolog-
ically exist in concentrations high enough to stimulate
MRGPRC.
2. Signaling Cascades and Physiologic Effects.
a. Preface. Rat and murine MRGPRC elicit their
effects primarily by the activation of G proteins. Both
receptors were shown to induce intracellular calcium
release via activation of PLCb (Han et al., 2002;
Grazzini et al., 2004). For murine MRGPRC coupling
to Gq/11 proteins seems to be essential, because
MRGPRC-dependent calcium signaling was absent in
murine embryonic fibroblasts derived from Gq/11-deficient
embryos (Han et al., 2002). In accordance with their
selective expression in small-diameter primary
sensory neurons, MRGPRC signaling in mice and
rats has been implicated in somatosensory detector
functions in the skin. Indeed, published data on the
in vivo effects of MRGPRC agonists detected pain-
enhancing, pruritogenic, but also analgesic effects
(summarized in Fig. 5).
b. Pain-enhancing effects. Intraplantar injections
of the MRGPRC-specific peptides BAM8–22 or Tyr6-g 2-
MSH6–12 into juvenile or adult rats dose dependently
resulted in acute nocifensive behavior, thermal hyper-
algesia, and mechanical allodynia (Grazzini et al.,
2004; Ndong et al., 2009). Likewise, intrathecal in-
jection of MRGPRC agonists into juvenile or adult rats
and Kunming mice also induced acute pain-like be-
havior and thermal hyperalgesia (Grazzini et al.,
2004; Chang et al., 2009; Wei et al., 2010). MRGPRC
may also be responsible for inflammatory pain,
because complete Freund’s adjuvant (CFA)–induced
thermal hyperalgesia was alleviated by RNAi-mediated
MRGPRC knockdown in rats (Ndong et al., 2009).
582 Solinski et al.

Fig. 5. Schematic presentation of signaling cascades, downstream targets, and physiologic effects proposed for rodent MRGPRC. Three physiologic
functions of MRGPRC in primary sensory DRG/trigeminal ganglia neurons have been identified: pruritogenic, pain-enhancing, and analgesic effects.
Itch is caused via PLCb-mediated activation of TRPA1. Acute pain, thermal hyperalgesia, and mechanical allodynia were found to depend on PKC-
mediated TRPV1 sensitization, peripheral CGRP release, and spinal nitric oxide (NO) production and NMDA receptor activity. Different pain-inducing
paradigms (marked with red bolts) lead to acute nociceptor activity as well as adaptive changes including CGRP induction, spinal NO production, and
windup of spinal dorsal horn neurons. These changes are occasionally accompanied by MRGPRC induction and increased abundance of BAM1–22 that
can lead to enhanced MRGPRC signaling. Analgesic effects of MRGPRC are partly triggered by engagement of MOP signaling that counteracts
adaptive changes in DRG and spinal neurons. Chronic application of morphine leads to tolerance, among others via induction of similar adaptive
changes as in pain paradigms. Rat MRGPRC have been shown to counteract these processes.

Notably, pain-enhancing effects of MRGPRC can only be
assessed at low agonist doses (e.g., up to 20 nmol of Tyr6-
g2-MSH6–12) and in a rigid time window of a maximum
of 20 minutes postinjection (Wei et al., 2010). This low
dose effect may be due to the activation of an analgesic
off-target at higher peptide concentrations, most likely
the Kyotorphin receptor, which masked MRGPRC-
induced pain, whereas rapid peptide degradation may
be responsible for the short-term response (Grazzini
et al., 2004; Wei et al., 2010).
Several publications identified heat-sensitive TRPV1
ion channels as major downstream targets of MRGPRC
responsible for its pain-enhancing effects (Honan and
McNaughton, 2007; Hager et al., 2008; Ndong et al.,
2009; Wilson et al., 2011). In rat primary sensory
neurons, BAM8–22 sensitized TRPV1 for its agonist
capsaicin (CAP) via a PKC-dependent pathway (Honan
and McNaughton, 2007). The PKC isoforms d, «, and z
were excluded to be part of this signaling cascade,
because these isoforms did not translocate to the
plasma membrane after MRGPRC stimulation. TRPV1
sensitization was also made responsible for an in-
creased CAP- or heat-induced calcitonin gene-related
peptide (CGRP) release from rat or mouse paw skin
after preincubation with BAM1–22 in conjunction with
naloxone, an opioid receptor antagonist that blocks
effects of BAM1–22 on opioid receptors but still allows
BAM1–22 to act on MRGPRC (Hager et al., 2008).
 MAS-Related G Protein–Coupled Receptors
Interestingly, BAM8–22 or g 2-MSH alone failed to
induce CGRP release, and, surprisingly, a combination
of BAM1–22 and naloxone that sensitized TRPV1 for
CAP still induced CGRP release from skin prepara-
tions derived from TRPV1-deficient mice. In rats,
MRGPRC-mediated thermal hyperalgesia was abro-
gated by a specific TRPV1 inhibitor (Ndong et al.,
2009), and coexpression of TRPV1 with murine
MRGPRC in NG108 cells resulted in augmented calcium
signals by BAM8–22 (Wilson et al., 2011). Thus, TRPV1
has been defined as a common target for cellular sig-
naling induced by MRGPRC, which might be of high
physiologic significance because coexpression of TRPV1
with MRGPRC in rodent primary sensory neurons was
frequently observed (Lembo et al., 2002; Hager et al.,
2008; Liu et al., 2009).
In addition to TRPV1, Chang and coworkers (2009)
identified the spinal N-methyl-D-aspartate (NMDA)
receptor and the nNOS as players in MRGPRC-
mediated thermal hyperalgesia, because the NMDA
receptor antagonists D -APV [ D -(2 )-2-amino-5-
phosphonopentanoic acid] and MK-801 [(5S,10R)-
(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,
10-imine] and the nNOS inhibitor N G-nitro-L-arginine
methyl ester were found to dose-dependently inhibit
the pronociceptive effects of Tyr6-g 2-MSH6–12 in
mice (Chang et al., 2009). Because nNOS inhibitors
also diminished NMDA-induced pain-like behavior
(Kitto et al., 1992), it remained unclear whether both
players are directly modulated by MRGPRC in the
same cell.
c. Analgesic effects. In contrast to pain-
enhancing effects of MRGPRC, several studies have
established analgesic effects of MRGPRC-specific ago-
nists (Hong et al., 2004; Zeng et al., 2004; Chen et al.,
2006, 2008, 2012; Cai et al., 2007a; Guan et al., 2010;
Jiang et al., 2013). Acute nocifensive behavior after
formalin or NMDA injection was found to be diminished
dose-dependently by BAM1–22 plus naloxone and by
BAM8–22 or Tyr6-g 2-MSH6–12 applied alone (Hong
et al., 2004; Zeng et al., 2004; Chen et al., 2006, 2008). In
parallel, spinal c-Fos immune reactivity, an established
marker of nociception (Bullitt, 1989), was reduced by
MRGPRC agonists. Thermal hyperalgesia after intra-
plantar formalin or CFA injection, quantified either by
tail flick or Hargreaves’s tests, were found to be
diminished by MRGPRC agonists (Hong et al., 2004;
Guan et al., 2010; Jiang et al., 2013). Interestingly,
CFA-induced thermal hyperalgesia was augmented in
the MRGPR cluster knockout mice (see section III.B.1)
that are deficient for MRGPRC (Guan et al., 2010).
Mechanical hyperalgesia, a hallmark of CFA-induced
inflammatory and spinal nerve ligation– or chronic
constriction injury–induced neuropathic pain, was
also reduced by application of MRGPRC agonists
(Cai et al., 2007a; Guan et al., 2010; Chen et al.,
2012; Jiang et al., 2013). MRGPRC upregulation and
583
increased abundance of its agonist BAM1–22 have
been observed during the process of CFA-induced
pain chronification (Cai et al., 2007a; Jiang et al.,
2013). Consequently, acute intrathecal application of
a BAM1–22-neutralizing antibody decreased CFA-
induced mechanical hyperalgesia, thus highlighting
ongoing MRGPRC signaling during inflammatory
pain (Cai et al., 2007a). After CFA injection, enhanced
MRGPRC signaling inhibited the induction of CGRP
in primary sensory and of nNOS in spinal projection
neurons (Jiang et al., 2013), which is believed to exert
long lasting effects on spinal sensitization [for details
see (Latremoliere and Woolf, 2009)]. This was attrib-
uted to the engagement of m-OR (MOP), as evidenced
by the lack of inhibition after preincubation with the
MOP inhibitor CTAP.
d. Pruritogenic effects. Concomitant to the obser-
vation that chloroquine-induced scratching behavior is
mediated largely by murine MRGPRA3 signaling,
intradermal injections of BAM8–22 into the nape of
the neck of mice also elicited a profound scratching
behavior (Liu et al., 2009). This behavioral result was
confirmed by several other studies, the proposed
murine MRGPRC agonist "SLIGRL" also elicited itch,
and agonist-induced scratching was diminished to
;25% in the MRGPR cluster knockout mouse (see
section III.B.1) that lacks MRGPRC (Liu et al., 2009,
2011; Wilson et al., 2011; Akiyama et al., 2012; Han
et al., 2013). However, intradermal injections into the
nape of the neck as a secure test for itch were
questioned because pain-inducing algogens and pruri-
togens elicit nearly indistinguishable reactions after
this application procedure (Shimada and LaMotte,
2008). Instead the “cheek model of itch” was proposed,
which is based on intradermal cheek injections and
enables discrimination of itch and pain. Cheek injec-
tions of pain-inducing algogens are followed by intense
wiping, whereas pruritogens elicit a vigorous scratch-
ing response. Therefore it is important to note that
BAM8–22 injected into the cheek of mice elicited only
scratching without any wiping reaction (Wilson et al.,
2011). The notion that murine MRGPRC cause itch
without pain is further accompanied by the finding
that ablation of MRGPRA3-positive primary sensory
neurons that almost all express MRGPRC (Zylka et al.,
2003; Liu et al., 2009) completely abolished BAM8–22-
or "SLIGRL"-induced scratching (Han et al., 2013). The
latter study is of particular interest because it showed
that MRGPRA3/MRGPRC-positive neurons are involved
in itch, but not pain perception in mice. Noteworthy,
activation of MRGPRC by BAM8–22, but not by
"SLIGRL" induced alloknesis in addition to acute
itch, which were both independent of histamine
signaling (Akiyama et al., 2012).
Wilson and coworkers (2011) analyzed downstream
effectors involved in MRGPRC-induced itching. Al-
though the percentage of primary sensory neurons that
584 Solinski et al.

elicited BAM8–22-induced calcium signals was dimin-
ished in TRPV1-deficient cells, action potentials after
BAM8–22 application were not altered. In contrast,
TRPA1 deficiency resulted in the reduction of both
responses, calcium signals and action potentials. Ac-
cordingly, BAM8–22-induced scratching responses were
absent only in TRPA1-deficient mice, whereas TRPV1
deficiency was dispensable.
E. MAS-Related G Protein–Coupled Receptors D
1. Pharmacology. The MRGPRD subfamily consists
of only one receptor subtype per species, which is
conserved in rodents and primates, and several ligands
have been implicated in MRGPRD pharmacology (see
Table 5). b-[3H]Alanine is able to bind to the human
MRGPRD after heterologous expression in CHO cells
(Shinohara et al., 2004). Measuring calcium signals in
these cells, an EC50 value of ;15 mM was determined
that is three to 4-fold below b-alanine concentrations
observed in rat sciatic nerve or cat brain samples
(Tallan et al., 1954; Marks et al., 1970). In line with
this observation for human MRGPRD, rat or mouse
MRGPRD also responded to b-alanine after heterolo-
gous expression in CHO cells with calcium signals and
EC50 values between 3 and 44 mM (Shinohara et al.,
2004; Zhang et al., 2007; Ajit et al., 2010). b-Alanine
induced signaling in MRGPRD-enriched fractions of
primary sensory neurons from rat (Crozier et al., 2007)
and calcium signals or action potentials in murine
primary sensory neurons (Liu et al., 2012). Thus,
b-alanine has been established as a ligand of MRGPRD
and could be responsible for their endogenous effects.
Interestingly, two other amino acids, GABA and
b-aminoisobutyric acid, have also been shown to induce
calcium signals via human MRGPRD, although with
lower potency compared with b-alanine (Shinohara
et al., 2004; Ajit et al., 2010; Uno et al., 2012).
However, because b-alanine and GABA also bind to
other proteins, including GPCR and ion channels
(Bowery, 1993; Macdonald and Olsen, 1994; Tiedje
et al., 2010), in vivo effects of both amino acids would
have to be interpreted with caution regarding the
molecular receptor entity.
In addition to amino acids, two peptides produced by
the renin-angiotensin system have also been identified
as MRGPRD ligands. Precisely, angiotensin-(1–7) and
alamandine [Ala1-angiotensin-(1–7)] were proposed to
activate MRGPRD (Gembardt et al., 2008; Lautner
et al., 2013). Angiotensin-(1–7) is also established to
bind the MAS1 receptor in vivo (Santos et al., 2003)
and is therefore not specific for MRGPRD. Further
screening approaches revealed novel exogenous com-
pounds that specifically activate or inhibit MRGPRD
activity (Zhang et al., 2007; Ajit et al., 2010; Uno et al.,
2012). These specific ligands will be very useful tools
for the analysis of the in vivo effects of MRGPRD in the
future.
2. Signaling Cascades and Physiologic Effects.
Several cellular studies agree that MRGPRD couple to
Gq/11 and pertussis toxin (PTX)–sensitive Gi/o proteins,
because MRGPRD induced PTX-insensitive calcium
releases and PTX-sensitive inhibition of forskolin-
induced adenylyl cyclase activation (Shinohara et al.,
2004; Ajit et al., 2010). Gi/o coupling links MRGPRD to
KCNQ2/3 channels, and inhibition of KCNQ2/3 activity
is mainly responsible for the inhibition of noninactivating
potassium currents (M-currents) induced by MRGPRD in

TABLE 5
Pharmacology of MAS-related G protein–coupled receptors D
MRGPRD subfamily members are listed by the ligands to which they have been assigned. All data were generated
after recombinant MRGPRD expression.

Receptor Ligand EC50 Readout System Reference

nM
hMRGPRD b-Alanine 15 calcium CHO Shinohara et al., 2004
4 calcium CHO Ajit et al., 2010
23 calcium HEK293 Uno et al., 2012
220 GTPgS HEK293 Uno et al., 2012
350 IP HEK293 Uno et al., 2012
GABA 191 calcium CHO Shinohara et al., 2004
53 calcium CHO Ajit et al., 2010
4000 calcium HEK293 Uno et al., 2012
BABA 53 calcium HEK293 Uno et al., 2012
820 GTPgS HEK293 Uno et al., 2012
380 IP HEK293 Uno et al., 2012
Angiotensin-(1–7) N. D. arachidonic acid COS Gembardt et al., 2008
Alamandine N. D. nitric oxide CHO Lautner et al., 2013
mMRGPRD b-Alanine 44 calcium CHO Shinohara et al., 2004
rMRGPRD b-Alanine 14 calcium CHO Shinohara et al., 2004
4 calcium CHO Ajit et al., 2010
1 calcium CHO Zhang et al., 2007
GABA 191 calcium CHO Shinohara et al., 2004
50 calcium CHO Ajit et al., 2010
L-Carnosine 34 calcium CHO Shinohara et al., 2004
BABA, b-aminoisobutyric acid; GTPgS, guanosine 59-3-O-(thio)triphosphate; h, human; IP, phosphoinositol pro-
duction; m, mouse; N.D., not determined; r, rat.
 MAS-Related G Protein–Coupled Receptors
primary sensory neurons (Crozier et al., 2007). This
signaling cascade was implicated to contribute to
MRGPRD-dependent enhanced neuronal activity
(Crozier et al., 2007). Likewise, decreased neuronal
activity was detected in MRGPRD-deficient mice (Rau
et al., 2009).
Just like other MRGPR members, MRGPRD also
specifically mark a discrete subset of primary sensory
neurons. Genetically engineered axonal tracers map
the stratum granulosum of the skin as the only pe-
ripheral target of MRGPRD-positive neurons (Zylka
et al., 2005). MRGPRD neurons form synapses in the
spinal lamina II (Zylka et al., 2005) and convey their
signal monosynaptically to almost all known classes of
spinal lamina II neurons, as evidenced by an optoge-
netical circuit mapping approach (Wang and Zylka,
2009). MRGPRD-positive neurons were classified as
nonpeptidergic C-fiber nociceptors that coexpress the
ionotropic ATP receptor P2X3, tetrodotoxin-insensitive
NaV channels, MOP, and partly the TRPV1 ion channel
(Shinohara et al., 2004; Zhang et al., 2005; Zylka et al.,
2005; Dussor et al., 2008). The functional analysis of
these cells for sensory transduction revealed a complex
picture. MRGPRD-deficient mice exhibit deficits in
the detection of mechanical, heat, and cold stimuli,
whereas ablation of MRGPRD-positive neurons only
impacted acute mechanical pain and mechanical
hyperalgesia in a CFA model of inflammatory pain,
whereas heat and cold transduction was completely
preserved (Cavanaugh et al., 2009; Rau et al., 2009).
In accordance with the latter finding, ablation of
MRGPRD-positive primary sensory neurons reduced
the firing rate and abundance of spinal dorsal horn
neurons responding to mechanical but not heat stimuli
(Zhang et al., 2013). Finally, stimulation of MRGPRD-
positive neurons with b-alanine only induced calcium
signals and action potentials in ;40% of genetically
labeled MRGPRD-expressing cells (Liu et al., 2012).
This response elicited itch in mice, because itch was
completely abrogated by MRGPRD deficiency. In vivo
electrophysiological recordings revealed that b-alanine–
activated neurons are heat- and mechanosensitive,
whereas the remaining heat-insensitive subset of
MRGRPD neurons that did not respond to b-alanine
was solely mechanosensitive. Thus, the latter pop-
ulation might be responsible for deficits in mechanical
pain that was observed after ablation of MRGPRD-
positive neurons (Cavanaugh et al., 2009). Interestingly,
intradermal application of b-alanine during a pilot
human study that comprised 11 individuals also resulted
in itch, pricking/stinging, and burning (Liu et al., 2012),
indicating that MRGPRD similarly affect itching in hu-
mans and rodents.
Transcripts of MRGPRD were also detected in other
tissues than in primary sensory neurons, including
arteries (Shinohara et al., 2004). Moreover, the angio-
tensinogen cleavage product alamandine circulates in
585
human blood and produces physiologic effects that
resemble those produced by angiotensin-(1–7), includ-
ing relaxation of aortic rings. These actions of
alamandine were not mediated via MAS1 or angioten-
sin II type 2 receptors (AT2R) but required MRGPRD
activation (Lautner et al., 2013). Surprisingly, the
abovementioned MRGPRD agonist b-alanine did not
relax aortic rings, but blocked the effects of alaman-
dine, suggesting that both MRGPRD agonists direct
MRGPRD-induced signaling to different pathways.
Effects of MRGPRD in primary sensory neurons and
the vasculature are summarized in Fig. 6.
F. MAS-Related G Protein–Coupled Receptors E to -H
The subfamilies MRGPRE to -H consist of only one
receptor per species and are conserved in rodents and
primates, except for MRGPRH that only exists in
rodents. So far, no member of these MRGPR has ever
been assigned to any ligand, which severely hampered
their functional characterization. A MRGPRE-deficient
mouse strain, which exhibited normal acute pain
responses in a hot plate assay but showed a trend
toward decreased nocifensive behavior in both phases
of the formalin test as well as statistical significant
deficits in the induction, but not maintenance, of
mechanical allodynia after chronic constriction injury,
was also established (Cox et al., 2008).
G. MAS-Related G Protein–Coupled Receptors X
1. Pharmacology.
a. MAS-related G protein–coupled receptors X1.
The human MRGPRX1 was the first primate-specific
MRGPR to be assigned to a ligand, and several ad-
ditional agonistic and antagonistic compounds have
been identified thereafter (see Table 6). MRGPRX1
have been shown to bind BAM peptides with high
affinity (KD value of BAM8–22 ;10 nM) after expres-
sion in HEK293 cells (Lembo et al., 2002). Likewise,
BAM peptides elicited intracellular calcium release in
the same cells (Lembo et al., 2002). In accord with
rodent MRGPRC, agonistic activity of BAM peptides
was completely preserved in an N-terminal truncated
form of BAM1–22 (BAM8–22). EC50 values of either
BAM8–22 or BAM1–22 and MRGPRX1 in calcium
assays varied between ;14 and ;800 nM (Lembo
et al., 2002; Burstein et al., 2006; Tatemoto et al., 2006;
Shemesh et al., 2008; Malik et al., 2009; Solinski et al.,
2013).
Despite sharing BAM peptides as agonists, human
MRGPRX1 and rodent MRGPRC also displayed phar-
macological differences (see Fig. 4B). For instance,
proneuropeptide-FF-A cleavage products or dynorphin-
14, which are partial agonists of MRGPRC, did not
activate MRGPRX1 at all, and importantly, g 2-MSH,
the most potent and efficacious agonist of rodent
MRGPRC, only faintly activated MRGPRX1 when
chimeric G proteins were coexpressed (Lembo et al.,
586 Solinski et al.

Fig. 6. Schematic presentation of signaling cascades, downstream targets, and physiologic effects proposed for MRGPRD in primary sensory DRG/
trigeminal ganglia (TG) neurons and blood vessels. b-Alanine–induced activation of MRGPRD leads to activation of Gi/o and Gq/11 proteins. KCNQ2/3
channels that conduct the important background M-current are inhibited by MRGPRD via Gi/o and possibly Gq/11 proteins. MRGPRD have been shown
to elicit pruritogenic sensations and to contribute to normal mechanical and thermal pain thresholds. These distinct actions might be deployed by
MRGPRD signaling in two distinct populations of primary sensory neurons. In blood vessels, MRGPRD are activated by the angiotensinogen
metabolite alamandine. Signaling cascades that are engaged by alamandine include the endothelium-derived production of nitric oxide (NO) that leads
to vasorelaxation.

2002; Solinski et al., 2013). Moreover, cyclic dimers of
the C-terminal part of g2-MSH were introduced to
antagonize BAM8–22-induced MRGPRX1 activation,
but do not bind rodent MRGPRC (Schmidt et al., 2009).
Therefore, the common RF(Y)G/amide consensus of
MRGPRC agonists does not apply to MRGPRX1. As
a consequence MRGPRC exhibit a high promiscuity
toward many ligands, whereas MRGPRX1 are much
more restrictive and solely bind BAM peptides, a
feature conserved to a certain degree in the MRGPRX1
of rhesus monkeys (Burstein et al., 2006).
In addition to potential endogenous agonists, exog-
enous agonists of MRGPRX1, e.g., tetracyclic benzimi-
dazoles, have been proposed (Malik et al., 2009).
Furthermore, 2,4-diaminopyrimidine derivates and 2,3-
disubstituted azabicyclooctanes antagonize BAM8–22-
induced signaling via MRGPRX1 (Kunapuli et al.,
2006; Bayrakdarian et al., 2011). Interestingly, the
MRGPRA3 ligand chloroquine also activated MRGPRX1,
although affinity was 1000-fold and efficacy ;2.5-fold
reduced compared with BAM8–22 (Liu et al., 2009).
Hence, in accordance with its evolutionary relation

TABLE 6
Pharmacology of human MAS-related G protein–coupled receptors X1
The potency of agonists is given as the EC50 value to induce calcium signaling in the given cellular system and the
potency of antagonists is given as the IC50 value to inhibit BAM8–22-induced calcium signaling in the given cellular
system. All data were generated after recombinant MRGPRX1 expression.

EC50 IC50 System Reference

nM
Agonist
BAM8–22 14 HEK293-Gaqi5 Lembo et al., 2002
25 HEK293 Burstein et al., 2006
40 HEK293 Tatemoto et al., 2006
8–50 CHO Kunapuli et al., 2006
150 HEK293 Solinski et al., 2013
BAM1–22 16 HEK293-Gaqi5 Lembo et al., 2002
800 HEK293 Burstein et al., 2006
80 CHO-Ga16 Shemesh et al., 2008
800 HEK293 Malik et al., 2009
Chloroquine 300,000 HEK293 Liu et al., 2009
Benzimidazole derivates 320 HEK293 Malik et al., 2009
Antagonist
Cyclic g 2-MSH6–12 derivates 12 HEK293-Gaqi5 Schmidt et al., 2009
Diaminopyrimidine derivates 14 HEK293-Gaqi5 Bayrakdarian et al., 2011
Azabicyclooctane derivates 100 CHO Kunapuli et al., 2006
 MAS-Related G Protein–Coupled Receptors 587

MRGPRX1 share some pharmacological character-
istics with rodent MRGPRC and MRGPRA subfamily
members but also display unique pharmacological
features.
b. MAS-related G protein–coupled receptors X2.
The MRGPRX2 subtype is clearly distinguishable from
MRGPRX1 regarding its pharmacology (see Table 7),
because MRGPRX2 do not bind BAM peptides
(Burstein et al., 2006). However, several other pep-
tidergic ligands have been proposed in different heter-
ologous or endogenous expression systems (Robas
et al., 2003; Kamohara et al., 2005; Burstein et al.,
2006; Tatemoto et al., 2006; Shemesh et al., 2008;
Malik et al., 2009; Kashem et al., 2011; Liu et al., 2011;
Subramanian et al., 2011a,b, 2013). The best charac-
terized MRGPRX2 ligand is the cortistatin-14 peptide
that activated MRGPRX2 with potencies in the
medium to high nanomolar range (Robas et al., 2003;
Kamohara et al., 2005; Burstein et al., 2006; Shemesh
et al., 2008; Malik et al., 2009). Notably, similar to
MRGPRX1, the ligand binding profile of MRGPRX2 is
conserved in rhesus monkeys, because cortistatin-14
also activates MRGPRX2 from this species (Burstein
et al., 2006; Malik et al., 2009).
MRGPRX2 show a broader expression pattern than
other MRGPRX, because MRGPRX2 expression was
detected in primary sensory neurons, several brain
areas, mast cells, and the adrenal medulla (Kamohara
et al., 2005). In line with this observation, proadreno-
medullin peptides that endogenously occur as side
products during adrenomedullin synthesis in the
adrenal medulla have also been shown to activate
MRGPRX2 (Kamohara et al., 2005). MRGPRX2 were
also shown to be involved in mast cell activation by
a set of structurally similar, endogenous (Tatemoto
et al., 2006; Subramanian et al., 2011a, 2013) or
exogenous (Kashem et al., 2011; Subramanian et al.,
2011b) basic secretagogues (see section III.G.2.d).
Finally, in concordance with murine MRGPRC, the
human PAR2-derived "SLIGKV"-peptide was shown to
activate MRGPRX2 after expression in CHO cells at
a concentration of 20 mM (Liu et al., 2011).
c. MAS-related G protein–coupled receptors X3
and -4. So far no study reported activation of MRGPRX3
and -4 by a given ligand. Therefore, data about signaling
induced by MRGPRX3 and -4 or their biologic role are
not available at present. For MRGPRX3 it has been
shown that expression of this receptor subtype under
control of the b-actin promoter in rats resulted in
increased proliferation of lens fiber cells and keratino-
cytes in basal and suprabasal layers of the skin (Kaisho
et al., 2005). With regard to the MRGPRX4, a screening
approach using human colorectal cancer cells revealed
that the MRGPRX4 protein is one of 15 mutational hot
spots in these cancer cells (Gylfe et al., 2013). Thus,
MRGPRX3 and -4 may behave as oncogenes in certain
cancers.
2. Signaling Cascades and Physiologic Effects.
a. Preface. MRGPRX1 and -2 function via acti-
vation of G proteins, as indicated by their ability to
induce guanosine 59-O-(3-[35S]thio)triphosphate incor-
poration into plasma membrane fractions (Kamohara
et al., 2005; Burstein et al., 2006; Tatemoto et al.,
2006). Both receptors further induced the release of
calcium from internal stores via PLCb-mediated pro-
duction of inositol-1,4,5-trisphosphate (Lembo et al.,

TABLE 7
Pharmacology of human MAS-related G protein–coupled receptors X2
Potency of the ligand is given as the EC50 value. The lowest concentration (LC) used to elicit a significant effect when measuring the respective readout is given. Data were
generated after recombinant MRGPRX2 expression, despite when endogenous expression is indicated by “(e)”.

Ligand EC50 LC Readout System Reference

nM
Cortistatin-14 25 calcium HEK293-Ga15 Robas et al., 2003
50 calcium CHO Kamohara et al., 2005
65 cAMP inhibition CHO Kamohara et al., 2005
30 GTPgS CHO Kamohara et al., 2005
2000 calcium HEK293 Burstein et al., 2006
610 calcium CHO-Ga16 Shemesh et al., 2008
500 calcium HEK293 Malik et al., 2009
PAMP1-12 60 calcium CHO Kamohara et al., 2005
40 cAMP inhibition CHO Kamohara et al., 2005
20 GTPgS CHO Kamohara et al., 2005
Substance P 8000 calcium HEK293 Tatemoto et al., 2006
Benzimidazole derivates 400 calcium HEK293 Malik et al., 2009
SLIGKVamide N.D. calcium CHO Liu et al., 2011
PMX-53 100 calcium LAD2 (e), RBL-2H3, human Subramanian et al., 2011b
10 degranulation mast cells (e)
E7 1000 calcium HMC-1 (e), LAD2 (e), human Kashem et al., 2011
10 degranulation mast cells (e)
Substance P 100 degranulation human mast cells (e) Tatemoto et al., 2006
LL-37 100 calcium LAD2 (e), HMC-1, RBL-2H3, Subramanian et al., 2011a
1000 degranulation human mast cells (e)
b-Defensins 100 calcium LAD2 (e), RBL-2H3, HEK293, murine Subramanian et al., 2013
100 degranulation mast cells, human mast cells (e)
GTPgS, guanosine 59-3-O-(thio)triphosphate; N.D., not determined.
588 Solinski et al.
2002; Robas et al., 2003; Breit et al., 2006; Solinski
et al., 2012). Different groups agree that activation of
Gq/11 proteins is involved in this process (Lembo et al.,
2002; Robas et al., 2003; Kamohara et al., 2005; Breit
et al., 2006; Burstein et al., 2006). However, apart from
its Gq/11 coupling, additional coupling to PTX-sensitive
Gi/o proteins was described for MRGPRX1 (Chen and
Ikeda, 2004; Gales et al., 2005; Burstein et al., 2006) or
-2 (Kamohara et al., 2005; Subramanian et al., 2013).
According to their expression pattern, MRGPRX1
and -2 may be involved in somatosensory detector
functions and plasticity of primary sensory neurons
(see sections III.G.2.b and III.G.2.c) and in mast cell
biology (see section III.G.2.d). However, despite specific
expression, data highlighting MRGPRX2-mediated
functions in primary sensory neurons are currently
elusive. Moreover, MRGPRX2 may also induce hypo-
tension and slow-wave sleep (see section III.G.2.e).
Figures 7 and 8 give an overview of effects induced by
MRGPRX1 or MRGPRX2, respectively.
b. Somatosensory functions. To shape an organ-
ism’s somatosensory performance, a GPCR needs to
transfer stimuli detected in the skin into enhanced
or reduced primary sensory neuron activity. Indeed,
MRGPRX1 have been shown to modulate the activity
of several distinct ion channels, thereby shaping
neuronal activity. After heterologous expression in rat
DRG, hippocampal, and superior cervical ganglion
neurons, MRGPRX1 inhibited voltage-gated calcium
channels, at least partly via PTX-sensitive Gi/o proteins,
leading to decreased synaptic transmission (Chen and
Ikeda, 2004). On the other hand, MRGPRX1 also
inhibited M-type potassium channels via Gq/11 proteins
in the same cells, thereby relieving the neuron from
a background potassium conductance that limits excit-
ability (Chen and Ikeda, 2004). In line with this notion,
human MRGPRX1 induced action potentials after
heterologous expression in murine DRG neurons (Liu
et al., 2009). The pain-enhancing TRPV1 might be
involved in MRGPRX1-induced sensory neuron excita-
tion, inasmuch as MRGPRX1 were shown to sensitize
and directly activate this pain sensor (Solinski et al.,
2012). Interestingly, MRGPRX1 engaged TRPV1 via two
distinct signaling pathways: sensitization took place via
PKC-mediated phosphorylation of serine residues 502
and 800 of the channel, whereas direct TRPV1 activa-
tion relied on diacylglycerol binding to channel regions
encompassing tyrosine 511 and phosphatidylinositol-
3,4-bisphosphate (PIP2) degradation. This multifaceted
modulation of TRPV1 activity is unique among the
GPCR superfamily (Chuang et al., 2001; Prescott and
Julius, 2003; Woo et al., 2008; Kim et al., 2009; Solinski
et al., 2012) and, thus, points to an important role of the
TRPV1-MRGPRX1 regulatory axis in somatosensation.
The fact that MRGPRX1 were shown to either
dampen or increase neuronal activity raises the
question about the mechanisms responsible for these
opposing effects. In this context it should be mentioned
that distinct effects of MRGPRX1 on neuronal activity
were observed in different cell types and that in-
hibition of neuronal activity apparently depends on Gi/o
and increased activity on Gq/11 signaling. Because G
protein coupling of a given GPCR might differ in
distinct cell types (Hermans, 2003; Kukkonen, 2004),
distinct G protein coupling of the MRGPRX1 in dif-
ferent cells might be the most straightforward expla-
nation for its opposing effects on neuronal activity.
Because of the lack of endogenous model systems,
effects of MRGPRX1 on neuronal activity have so far
only been analyzed after overexpression of the
MRGPRX1 protein also affecting its G protein coupling
profile (Kenakin, 1997, 2006). Thus, at this point it is
not clear whether MRGPRX1 can act analgetically by
decreasing neuronal activity under certain conditions
and algetically by increasing neuronal activity in
different circumstances. Alternatively, the receptors
may even induce both effects at the same time, such
that the net effect in a given cell would depend on
which of the two signaling pathways dominates the
other. It will be an important task for the future to
analyze whether and how MRGPRX1 regulate the
activity of primary sensory neurons of primates and,
thus, nociception in vivo. These experiments are dif-
ficult to perform, because functional primary sensory
neurons from humans are not available and rodents do
not harbor MRGPRX1-encoding genes. However, ro-
dent MRGPRC and human MRGPRX1 both bind
BAM8–22, which has been shown to induce pain-
enhancing, analgesic, and pruritogenic effects in mice
or rats (see section III.D.2). Interestingly, in a seminal
study in which BAM8–22 was applied to the skin of
healthy human volunteers using cowhage spicules,
acute itching accompanied by nociceptive sensations
(pricking, stinging, and burning) was reported (Sikand
et al., 2011), thus showing that BAM8–22 acts
similarly in rodents and humans and that MRGPRX1
function as somatosensory receptors with nociceptive
and pruriceptive actions. However, because application
by cowhage spicules is more likely to elicit itch
compared with pain (Sikand et al., 2009), more work
is needed to determine unequivocally if MRGPRX1 ex-
ert acute functions in pruriceptive and/or nociceptive
primary sensory neurons in vivo.
c. Primary sensory neuron plasticity.
Small-diameter primary sensory neurons respond to
various acute or chronic stimuli, thereby showing high
plastic changes in connectivity, morphology, or re-
ceptor expression pattern [see Woolf and Ma (2007) for
further reading]. Interestingly, pathways that were
implicated in adaptive responses of primary sensory
neurons are well established players of proliferative
signaling in dividing cells. One example is the extra-
cellular signal-regulated kinase (ERK)-1/2 pathway
that was implicated in adaptive responses of the
 MAS-Related G Protein–Coupled Receptors 589

Fig. 7. Schematic presentation of signaling cascades, downstream targets, and physiologic effects proposed for human MRGPRX1 in primary sensory
DRG/trigeminal ganglia (TG) neurons and mast cells. (A) Application of BAM8–22 into human skin elicits histamine-independent pruriceptive and
nociceptive sensations. Recombinant human MRGPRX1 inhibit voltage-dependent calcium channels via Gi/o proteins and M-current-conducting
KCNQ2/3 channels via Gq/11 proteins. TRPV1 activity is enhanced by recombinant MRGPRX1 through PKC, diacylglycerol (DAG), and PIP2. (B) Effects
of sustained MRGPRX1 activation in recombinant cell systems include ERK-1/2 and NFAT activation, which induces c-Fos, early growth response
protein 1 (EGR-1), and CCR2 expression. In a human mast cell line endogenously expressing MRGPRX1, BAM8–22 enhanced the release of the CCR2
agonist chemokine ligand 2 (CCL2).

nociceptive system to chronic inflammation (Ko et al.,
2005; Wang et al., 2006a; Utreras et al., 2009; Su et al.,
2010; Romero et al., 2012).
Several indications point to the engagement of
nuclear signaling pathways by MRGPRX, leading to
transcriptional modulation. Overexpression of MRGPRX1
or -4 increased proliferation of 3T3 fibroblasts,
highlighting considerable constitutive activity of these
MRGPRX after recombinant expression (Burstein et al.,
2006). We recently found that after expression in
primary sensory neuron–derived F11 cells, MRGPRX1
induced activation of ERK-1/2 and serum response
element–dependent transcription as well as ERK-1/2–
dependent expression of the immediate early genes
c-Fos and early growth response protein 1 (Solinski
et al., 2013). Because of the published pathophysiologic
importance of this pathway in rodents and humans (Ko
et al., 2005; Wang et al., 2006a; Utreras et al., 2009; Su
et al., 2010; Romero et al., 2012), MRGPRX1 may
represent interesting targets in chronic inflammatory
pain if this pathway is also activated by MRGPRX1 in
vivo as well.
590 Solinski et al.

Fig. 8. Schematic presentation of signaling cascades, down-stream targets, and physiologic effects proposed for human MRGPRX2 in mast cells and
potential effects in chromaffin cells and hippocampal neurons. (A) Several agonists have been implicated in mast cell degranulation via activation of
MRGPRX2 including the C5a receptor antagonist PMX-53, the C3a receptor super-agonist E7, substance P (SP), the cathelicidin LL-37, and
b-defensins. PTX-sensitive Gi/o proteins via PKC and PLCb-activating G proteins, presumably Gq/11, elicit IgE-independent degranulation of connective
tissue type mast cells. (B) MRGPRX2 have been proposed to cause hypotensive and slow-wave sleep inducing effects. Hypotension might be mediated
via adrenomedullary PAMP1–20-induced inhibition of (nor)epinephrine release. Sleep phase modulation is conveyed by cortistatin-14 (COR-14) and
potentially engages MRGPRX2-induced reduction of hippocampus excitability.

It is interesting to mention that MRGPRX1 not only
signal to the nucleus via classic proliferative pathways
but also induced transcriptional alterations in primary
sensory neurons via the calcineurin-dependent tran-
scription factor nuclear factor of activated T cells
(NFAT) (Solinski et al., 2013). Activation of NFAT
was already implicated in several plastic changes of
primary sensory neurons, for instance axonal growth
(Graef et al., 2003; Nguyen and Di Giovanni, 2008) and
activity-dependent transcription (Groth et al., 2007;
Jackson et al., 2007; Jung and Miller, 2008). Moreover,
BAM8–22-induced NFAT activation led to increased
expression of chemokine receptors 2 (CCR2) in primary
sensory neurons after recombinant MRGPRX1 expres-
sion (Solinski et al., 2013). CCR2 were consistently
found to be upregulated in primary sensory neurons
under neuropathic conditions and to cause pain chron-
ification (Abbadie et al., 2003; Sun et al., 2006; Bhangoo
et al., 2007, 2009; White et al., 2007; Jung et al., 2009;
Fu et al., 2010; Serrano et al., 2010; Wang et al., 2010).
Thus, MRGPRX1 may also be interesting targets in
chronic neuropathic pain.
 MAS-Related G Protein–Coupled Receptors
d. Mast cell biology. Mast cells are long-lived
mononuclear cells that reside in tissues near external
surfaces, e.g., in skin or mucosa, and thereby are
among the first cells of the immune system to respond
to pathogens or allergens (Galli et al., 2011). Mast cells
fulfill their tasks in innate and adaptive immune
responses by secretion of a plethora of mediators
(degranulation), invaluable for host defense (Galli
et al., 2011). Mast cell degranulation is caused by
IgE-dependent and -independent processes (Metcalfe
et al., 1997; Ferry et al., 2002). A very heterogeneous
group of agents, either released endogenously by
immune cells, neurons, and epithelial cells, or applied
exogenously, mediates IgE-independent mast cell de-
granulation and is referred to as secretagogues (Sharma
et al., 2002). However, underlying signaling pathways
are diverse and a matter of debate.
Endogenously expressed MRGPRX2 were proposed
as receptors for different secretagogues in cultured
human primary mast cells and in the differentiated
human mast cell line LAD2 (Tatemoto et al., 2006;
Kashem et al., 2011; Subramanian et al., 2011a,b,
2013). Early during infection, a first line of mast cell
activation is thought to arise from complement-
mediated anaphylatoxin production (e.g., C3a or C5a)
(Kashem et al., 2011; Subramanian et al., 2011b).
Although MRGPRX2 are not involved in mast cell
activation provoked by endogenous anaphylatoxins,
mast cell degranulation induced by a synthetic C5a
receptor antagonist (PMX-53) or a C3a receptor super-
agonist (E7) required MRGPRX2 expression (Kashem
et al., 2011; Subramanian et al., 2011b). Later on
during infection, epithelial cells can secrete b-defensins
or cathelicidins, both inducing mast cell degranulation
via MRGPRX2 (Subramanian et al., 2011a, 2013).
Hence, MRGPRX2 can integrate paracrine input from
various cell types by detecting alterations of the local
milieu and inducing mast cell degranulation.
Degranulation of mast cells after MRGPRX2 activa-
tion depends on PTX-sensitive G proteins, irrespective
of the MRGPRX2 ligand under investigation (Tatemoto
et al., 2006; Kashem et al., 2011; Subramanian et al.,
2011a,b, 2013). However, MRGPRX2 activation by
LL-37 or human b-defensin-3 involves two different
signaling cascades that act synergistically on degran-
ulation, but only one of which depends on Gi/o-induced
PKC activation (Subramanian et al., 2011a, 2013). The
second PTX-insensitive pathway involved calcium in-
flux and release, as suggested by the inhibitory actions
of the calcium release–activated calcium channel
blocker, lathanium (La 3+), and the inositol 1,4,5-
trisphosphate receptor and canonical TRP blocker,
2-aminoethoxydiphenyl borate (Subramanian et al.,
2011a, 2013).
Because some basic secretagogues can be secreted by
primary sensory neurons, MRGPRX2 may also en-
hance neuron-to-mast cell signaling, thereby shaping
591
neuroimmune interactions. In congruence with this
concept, MRGPRX1 were implicated in the secretion of
chemokine ligand 2 from LAD2 mast cells after
stimulation with BAM8–22 (Solinski et al., 2013).
Given that MRGPRX1 induced the expression of the
chemokine ligand 2 receptor CCR2 in primary sensory
neurons (Solinski et al., 2013), it is appealing to
hypothesize that MRGPRX1 could additionally en-
hance mast-cell-to-neuron signaling. Because mast
cells contribute to chronic pain (Zuo et al., 2003),
inhibition of such a paracrine signaling circuit
would be an interesting approach for future analgesic
therapy.
e. Putative role of MAS-related G protein–coupled
receptors X2 in sleep and blood pressure regulation.
PAMP1-20 is a potent hypotensive peptide, functioning
mainly via inhibition of norepinephrine and epineph-
rine release from sympathetic neurons or adrenal
chromaffin cells, respectively (Shimosawa et al., 1995;
Kobayashi et al., 2001). On the protein level, PAMP1-
20 is mainly distributed in adrenal medulla and atrium
(Washimine et al., 1994). Because MRGPRX2 proteins
have been detected in adrenal chromaffin cells and
because PAMP1-20 activated MRGPRX2 in heterolo-
gous expression systems, Kamohara and coworkers
(2005) proposed that epinephrine-dependent hypoten-
sive functions of PAMP1-20 might be mediated by
MRGPRX2 signaling.
Cortistatin-14 activates all five somatostatin receptor
(SSTR1-5) subtypes, but some effects of cortistatin-14
appear to be clearly independent of these receptors and
are not caused by somatostatin (de Lecea, 2008). Among
these effects is the induction of slow-wave sleep that
might be partly mediated by inhibition of hippocampal
neurons. Because MRGPRX2 are expressed in hippo-
campal neurons of layer CA2-4 and because MRGPRX2
responded specifically to cortistatin-14, Robas and co-
workers (2003) put forward the proposal that MRGPRX2
may be involved in cortistatin-14–mediated slow-wave
sleep induction.
IV. Agonist-Promoted Internalization and
Desensitization of MAS-Related
G Protein–Coupled Receptors
GPCR signaling is commonly regulated by agonist-
induced receptor phosphorylation that leads to uncou-
pling from cognate G proteins (desensitization) and
subsequent receptor internalization (endocytosis), mostly
via association with b-arrestins (Ferguson, 2001). In
addition, b-arrestins have been shown to induce
G protein-independent signaling pathways, further
increasing the repertoire of GPCR-mediated signaling
(Shenoy and Lefkowitz, 2003). Only a few GPCR have
been shown to resist agonist-promoted processes that
negatively regulate receptor activity, including k-OR,
592 Solinski et al.
b3-adrenoceptors, and SSTR4 (Nantel et al., 1993; Chu
et al., 1997; Csaba and Dournaud, 2001).
Detailed analysis of the subcellular distribution of
human MRGPRD fused to green fluorescent protein
(GFP) in CHO cells after a 30-minute period of
stimulation with saturating concentrations of b-alanine
(Shinohara et al., 2004) revealed translocation of the
receptor protein from the plasma membrane to punctate
intracellular vesicles, indicative of ligand-induced
internalization. Dose-dependent internalization of
c-myc–tagged rat MRGPRD by b-alanine after recombi-
nant expression in HEK293 cells was also identified,
using either a qualitative immunofluorescence or a
quantitative enzyme-linked immunosorbent assay ap-
proach (Milasta et al., 2006). Thus, MRGPRD, the only
MRGPR conserved in rodents and primates and as-
signed to an agonist, apparently belongs to the group of
endocytosis-prone GPCR.
After recombinant expression of murine MRGPRA1
or MRGPRA4 in HEK293 cells, Dong and coworkers
(2001) observed a prominent decline in ligand-induced
calcium signals after repetitive short term stimulation
with FLRFamide or NPAF, indicating agonist-
promoted desensitization. Desensitization was revers-
ible by ligand wash-out over ;20 minutes and most
likely occurred because of phosphorylation and endo-
cytosis of the respective receptor protein. In fact, in
HEK293 cells murine MRGPRA1 fused to GFP was
internalized to punctate intracellular vesicles after 30
minutes of agonist stimulation (Han et al., 2002).
Likewise, MRGPRC-GFP fusion proteins also in-
ternalized after 30 minutes of stimulation by saturat-
ing concentrations of g2-MSH in HEK293 cells (Han
et al., 2002). This work was corroborated by the finding
that 1) other full agonists of murine MRGPRC in-
cluding BAM8–22 are able to induce the same extent of
MRGPRC endocytosis; 2) endocytosis is not confined to
HEK293 cells, but also detectable in several DRG
neuron-derived cell lines; 3) b-arrestins are indispens-
able for proper ligand-induced MRGPRC endocytosis in
Cos7 cells (Solinski et al., 2010). Notably, rat MRGPRC
was also found to be sensitive to BAM8–22- or
g2-MSH–induced endocytosis in the same cell models
(Solinski et al., 2010). In sharp contrast to rodent
BAM8–22-sensitive MRGPRC, human MRGPRX1
resisted BAM8–22-induced desensitization and endo-
cytosis after recombinant expression in different
cellular systems, including primary sensory neuron-
derived F11 and ND-C cells (Solinski et al., 2010).
Thus, identical ligands regulate MRGPRC and MRGPRX1
endocytosis in diametrically opposed ways. Moreover,
similar to MRGPRX1, ligand-induced phosphorylation
and internalization of the MRGPRX2 was not detectable
(Subramanian et al., 2011a). Thus, absence of agonist-
promoted receptor desensitization appears as a common
feature among MRGPRX, defining the MRGPRX sub-
family as the first among all GPCR families to harbor
more than one endocytosis resistant member. Whether
MRGPRX3 and -4 also resist agonist-mediated de-
sensitization still needs to be confirmed in the future.
Notably, serine residues in the C-terminal tail of a given
GPCR are profoundly involved in agonist-induced re-
ceptor regulation (Ferguson, 2001) and, as already
mentioned, C-terminal tails of MRGPRX developed
under strong positive selection pressure during evo-
lution. It is tempting to speculate that considerable
physiologic benefits have been earned by enriching
the lack of receptor desensitization and endocytosis
among MRGPRX. A summary of the available litera-
ture analyzing ligand-induced regulation of MRGPR
is given in Table 8.
V. Functional Interactions of MAS-Related
G Protein–Coupled Receptors with Other
Receptor Families
A. Heteromultimerization among MAS-Related
G Protein–Coupled Receptors and Receptor Subtypes
of Other GPCR Families
Heteromultimerization among GPCR has been pro-
posed to alter ligand binding, G protein coupling,
protein trafficking, and agonist-promoted desensitiza-
tion of the receptors involved (Bulenger et al., 2005).
Thus, direct protein-protein interactions due to re-
ceptor multimerization represent an important mech-
anism by which distinct hormone systems interact with
each other (Jordan et al., 2001; Torvinen et al., 2005;
Parenty et al., 2008). Because MRGPRX1 and OR
share a common ligand, the BAM1–22 peptide, are
coexpressed in primary sensory neurons, and both
affect pain modulation, functional interactions be-
tween MRGPRX1 and the d-OR (DOP) due to multi-
merization have been analyzed. Bioluminescence
resonance energy transfer experiments revealed that
MRGPRX1 interacted with human DOP in living
HEK293 cells stably overexpressing each receptor
(Breit et al., 2006). Individual activation of either
DOP (with Leu-Enkephalin) or MRGPRX1 (with
BAM8–22) allowed both receptors to modulate their
respective signaling pathways. In contrast, the DOP/
MRGPRX1 bivalent agonist BAM1–22, which activated
each receptor expressed individually, fully activated
the MRGPRX1 but did not promote DOP-mediated
signaling within the heteromultimer. Similarly, concom-
itant activation of the DOP/MRGPRX1 heteromultimer
by selective DOP and MRGPRX1 agonists (Leu-
Enkephalin and BAM8–22) promoted MRGPRX1 but
not DOP signaling. Furthermore, coexpression of the
endocytosis resistant MRGPRX1 inhibited agonist-
promoted internalization of the endocytosis-prone
DOP. Overall, these data suggest that MRGPRX1
signaling within the DOP/MRGPRX1 heteromultimer
acts in a dominant-negative fashion on DOP signaling
 MAS-Related G Protein–Coupled Receptors 593
TABLE 8
Ligand-promoted desensitization and endocytosis of MAS-related G protein–coupled receptors
MRGPR that have been analyzed for their ligand-induced regulation are listed in conjunction with the respective ligand and expression system
used.

Receptor Ligand Desensitization Endocytosis System Reference

mMRGPRA1 FLRFamide YES YES HEK293 Dong et al., 2001

Han et al., 2002
mMRGPRA4 NPAF YES N.D. HEK293 Dong et al., 2001
mMRGPRC g2-MSH YES YES HEK293, F11, ND-C, Cos7 Han et al., 2002
BAM8–22 Solinski et al., 2010
Dyn-14
rMRGPRC g2-MSH BAM8–22 N.D. YES HEK293, F11, ND-C Solinski et al., 2010
hMRGPRD b-Alanine N.D. YES CHO Shinohara et al., 2004
rMRGPRD b-Alanine N.D. YES HEK293 Milasta et al., 2006
hMRGPRX1 BAM8–-22 NO NO HEK293, F11, ND-C, Cos7 Solinski et al., 2010
hMRGPRX2 Cortistatin-14 NO NO HEK293 Subramanian et al., 2011a
LL-37 LAD2, HMC-1
h, human; m, mouse; N.D., not determined; r, rat.

and thus, could affect the fine tuning of pain sensation
regulated by DOP.
MRGPR have not only been shown to interact with
members of other GPCR families, but also among each
other. The rat MRGPRD and -E subtypes form multi-
meric complexes when expressed in HEK293 cells or
peripheral nociceptive neurons, as previously indicated
by coimmunoprecipitation and time-resolved fluores-
cence resonance energy transfer (Milasta et al., 2006).
These interactions increased the potency of b-alanine
to phosphorylate ERK-1/2, maintained the capacity of
b-alanine to elevate intracellular calcium concentra-
tions, and inhibited b-alanine–induced internalization
of MRGPRD. Given the large number of MRGPR
family members and assuming that heteromultimeric
interactions also exist between other subtypes, one
would predict an immense variety of putative binding
sites for MRGPR-selective ligands, expanding the
impact of this receptor family on many physiologic
pathways even further.
B. Inhibition of Tolerance to Morphine
As mentioned above, BAM-sensitive, human
MRGPRX1 functionally interact with the opioid system
based on receptor multimerization. Interestingly,
multimerization-independent interactions between
BAM-sensitive MRGPRC and opioid receptors have
also been described in vivo. A major problem of chronic
pain treatment with morphine is the development of
tolerance characterized by declining analgesic effects
after repeated dosing. The mechanisms that cause the
underlying adaptation processes are manifold and
not completely understood [for further reading see
Williams et al. (2013)]. In rats, tolerance to morphine can
be generated by its daily application over six to seven
days. Interestingly, after intrathecal application of
MRGPRC-specific agonists in rats, morphine regained
;50% of its analgesic actions in formerly morphine-
tolerant animals on the consecutive day (Jiang et al.,
2006; Cai et al., 2007b; Chen et al., 2010b). MRGPRC
agonists were not only able to restore analgesic efficacy
after tolerance to morphine had already been estab-
lished, intrathecally applied BAM8–22 or Tyr6-g 2-
MSH6–12 on every second day during induction of
morphine tolerance dose-dependently inhibited the de-
velopment of morphine tolerance (Cai et al., 2007b; Chen
et al., 2010b). Compensatory upregulation of pain-
enhancing molecular players in the DRG and spinal
cord including PKCg-dependent upregulation of nNOS
or CGRP is involved in the development of morphine
tolerance (Menard et al., 1996; Kolesnikov et al., 1997;
Granados-Soto et al., 2000). Because MRGPRC agonists

TABLE 9
Ligand sharing between MAS-related G protein–coupled receptors and members of other GPCR families
Ligands that have been implicated to bind and activate both, MRGPR subtypes and other GPCR, are given.

Ligand MRGPR Reference Other GPCR Reference

BAM1–22 r/mMRGPRC hMRGPRX1 Lembo et al., 2002 DOP, MOP, KOP Quirion and Weiss,1983
Han et al., 2002
Grazzini et al., 2004
NPFF mMRGPRC Han et al., 2002 NPFF1R, NPFF2R Mollereau et al., 2002
g 2-MSH r/mMRGPRC Han et al., 2002 MC3R, MC4R Roselli-Rehfuss et al., 1993
Grazzini et al., 2004 Oosterom et al., 1999
GABA h/rMRGPRD Shinohara et al., 2004 GABAB Bowery et al., 1980
Kaupmann et al., 1997
Angiotensin-(1–7) hMRGPRD Gembardt, 2008 MAS1, AT2R Santos et al., 2003
Walters, 2005
Cortistatin-14 hMRGPRX2 Robas, 2003 SSTR1-5 de Lecea et al., 1996
Fukusumi et al., 1997
h, human; KOP, k-OR; m, mouse; MC3R, melanocortin 3 receptor; r, rat.
594 Solinski et al.
blocked enhanced PKCg activity in the spinal cord and
diminished nNOS and CGRP immune reactivity in DRG
and spinal cord slices, this signaling cascade might
inhibit morphine tolerance by MRGPRC (Chen et al.,
2010b).
VI. Conclusions and Perspectives
Over the last decade, our knowledge about MRGPR
has been significantly extended: 1) the murine
MRGPRA3 subtype has been identified as the itch-
mediating chloroquine receptor; 2) rodent MRGPRC
have also been shown to be involved in itching and
MRGPRC have been made responsible for analgesic
and pain-enhancing effects; 3) MRGPRD enhance
neuronal excitability and induce vasorelaxation; and
4) human MRGPRX also modulate excitability of
primary sensory neurons and gene expression in these
cells. However, because of the lack of suitable model
systems, these data are solely based on experiments
obtained after heterologous expression of the MRGPRX1
protein. In mast cells, endogenously expressing the
MRGPRX1 and -2 subtype effects on degranulation and
mediator release have been found. Adaptive evolu-
tionary development and resistance to agonist-
promoted desensitization/endocytosis further highlight
the extraordinary features of the primate-specific
MRGPRX subfamily. Notably, MRGPRX can be consid-
ered as valuable drug targets with low or even no off-
target effects because of their restricted expression in
primary sensory neurons and mast cells.
However, our knowledge about the MRGPR family is
still superficial and central questions need to be
addressed: 1) to what extent do MRGPR serve as
markers for primary sensory neurons that execute
specific functions; 2) what is the difference between
distinct rodent species in this regard; 3) what is the
physiologic role of distinct MRGPR in these specialized
neurons? Consider, in particular, that some subtypes of
the MRGPRA and -X subfamilies are not yet analyzed,
mainly because not a single ligand for these receptors
is available. Similarly, progress in the work with
MRGPRE to -H receptors is significantly hindered by
the lack of agonists. Moreover, it will be essential to
define the signaling events responsible for the effects of
MRGPRC agonists on nociception. Interestingly, hu-
man MRGPRX1 have been shown to enhance or inhibit
signaling pathways that supposedly modify neuronal
activity, suggesting that MRGPRX1 might also exert
proalgetic and analgesic effects in humans. In this
context it will be important to analyze to which extent
data obtained from BAM8–22-sensitive rodent MRGPRC
can be extended to BAM8–22-sensitive human MRGPRX1.
Convincing evidence that rodents can in fact be used
as a native model systems for human BAM8–22-
sensitive MRGPRX1 would propel the "deorphaniza-
tion" process of these receptors forward and would
simplify the development of drugs tested in established
animal models but intended to target human
MRGPRX1. In parallel, it would be of interest to
develop primate- or even human-based DRG-derived
cell models that endogenously express MRGPRX1.
Considering mast cells, a step forward has been made
at this point, because the human mast cell line LAD2
has been shown to endogenously express MRGPRX1
and -2. Furthermore, the problem of ligand-sharing
between MRGPR and subtypes of other GPCR families,
observed for many endogenous ligands (see Table 9), is
a major drawback in the field. Thus, screening efforts
yielding MRGPR subtype–specific agonists and antag-
onists would significantly help attain a better un-
derstanding of the physiologic pathways engaged by
MRGPR in vivo.
Acknowledgments
The authors thank Dr. Alexander Dietrich (Walther-Straub-
Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-
Universität München, Munich, Germany) for critical reading of the
manuscript.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: Solinski,
Gudermann, Breit.
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