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PHARMACOLOGICAL REVIEWS
Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/pr.113.008136
Pharmacol Rev 66:1080–1105, October 2014
ASSOCIATE EDITOR: DIANNE M. PEREZ
Mas and Its Related G Protein–Coupled
Receptors, Mrgprs s
Michael Bader, Natalia Alenina, Miguel A. Andrade-Navarro, and Robson A. Santos
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany (M.B., N.A., M.A.A.-N.); Charité-University Medicine, Berlin, Germany
(M.B.); Institute for Biology, University of Lübeck, Lübeck, Germany (M.B.); and Department of Physiology and Biophysics, Federal
University of Minas Gerais, Belo Horizonte, Brazil (M.B., N.A., R.A.S.)
Address correspondence to: Prof. Dr. Michael Bader, Max-Delbrück-Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, 13125
Berlin, Germany. E-mail: [email protected]
dx.doi.org/10.1124/pr.113.008136.
s This article has supplemental material available at pharmrev.aspetjournals.org.
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Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081
I. Introduction and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081
II. Evolution of Mas-Related G Protein–Coupled Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1083
III. Gene Structure and Expression of Mas-Related G Protein–Coupled Receptors . . . . . . . . . . . . . . . 1083
A. Chromosomal Localization and Gene Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1083
B. Expression of Mas-Related G Protein–Coupled Receptors in Dorsal Root Ganglion
Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1085
C. Expression of Mas-Related G Protein–Coupled Receptors in Mast Cells . . . . . . . . . . . . . . . . . . 1086
D. Expression of Mas-Related G Protein–Coupled Receptors in Other Tissues . . . . . . . . . . . . . . . 1087
E. Expression of Mas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1088
IV. Structure, Ligands, and Signaling of Mas-Related G Protein–Coupled Receptors . . . . . . . . . . . . . 1088
A. Mas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1089
B. hMRGPRX1 (hSNSR4). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1091
C. rMrgprX1 (rSNSR1, rMrgC), mMrgprX1 (mMrgC11) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1091
D. mMrgprA1, mMrgprA4, mMrgprX1 (mMrgC11) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1092
E. mMrgprA3, rMrgprA, hMRGPRX1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1092
F. hMRGPRX2, rMrgprB3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1092
G. hMRGPRX3 (hSNSR1), hMRGPRX4 (hSNSR6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1093
H. rMrgprA (rMrgprX3), mMrgprA9, mMrgprA10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1093
I. MrgprD, MrgprE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1093
J. MrgprF (RTA), MAS1L (mrg) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1093
K. MrgprG, MrgprH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1094
L. Receptor Hetero-Oligomerization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1094
V. Physiologic and Pathophysiologic Functions of Mas-Related G Protein–Coupled Receptors. . . . 1094
A. Mas-Related G Protein–Coupled Receptors in Sensory Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . 1094
B. Mas-Related G Protein–Coupled Receptors in Mast Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1095
C. Mas-Related G Protein–Coupled Receptors in Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1096
D. Mas-Related G Protein–Coupled Receptors and Mas in Cardiovascular Organs. . . . . . . . . . . 1096
1. Mas in Heart. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1096
2. Mas in Blood Vessels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1097
3. Mas in Cardiovascular Centers of the Brain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1098
4. Mas in Kidney.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1098
E. Mas in Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1098
F. Mas in Behavior. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1099
G. Mas in Reproductive Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1099
VI. Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1099
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1100
Mas-Related Genes
1081
Abstract——The Mas-related G protein–coupled receptors
(Mrgprs or Mas-related genes) comprise a subfamily of
receptors named after the first discovered member,
Mas. For most Mrgprs, pruriception seems to be the
major function based on the following observations: 1)
they are relatively promiscuous in their ligand specificity
with best affinities for itch-inducing substances; 2) they
are expressed in sensory neurons and mast cells in the
skin, the main cellular components of pruriception; and 3)
they appear in evolution first in tetrapods, which have
arms and legs necessary for scratching to remove parasites
or other noxious substances from the skin before they
create harm. Because parasites coevolved with hosts,
each species faced different parasitic challenges, which
may explain another striking observation, the multiple
independent duplication and expansion events of Mrgpr
genes in different species as a consequence of parallel
adaptive evolution. Their predominant expression in
dorsal root ganglia anticipates additional functions of
Mrgprs in nociception. Some Mrgprs have endogenous
ligands, such as b-alanine, alamandine, adenine, RF-amide
peptides, or salusin-b. However, because the functions of
these agonists are still elusive, the physiologic role of the
respective Mrgprs needs to be clarified. The best studied
Mrgpr is Mas itself. It was shown to be a receptor for
angiotensin-1–7 and to exert mainly protective actions
in cardiovascular and metabolic diseases. This review
summarizes the current knowledge about Mrgprs, their
evolution, their ligands, their possible physiologic
functions, and their therapeutic potential.
I. Introduction and History
responsible for the activation of the MAS gene in the
tumorigenicity assay. Thus, Mas is not an oncogene and
has never been found amplified in a primary tumor but
can transform cells when artificially overexpressed.
The first attempts to clarify the function of the Mas
protein were done by Jackson et al. (1988) by its transient
expression in Xenopus frog oocytes and stable transfection
of a mammalian cell line. Under voltage-clamp conditions, oocytes injected with Mas RNA exhibited a
dose-dependent induction of an inward current in response to the angiotensins (Ang) I, II, and III, whereas
in the Mas-transfected cells, stimulation with Ang II
and III led to the mobilization of intracellular Ca2+ and
to the initiation of DNA synthesis. On the basis of these
results Mas was proposed to be a functional angiotensin
receptor—a molecule whose identification had been
pending for years. Although a number of further studies
were in agreement with these assumptions (Jackson
and Hanley, 1989; McGillis et al., 1989; Poyner et al.,
1990; Andrawis et al., 1992), the activation of inward
currents by Ang in Mas-mRNA injected oocytes was
not inhibited by Ang antagonists (Jackson et al., 1988).
Moreover, Ambroz et al. (1991) could show that the
intracellular Ca2+ increase in Mas-transfected cells after
Ang II treatment was only observed in cells already
expressing endogenously Ang II receptors. Therefore,
doubts arose as to whether the Mas gene product, per
se, is an Ang II receptor. Moreover, cloning of the Ang II
receptor type 1 (AT1) in 1991 (Murphy et al., 1991;
Sasaki et al., 1991) did not favor the original hypothesis
of Ang II being a ligand for Mas. The later identification
of Ang-(1–7) as a Mas agonist in 2003 and of the direct
interaction between Mas and AT1 receptors in 2005
(Kostenis et al., 2005; Santos et al., 2007) partly explained
the original observations of Jackson et al. (1988) in
Mas was described more than 25 years ago (Young
et al., 1986) and later became eponymous for the whole
family of Mas-related G protein–coupled receptors (Mrgprs),
formerly called Mas-related genes (Mrgs). Intriguingly, the
first three main discoveries about this gene turned out
to be erroneous, mostly due to the fact that the quantity
of collected data and the general knowledge at that time
obscured the interpretation of the obtained results and
led to inappropriate conclusions.
The human MAS gene was originally isolated from
DNA of a human epidermoid carcinoma cell line because
of its ability to transform NIH3T3 cells upon transfection (Young et al., 1986) and therefore was thought to
be a proto-oncogene. Because computer analysis of the
MAS amino acid sequence suggested that the protein
belongs to the class of G protein–coupled receptors
(GPCRs) with seven transmembrane domains (Young
et al., 1986), this provided the first direct evidence
for an oncogenic activity of a GPCR. Although such
tumorigenicity was confirmed in independent experiments (Janssen et al., 1988; van’t Veer et al., 1993), the
oncogenic potential of Mas was challenged. In these
experiments, the transfected cells or the tertiary tumor
in nude mice contained amplified MAS sequences
characterized by rearrangements in 59- and 39-noncoding
regions, such as an insertion of human centromeric
a-satellite repeat DNA (van’t Veer et al., 1993). However,
the original tumor DNA used in the first round of transfection was neither rearranged nor amplified or mutated
in the MAS coding sequence and therefore cannot be
considered as the driving cause for tumor development.
Presumably, the MAS 59-region represents a hot spot of
recombination, and the rearrangement of the 59-noncoding
sequence, which occurred during transfection, was
ABBREVIATIONS: AKT, protein kinase B; Ang, angiotensin; AT1, Ang II receptor type 1; BAM, bovine adrenal medulla; CGRP, calcitonin
gene-related peptide; DRG, dorsal root ganglion; eNOS, endothelial nitric oxide synthase; GPCR, G protein–coupled receptor; KO, knockout;
Mrg, Mas-related gene; Mrgpr, Mas-related G protein–coupled receptor, MSH, melanocyte-stimulating hormone; PACAP, pituitary adenylate
cyclase–activating peptide; POMC, proopiomelanocortin; RAS, renin-angiotensin system; RTA, rat thoracic aorta; siRNA, small interfering
RNA; SNSR, sensory neuron-specific receptor; TM, transmembrane; TRPA1, transient receptor potential ankyrin 1; TRPV1, transient
receptor potential vanilloid 1; VSMC, vascular smooth muscle cells.
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Bader et al.
Xenopus oocytes and clarified that Mas is not an Ang
II receptor, per se, but a modulator of AT1 signaling.
The third error published about the Mas gene after
the oncogene and the Ang II receptor assignments was
its maternal imprinting in mice during embryonic
development and for some organs, such as tongue and
heart, in adults (Villar and Pedersen, 1994) as well as
in human breast tissue (Miller et al., 1997). In genomic
imprinting, one of the two parental alleles of an
autosomal gene is silenced epigenetically by a cis-acting
mechanism. The Mas gene is located in close proximity
to the imprinted Igf2r gene in the mouse and human
genomes (Barlow et al., 1991; Schweifer et al., 1997).
Imprinting of the maternally expressed Igf2r gene is
controlled by an intronic imprint control element that
contains the promoter of the long noncoding RNA, Airn
(antisense Igf2r RNA noncoding), which overlaps the
silenced paternal Igf2r promoter and partially the Mas
gene in an antisense orientation (Wutz et al., 1997; Lyle
et al., 2000). However, our work with Mas-deficient mice
clearly demonstrated that Mas is biallelically expressed
(Alenina et al., 2002a). Thus, because of the lack of
strand selectivity in the reverse-transcription polymerase
chain reaction assays used by Villar and Pedersen (1994)
and Miller et al. (1997), the maternally imprinted RNA
detected by them was most probably not the coding
mRNA but the antisense RNA of the Mas gene as part
of Airn. Altogether, these data demonstrate that Mas
antisense RNA but not the mRNA underlies monoallelic
expression in mouse and human.
The expansion of the Mrgpr family started in 1990
with cloning of the rat thoracic aorta (RTA) gene (Ross
et al., 1990) and of the mas-related gene (mrg, now
called Mas1L) (Monnot et al., 1991) with 34 and 35%
homology to Mas, respectively, both identified by
screening of cDNA libraries with probes either for the
conserved part of the M1 muscarinic receptor or for Mas
itself. Ten years later, a comparative analysis of the
transcriptome of dorsal root ganglia (DRG) of Ngn1-deficient
mice, which lack a subclass of nociceptive neurons,
enlarged the family by several genes expressed in such
neurons, which were called Mas-related genes A, MrgA
(Dong et al., 2001). Subsequent screening of murine
DRG cDNA libraries and bioinformatic analysis of
databases led to the identification of approximately
50 Mrgs (also called SNSR or sensory neuron-specific
receptors by some authors) in mouse, rat, human, and
macaque (Dong et al., 2001; Wittenberger et al., 2001;
Lembo et al., 2002; Takeda et al., 2002; Zhang et al.,
2005), which are now divided into several subfamilies
and have been renamed according to a new nomenclature (http://www.guidetopharmacology.org): MrgprA, B,
C, D, E, F, G, H, and a primate-specific MRGPRX
subfamily (Table 1). Interestingly, different subfamilies
consist of multiple duplicated genes in different species.
Primates have several MRGPRX genes, whereas rodents
have multiple MrgprA, B, and C genes, and even different rodent species differ in the amount of genes per
subfamily (Dong et al., 2001; Zylka et al., 2003).
Although being structurally homologous to Mas, which
is widely recognized as a component of the reninangiotensin system (RAS), the function of most other
Mrgprs turned out to be unrelated to the RAS with
a broad range of peptidic and nonpeptidic ligands and
implications in the perception of itch and pain.
Nevertheless, our recent discovery of the putative new
RAS peptide, alamandine [Ala1-Ang-(1–7)], which binds
and activates mMrgprD (Lautner et al., 2013), suggests
that the regulation of the RAS and of the cardiovascular
system is not restricted to Mas among the Mrgprs.
In the following, we will summarize the current
knowledge about Mrgprs, in particular about their evolution, their ligands, their possible physiologic functions, and
their therapeutic potential. Despite being the most extensive, the part about Mas is far from being comprehensive, because the amount of literature about the
TABLE 1
Mrgpr genes in different subfamilies and species
Original names are given in parentheses. The subfamilies are grouped by evolutionary relationship (see Fig. 1).
Genes
Subfamily
Mouse
Rat
Human
mMrgprA1, mMrgprA2, mMrgprA3,
mMrgprA4, mMrgprA5, mMrgprA6, mMrgprA7,
mMrgprA8, mMrgprA9, mMrgprA10
mMrgprB1, mMrgprB2, mMrgprB3, mMrgprB4,
mMrgprB5, mMrgprB8, mMrgprX2
mMrgprX1 (mMrgC11)
—
rMrgprA
—
rMrgprB2, rMrgprB3, rMrgprB4,
rMrgprB5, rMrgprX2, rMrgprX2-like
rMrgprX1 (rMrgC)
—
—
MrgprD
MrgprE
MrgprF
MrgprG
Mas1L
mMrgprD
mMrgprE
mMrgprF
mMrgprG
rMrgprD
rMrgprE
rMrgprF (RTA)
rMrgprG
MrgprH
Mas
mMrgprH (GPR90)
mMas
MrgprA
MrgprB
MrgprC
MrgprX
—
rMrgprH
rMas
—
—
hMRGPRX1, hMRGPRX2,
hMRGPRX3, hMRGPRX4
hMRGPRD
hMRGPRE
hMRGPRF
hMRGPRG
hMAS1L (mrg)
—
hMAS
Mas-Related Genes
functions of this protein exceeds the scope of a review about
all Mrgprs. For more information about the eponymous
gene of the Mrgpr family, the reader is referred to more
comprehensive recent reviews (Xia and Lazartigues, 2010;
Rabelo et al., 2011; Xu et al., 2011; Ferreira et al., 2012;
Bader, 2013; Passos-Silva et al., 2013; Santos et al., 2013a;
Simoes e Silva et al., 2013; Regenhardt et al., 2014a).
II. Evolution of Mas-Related G
Protein–Coupled Receptors
Mrgprs belong to the class A family of the superfamily
containing all GPCRs with more than 800 genes [in the
human genome (Fredriksson et al., 2003)]. We did not
find members of the Mrgpr family outside Tetrapoda,
neither in the complete genomes of bony fish (e.g., zebrafish,
Danio rerio) nor in sequences from incompletely sequenced
species, such as coelacanth or lungfishes, which are closer
relatives to Tetrapoda than bony fish. Thus, we conclude
that this family emerged after the divergence of bony fish
from the line leading to Tetrapoda.
To pinpoint the different groups of sequences within
the Mrgprs, we constructed a phylogenetic tree with
the sequences of 10 human members and all homologs
from a selection of completely sequenced Tetrapoda
species (see Supplemental Table 1 for details). Following
Dong et al. (2001), we used the Frp1 orthologs from
mouse and the frog Xenopus, which, as members of
a related subfamily, work as outliers and help to find
the root of the branch containing the Mas related
sequences (Fig. 1).
Here we summarize the most outstanding features
derived from the tree, which are mainly confirming
earlier publications (Dong et al., 2001; Lembo et al.,
2002; Choi and Lahn, 2003; Zylka et al., 2003; Yang
et al., 2005; Zhang et al., 2005). The branches of the
tree have been swapped to present these facts roughly
from top to bottom.
The top branch contains the two outliers (Frp1)
followed by a frog-specific branch (5 members). The
next branch includes Mas and rodent (but no primate)
MrgprH genes, and seems to be the oldest one because
it includes a frog and two chicken members. Thus, the
name Mas-related G protein–coupled receptors, Mrgprs,
for the whole family, which historically arose since Mas
was the first gene discovered, is also correct in evolutionary terms. The next branch contains three duplicated chicken members, indicating that it evolved in the
lineage leading to aves. Then there are two branches
that emerged later (no frog or chicken members): one
that includes MrgprD, MAS1L, MrgprF, MrgprE, and
MrgprG, and another with the human MRGPRX and
the rodent-specific MrgprA, B, and C genes. The MrgprC
subfamily, however, consists only of pseudogenes in mouse,
except mMrgC11 (Zylka et al., 2003). The two functional
genes rMrgC and mMrgC11 have been renamed rMrgprX1
and mMrgprX1, respectively, despite the fact that they are
1083
not particularly close homologs of the human hMRGPRX1
gene (Fig. 1). Regardless, the Mrgpr gene family contains
more pseudogenes than functional genes.
The most striking feature, however, is the expansion
of Mrgpr subfamilies by repeated duplication events,
which seem to have happened mainly independently in
different branches of vertebrate evolution (Dong et al.,
2001; Lembo et al., 2002). Whereas the A and B
subfamilies were expanded in both the mouse and rat
lineages, the C family got larger only in the mouse
lineage; however, most of the duplicated genes of this
subfamily were inactivated at some point (Zylka et al.,
2003). In primates, the MRGPRX family was expanded
(Choi and Lahn, 2003; Yang et al., 2005; Burstein
et al., 2006). But such massive expansion can also be
found in other Tetrapod species. Marsupials, such as
the opossum and the Tasmanian devil, each have more
than 20 Mrgpr-like genes, and reptiles, such as the
Chinese soft turtle and the Anole lizard, have more
than 15 orthologs, which are more homologous to each
other in each species than to any other Tetrapod Mrgpr
gene (ENSEMBL GeneTree ENSGT00550000074531).
This massive duplication of Mrgpr genes may have
been facilitated by insertion of retrotransposons close
to the ancestral gene, because it was observed in the
rat and mouse MrgprA–C clusters (Dong et al., 2001;
Zylka et al., 2003). However, such a mechanism will
most probably not explain the effect for all vertebrates.
Instead, the species-specific expansion of certain Mrgpr
subfamilies strongly supports the notion that there was
a powerful evolutionary drive to gain more Mrgprs,
which was independently acting in several Tetrapod
lineages. Accordingly, it has been suggested that the
evolution of Mrgprs was adaptive by showing that the
amino acid alterations mainly affected the extracellular
domains of the receptors and thereby, most likely, the
ligand binding sites (Choi and Lahn, 2003; Yang et al.,
2005; Fatakia et al., 2011). Thus, the receptors seem to
have been optimized for the different sets of ligands
each species was independently facing in its environment. Later in this review, we will present a hypothesis
that may explain this phenomenon as well as the appearance of the whole Mrgpr family only in Tetrapods.
III. Gene Structure and Expression of
Mas-Related G Protein–Coupled Receptors
A. Chromosomal Localization and Gene Structure
All genes for Mrgprs other than Mas, Mas1L, and
MrgprH are located on a single chromosome in human
(chromosome 11), rat (chromosome 1), and mouse
(chromosome 7), forming in each case two large clusters
in syntenic regions as a result of the evolutionary recent
repeated duplications (Fig. 2). In rat, the genes for Mas and
MrgprH are also on chromosome 1, but in mouse these two
genes are located on chromosome 17, and the human MAS
and MAS1L genes are situated on chromosome 6, with
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Bader et al.
Fig. 1. Phylogenetic tree of the 10 human MRGPR proteins, together with Mrgpr proteins from the selected Tetrapoda species Pan troglodytes (gorilla,
10 sequences), Macacca mulatta (rhesus monkey, 10 sequences), Mus musculus (mouse, 25 sequences), Rattus norvegicus (rat, 18 sequences), Gallus
gallus (chicken, 4 sequences), Xenopus tropicalis (frog, 6 sequences), and two sequences from the related Frp1 subfamily (from frog and mouse) that
were used as homologous outliers to fix the root of the Mrgpr subfamily. Genes without associated protein products in the database (e.g., pseudogenes)
were not included in the analysis. The 10 human MRGPR protein sequences were obtained from the NCBI Protein database (NCBI Resource
Coordinators, 2013). All known genes of the same family were obtained for selected Tetrapoda species, via Homologene groups of the human proteins
(NCBI Resource Coordinators, 2013) and through sequence searches (list of database identifiers in Supplemental Table 1). The homology to the Mrgpr
subfamily was tested using reciprocal sequence searches to the human proteome to verify that the closest hit was not a protein from another close
GPCR subfamily. Ultimately, an iterative process of producing increasingly large multiple sequence alignments and the corresponding phylogenetic tree
allowed to discriminate genes that did not belong to the Mrgpr subfamily. The final selection of genes (Supplemental Table 1) included 73 additional Tetrapoda
sequences and 2 Frp1 members from a related GPCR subfamily that were used as outliers. The multiple sequence alignments were produced using the
MUSCLE server at European Bioinformatics Institute (Edgar, 2004) and the phylogenetic tree was constructed using ClustalX (Larkin et al., 2007).
Mas-Related Genes
MAS1L in a region associated with diabetes type 1 (Aly
et al., 2008).
Nearly all coding sequences of Mas and Mrgprs are
present on a single exon, only hMRGPRF, rMrgprG,
mMrgprx1, mMrgprB1, and all mMrgprAs, except
mMrgprA4, have two coding exons, and mMrgprF and
rMrgprF have 3. Nevertheless, in all genes there are
several 59-noncoding exons, with Mas itself carrying
more than 10 of them (N. Alenina, unpublished data).
B. Expression of Mas-Related G Protein–Coupled
Receptors in Dorsal Root Ganglion Neurons
The Mrgpr family was originally discovered by its
expression in DRGs (Dong et al., 2001; Lembo et al.,
2002). All members are expressed there and in the
trigeminal ganglia in small diameter nociceptive neurons
in mice; however, Mas, as well as MrgprF, G, and H are
expressed at low levels (Cox et al., 2008; Costa et al.,
2012; Avula et al., 2013; Cao et al., 2013a). mMrgprexpressing neurons are nonpeptidergic, characterized
by the presence of immunoreactivity for the Griffonia
simplicifolia lectin IB4 and the glial-cell line–derived
neurotrophic factor coreceptor c-Ret and the absence of
transient receptor potential vanilloid 1 (TRPV1), substance P, and, in most but not all cases, of calcitonin
gene-related peptide (CGRP) (Dong et al., 2001; Han
1085
et al., 2013) (Fig. 3). These neurons are unmyelinated
and project to the superficial layer of the epidermis and
the stratum granulosum, and in the dorsal horn of the
spinal cord, the axons project to the inner lamina II (Fig. 3)
(Zylka et al., 2005). They are developed from neural crest
precursors in the last trimester of mouse embryogenesis
and the first weeks of life (Lallemend and Ernfors, 2012).
The transcription factors Runx1 and tlx3 are essential
for the development of this class of neurons and directly
regulate the expression of mMrgprD and mMrgprX1
starting around embryonic day 16 (Chen et al., 2006;
Luo et al., 2007; Liu et al., 2008; Lopes et al., 2012; Xu
et al., 2013). Runx1 also induces c-Ret in some neurons,
which, in turn, switch on the expression of mMrgprA1,
mMrgprA3, and mMrgprB4 in this subset of cells (Luo
et al., 2007; Franck et al., 2011). After birth, Runx1
becomes a repressor of mMrgprAs, mMrgprB4, and
mMrgprX1 and is turned off in this cell lineage,
although it remains expressed in the mMrgprD subclass
of neurons where it consequently leads to the exclusive
expression of mMrgprD (Liu et al., 2008). Consequently,
two subclasses of small-diameter nonpeptidergic neurons are defined in the adult mouse, the D compartment
(30% of DRG neurons) expressing MrgprD and the A/B/C
compartment (3–5% of DRG neurons) expressing the
other Mrgprs (Fig. 3) (Liu et al., 2008; Abdel Samad
Fig. 2. Chromosomal (Chr.) localizations of Mrgpr genes in human, rat, and mouse. The genes are shown as arrows in their direction of transcription.
The deleted fragment in the cluster-knockout mice (Liu et al., 2009) is marked.
1086
Bader et al.
Fig. 3. DRG neurons expressing Mrgprs project to the stratum granulosum of the epidermis and to the lamina II of the dorsal horn of the spinal cord
(green). At least two different classes of Mrgpr expressing neurons exist, the ones expressing MrgprA and X1, and the ones expressing MrgprD. These
cells all express IB4, but normally not CGRP, in contrast to nociceptive neurons that express CGRP but not IB4, which project to deeper layers of the
skin and to lamina I of the spinal cord dorsal horn (blue). The lower panel presents confocal microscopy images of MrgprD expressing cell bodies in the
DRGs (A–D) and axons in the spinal cord (E–H) labeled by transgenic expression of Venus (Table 2), showing coexpression with IB4 and mutual
exclusive expression of CGRP (from Wang and Zylka, 2009). Examples of Mrgprd-Venus neurons that bind IB4 are indicated (arrowheads). Arrows
mark the ventral boundary of lamina II. Inset in (H) is magnified at the bottom right. Scale bars: A–D, 50 mm; E–H, 200 mm.
et al., 2010). The Mrgprs, however, are not essential
for the development and projection of these neurons,
because mice deficient for mMrgprD (Zylka et al., 2005)
(Table 2) and cluster-knockout mice lacking 12 Mrgpr
genes (Liu et al., 2009) (Fig. 2; Table 2) show a normal
amount and wiring of the corresponding cells.
The MrgprA/B/C compartment can be further subdivided
into subsets of cells by the differential expression and
coexpression of mMrgprAs, Bs, and X1 (Dong et al., 2001).
A recent transgenic mouse study defined an mMrgprA3expressing subset, which was peptidergic and expressed
CGRP (Han et al., 2013). In addition, first evidence
showed that Smad4 and Bone morphogenetic peptide
signaling is essential for the expression of mMrgprB4
in another subset of neurons (Liu et al., 2008). Mouse
MrgprX1 is always coexpressed with an MrgprA and
never with MrgprBs in DRG neurons (Liu et al., 2008).
In the rat, the situation is different (Lembo et al., 2002;
Zylka et al., 2003): rMrgprX1 is never coexpressed with
rMrgprA and rMrgprD is always coexpressed with
rMrgprA and, in some cells, with rMrgprB4. Furthermore, rMrgprX1-positive DRG neurons in the rat are
also peptidergic (Lembo et al., 2002; Hager et al., 2008).
In humans and macaques also, all the MRGPRX
orthologs and MRGPRD are found exclusively expressed
in DRG neurons (Dong et al., 2001; Lembo et al., 2002;
Robas et al., 2003; Zhang et al., 2005), but not much is
known about the characteristics of these cells, despite
the fact that in these species a significant proportion of
them also seem to be peptidergic and express TRPV1,
CGRP, and substance P (Zhang et al., 2005).
C. Expression of Mas-Related G Protein–Coupled
Receptors in Mast Cells
A second major cell type expressing several Mrgprs
in considerable amounts in addition to DRG neurons
is the mast cell. In humans, mainly hMRGPRX2 is
expressed in human umbilical cord mast cells as well
as small amounts of hMRGPRX1, but not hMRGPRX3
and hMRGPRX4 (Tatemoto et al., 2006; Subramanian
et al., 2011b). In rats, expression of rMrgprB3 and B8
was detectable in peritoneal mast cells but only low
levels of rMrgprB1, B2, B6, and B9 were detected, and
neither rMrgprA, rMrgprX1, nor any other rMrgprB
1087
Mas-Related Genes
TABLE 2
Animal models with alterations in an Mrgpr
Name/Affected Mrgpr
mMas-KO
rMas-KO
rMas-opsin-TG
mMrgprA1, A2, A3, A4, A10,
A12, A14, A16, A19, B4,
B5, C11 KO
mMrgprA1-tetO-TG
Genetic Alteration
References
Knockout mouse
Walther et al., 1998
Knockout rat (generated with zinc finger
http://rgd.mcw.edu/rgdweb/report/gene/main.
nucleases)
html?id=5131946
Transgenic mouse with rMas gene under opsin Xu et al., 2000
promoter control
Knockout mouse for gene cluster
Liu et al., 2009
+
—
Transgenic mouse with mMrgprA1 gene under
tet operator control
+
mMrgprA2-KO
Knockout mouse
mMrgprA3-Cre
Transgenic mouse with Cre-recombinase
inserted in mMrgprA3-BAC
Knockin of placental alkaline phosphatase,
knockout
Knockin of Farnesyl-enhanced GFP-2A-Flprecombinase, knockout
Knockin of tdTomato-2A-Cre-recombinase,
knockout
Knockout mouse
mMrgprB4-PLAP
mMrgprB4- eGFPf -2A-Flp
mMrgprB4- tdTomato-2ANLSCre
mMrgprD-KO
Phenotype
Described
mMrgprD-DTA-IRES- eGFPf Diphtheria-toxin A and Farnesyl-enhanced
GFP knockin mouse, knockout
mMrgprD-DTR-IRES- eGFPf Diphtheria-toxin receptor and Farnesylenhanced GFP knockin mouse, knockout
mMrgprD-eGFP-Cre
eGFP-Cre-recombinase knockin mouse,
knockout
mMrgprDD-eGFPf
Farnesyl-enhanced GFP knockin mouse,
knockout
mMrgprD-eGFPf
IRES-Farnesyl-enhanced GFP knockin mouse,
no knockout
mMrgprDD-PLAP
Knockin of placental alkaline phosphatase,
knockout
mMrgprDD-ChR2-Venus
Knockin of channel rhodopsin-Venus, knockout
mMrgprE-KO
Knockout mouse
mMrgprF-KO
Knockout mouse
mMrgprG-KO
Knockout mouse
mMrgprH-KO
Knockout mouse
mMrgprx1-KO
Knockout mouse
hMRGPRX3-actin-TG
Transgenic rat with hMRGPRX3 gene under
chicken actin promoter control
http://www.mmrrc.org/catalog/sds.php?
mmrrc_id=29882/029882.html; Fiacco et al.,
2007
http://kodatabase.taconic.com/database.php?
id=TF0436
Han et al., 2013
+
+
—
+
Liu et al., 2007
+
Vrontou et al., 2013
+
Vrontou et al., 2013
+
http://kodatabase.taconic.com/database.php?
id=TF0437
Cavanaugh et al., 2009
—
Cavanaugh et al., 2009
+
Rau et al., 2009
+
Zylka et al., 2005
+
Zylka et al., 2005
+
Zylka et al., 2005
+
Wang and Zylka, 2009
Cox et al., 2008
http://kodatabase.taconic.com/database.php?
id=TF1876
http://kodatabase.taconic.com/database.php?
id=TF0924
http://kodatabase.taconic.com/database.php?
id=TF0274
http://kodatabase.taconic.com/database.php?
id=TF2901
Kaisho et al., 2005
+
+
—
+
—
—
—
+
DTA, diphtheria-toxin A; DTR, diphtheria-toxin receptor; GFP, green fluorescent protein; IRES, internal ribosomal entry site; PLA, phospholipase A.
was expressed in this cell type (Tatemoto et al., 2006).
In contrast, rMrgprX1 was detected by immunohistochemistry in skin mast cells (Hager et al., 2008).
mMrgprB10 was found in mucosal mast cells in mouse
intestine in inflammatory conditions (Avula et al., 2013).
The presence of other Mrgprs (A and E–H) was not tested
in mouse mast cells other than in one study showing the
absence of mMrgprA3 transcripts (Liu et al., 2009).
D. Expression of Mas-Related G Protein–Coupled
Receptors in Other Tissues
Although most mouse MrgprAs, MrgprBs, and MrgprX1
seem to be exclusively expressed either in DRG neurons
(Dong et al., 2001) or in mast cells, some Mrgprs have been
shown to be expressed also in other tissues. Only low
expression of mMrgprB3 and mMrgprB8 has been described for bladder and testis, respectively (Huang et al.,
2013). Human and mouse MrgprE are widely expressed in
neurons in the brain, and hMRGPRE is expressed also in
placenta but not in any other human tissue (Zhang et al.,
2005; Cox et al., 2008). rMrgprE is mainly expressed in
DRG but also in the brain, spinal cord, and testis (Milasta
et al., 2006). hMRGPRF, rMrgprF (originally named RTA),
and mMrgprF were shown to be mainly expressed in
cerebellum and smooth muscle–containing tissues, such as
large vessels (Ross et al., 1990) (Patent WO2003004528A1,
2001). More recently, hMRGPRF was found to be drastically
upregulated in monocyte-to-macrophage differentiation
(Hohenhaus et al., 2013). mMrgprH was originally
discovered as GPR90 in 2001, and its mRNA was detected
by Northern blot mainly in heart but less so in kidney
(Wittenberger et al., 2001). hMRGPRX2 expression was
highest in DRGs and could also be found in testis and gut,
but less so in several other tissues (Robas et al., 2003;
1088
Bader et al.
Allia et al., 2005; van Hagen et al., 2008). This was in
contrast to the original description of the hMRGPRX
family showing no expression in any other tissue than in
DRGs (Lembo et al., 2002). rMrgprD expression was also
found outside the main expressing tissue, DRG, in tissues
such as testis, urinary bladder, uterus, and arteries, but
less so in other tissues, including cerebellum, gut, and fat
(Shinohara et al., 2004; Milasta et al., 2006). Rat MrgprX1
and rMrgprA were also most highly expressed in DRG
and trigeminal ganglia but also broadly in nearly every
tested tissue (Bender et al., 2002; Gustafson et al., 2005).
In these studies, quantitative reverse-transcription polymerase chain reaction was used with total RNA from the
tissues; thus, the expressing cell type was not distinguished
and mast cells cannot be excluded. However, the localization of rMrgprA expression in the vasculature and in the
collecting duct of the kidney was recently reported in detail,
and an inhibition of vasopressin signaling by rMrgprA was
postulated (Kishore et al., 2013).
One group has systematically analyzed the expression of all Mrgprs in mouse intestine (Avula et al.,
2011, 2013). With a few exceptions, they are all expressed
on different types of enteric neurons in varying levels;
however, the function of Mrgprs in these cells is elusive.
Under normal conditions, mMrgprA4 and mMrgprA7 are
most highly expressed, and in inflammation, mMrgprA2,
mMrgprA5, mMrgprB2, mMrgprB10, mMrgprX1, and
mMrgprD get upregulated, which is partly due to the
invasion of mast cells (mMrgprB10) (Avula et al., 2013).
E. Expression of Mas
The founder gene of the Mrgpr family, Mas, is more
broadly expressed than most other Mrgprs. Highest
expression is found in brain and testis but low levels
are also detected in other organs. In the rat and mouse
brain, Mas transcripts are localized in the hippocampus and cerebral cortex (in particular in the dentate
gyrus), the CA3 and CA4 areas of the hippocampus, the
olfactory tubercle, the piriform cortex, and the olfactory
bulb, but also at lower levels all over the neocortex and
especially in the frontal lobe (Young et al., 1988;
Bunnemann et al., 1990). More recently, Mas expression
was also discovered in regions of the rat brain important
for cardiovascular regulation (Becker et al., 2007) and in
the monkey retina (Kitaoka et al., 1994). Mas expression in the brain starts in the rat at postnatal day 1,
with a pattern similar to that seen in the adult (Martin
et al., 1992), and stays plastic because it is transiently
upregulated after seizure episodes in the hippocampus
(Martin and Hockfield, 1993).
In the rodent testis, Mas expression is not detectable
in newborn animals, but rather, starts a few weeks
after birth and continuously increases during puberty
(Metzger et al., 1995; Alenina et al., 2002b). On the
cellular level, Mas is confined to Leydig and Sertoli
cells, with a clear preference for Leydig cells (Alenina
et al., 2002b).
However, Mas expression was also discovered in other
tissues of mice and rats, such as heart, kidney, lung,
liver, spleen, tongue, and skeletal muscle (Villar and
Pedersen, 1994; Metzger et al., 1995; Ferrario et al.,
2005; Alenina et al., 2008). This ubiquitous low-level
presence of Mas mRNA may partly be due to its expression in the endothelial layer of vessels in different
organs, as has been shown for brain (Kumar et al., 1996),
heart (Alenina et al., 2008), and corpus cavernosum
(Goncalves et al., 2007), supporting an important role of
this protein in the function of the endothelium.
IV. Structure, Ligands, and Signaling of MasRelated G Protein–Coupled Receptors
Several receptors of the Mrgpr family, when being
analyzed for ligand-induced activation, showed a high
constitutive activity (Chen and Ikeda, 2004; Burstein
et al., 2006; Uno et al., 2012). The structural reason
for this ligand-independent signaling is not yet clear
because there is no experimentally generated threedimensional structure for any of the Mrgpr family
members. Three-dimensional structures of Mas, rMrgprA,
and mMrgprX1 have been predicted from the comparison
with the rhodopsin structure (Heo et al., 2007a,b; Prokop
et al., 2013). However, later trials to validate the deduced
predictions for the agonist binding sites of rMrgprA by
specifically mutating them failed, raising doubts on the
validity of the structure predictions based on remotely
related homologs (Knospe et al., 2013). Nevertheless, it
is obvious from the primary sequence that all Mrgprs
lack otherwise highly conserved features among class A
GPCRs, which may be involved in blocking agonistindependent activity: the Cys-Cys bridge from transmembrane domain (TM) 3 to extracellular domain 2
(Knospe et al., 2013; Prokop et al., 2013) and the so-called
“ionic-lock” from the amino acids E/DRY in TM3 to
a glutamate in TM6 (Rosenbaum et al., 2009). This ionic
lock has been shown to inhibit activation and constitutive
activity in rhodopsin and some class A GPCRs (Scheer
et al., 1997; Palczewski et al., 2000), but not in others
(Schneider et al., 2010). However, in the absence of a
crystal structure of any Mrgpr, the molecular basis of the
intrinsic activity of these receptors cannot be resolved.
Despite their high constitutive activity, Mrgprs can
be further stimulated by agonists, but for most of them,
only a single agonist could be identified. Moreover,
even closely related members of the Mrgpr family
showed quite divergent ligand specificities. However,
all available data about agonists are based on gain-offunction experiments in transfected cells. There currently is no proof that any of the described agonists is
the natural ligand of the respective Mrgpr. Furthermore, in most cases, the effective concentrations of the
agonists are hardly reached in vivo. Thus, nearly all of
the Mrgprs still have to be deorphanized. The only
exceptions may be Mas itself and MrgprD, for which
Mas-Related Genes
angiotensin (Ang)-(1–7) and b-alanine, respectively,
have been described as agonists, the physiologic effects
of which are absent in corresponding knockout mice
(Santos et al., 2003; Rau et al., 2009) (Tables 2 and 3).
For mMrgprX1, the absence of effect of its possible
endogenous ligand bovine adrenal medulla (BAM)
peptide 8–22 has been shown in the cluster knockout
mice (Fig. 2; Table 2) lacking 12 Mrgpr genes, including
the one for mMrgprX1; thus, the data are not totally
conclusive (Guan et al., 2010). For no other Mrgpr has
the absence of effect of a naturally existing ligand in
a deficient animal model been shown. For hMRGPRX2,
the knockdown by short hairpin RNA in a mast cell line
also leads to an attenuation of the effects of known
agonists, but again, these experiments are done in cell
culture with relatively high agonist concentrations and
do not reflect the in vivo situation (Subramanian et al.,
2013). Therefore, and based on the high constitutive
activity of Mrgprs, the hypothesis has been coined that
their basic state is the active one and that their yet
unknown natural ligands should be inverse agonists,
which inhibit their activity (Burstein et al., 2006).
Nevertheless, no screen of naturally occurring inverse
agonists has yet been performed, and thus, we may
still miss the physiologic ligands of Mrgprs.
Another possibility is that Mrgprs were not evolved
for the recognition of endogenous ligands but for eliciting
itch or pain for warning an organism of noxious
substances released to its skin by toxic plants or animals
or by parasitic infections. Evidence for this hypothesis are
1) their relatively high promiscuity with best affinities for
itch-inducing substances; 2) their nearly exclusive expression in sensory neurons and mast cells in the skin, the
main cellular components of the itch pathway; and 3) their
evolutionary appearance in tetrapods together with arms
and legs that allow scratching. Fish cannot fight skin
invaders by scratching, thus, itch-inducing sensory receptors would be useless. However, on land, it is a great
advantage to be able to remove parasites or other noxious
substances before they create more harm. Because the
parasites coevolved with the Tetrapods, and as a consequence each species faced different parasitic challenges,
the parallel adaptive evolution of the Mrgprs by multiple
independent duplication and expansion events in different
species could also be explained. Nevertheless, some of the
Mrgprs have endogenous ligands and elicit effects beyond
itch and pain. Thus, the evolution of the Mrgprs may not
have only been driven by the invention of itch.
In the following, the agonists described for the different Mrgprs are listed (Table 3).
A. Mas
After the initial erroneous description of Ang II as
Mas-agonist (Jackson et al., 1988) (see section I), we
characterized Ang-(1–7) as functional ligand for this
GPCR (Santos et al., 2003). In this study, specific
binding of [125I]Ang-(1–7) to Mas-transfected cells was
1089
reported, and the low, but significant specific binding
of [125I]Ang-(1–7) to kidney sections was annulled by
genetic deletion of Mas. Specific binding of [125I]Ang-(1–7)
was also reported in cells transfected with fluorescently
labeled Mas (Gironacci et al., 2011). In these two studies,
the Ang-(1–7) antagonist, A-779 (Santos et al., 1994),
competed for the binding of [125I]Ang-(1–7) to the
Mas-transfected cells with an IC50 close to the EC50 of
[125I]Ang-(1–7). Nevertheless, standard pharmacologic
binding experiments with [125I]Ang-(1–7) are limited by
the high nonspecific binding of the radiolabeled peptide,
possibly due to the introduction of the bulky iodine
isotope in the peptide sequence, which may favor low
affinity binding to Mas-unrelated sites. In contrast, the
nonspecific binding observed in experiments using
fluorescent Ang-(1–7) is very low. By using this method,
a significant, specific binding of Fam-Ang-(1–7) to Mastransfected CHO cells has been reported (Pinheiro et al.,
2004; Savergnini et al., 2010; Jankowski et al., 2011).
Accordingly, specific binding of Fam-Ang-(1-7) to platelets (Fraga-Silva et al., 2008) and Leydig cells in testes
(Leal et al., 2009) is absent in Mas-knockout (KO) mice.
The blockade of the effects of the Ang-(1–7) mimic,
AVE0991, by A-779 (Wiemer et al., 2002) and the functional
effects produced by Ang-(1–7) in Mas-transfected cells
(Santos et al., 2003; Pinheiro et al., 2004; Sampaio et al.,
2007b) are in keeping with the significant body of evidence
that Ang-(1–7) is an endogenous ligand for Mas. A recent
in silico docking experiment explains the different
affinities of Mas for Ang II and Ang-(1–7) (Prokop et al.,
2013): the additional Phe8 of Ang II compared with
Ang-(1–7) interacts with amino acid residues of the
AT1 and AT2 receptors, which lack in Mas.
As further evidence for the specific interaction of
Ang-(1–7) with Mas, we showed that Mas-KO mice lost
responsiveness to Ang-(1–7) with respect to vasodilation,
antidiuresis, and antithrombosis (Santos et al., 2003;
Fraga da Silva et al., 2008, 2011). Likewise, the
antihypertrophic effect of Ang-(1–7) in cardiomyocytes
was blunted by downregulation of Mas by antisense
RNA (Tallant et al., 2005). Nevertheless, there are
reports about other receptors for Ang-(1–7), such as the
Ang II AT2 receptor (De Souza et al., 2004; Walters
et al., 2005) and other natural ligands for Mas (Dong
et al., 2001; Gembardt et al., 2008; Jankowski et al., 2011).
Thus, it is not clear whether the interaction between
Ang-(1–7) and Mas is mutually exclusive.
It should be pointed out that all the described effects
of Ang-(1–7) or AVE0991 in Mas-transfected cells are
related to a Ca2+-independent activation of nitric oxide
synthase (via the phosphatidylinositol 3-kinase/protein
kinase B [AKT] pathway) or to activation of phospholipase A2 (Santos et al., 2003; Pinheiro et al., 2004;
Sampaio et al., 2007b; Savergnini et al., 2013; Than
et al., 2013) (Fig. 4). Our recent phosphoproteomic study
identified several further possible signaling pathways
of cells induced by Ang-(1–7) stimulation, such as the
1090
Bader et al.
TABLE 3
Ligands of Mrgprs
Some structures are shown on the bottom of the table. The EC50 values are different depending on the assay system used in each publication and can only be taken as
estimation concerning the specificity of the receptors.
Mrgpr
Ligands
Structure or Peptide Sequence
EC50
References
mM
Mas
mMrgprA1
mMrgprA3
mMrgprA4
mMrgprA9
mMrgprA10
rMrgprA
mMrgprX1
rMrgprX1
MrgprD (rat, mouse,
and human)
hMRGPRX7
SNSR3
hMRGPRX1
hMRGPRX2
Angiotensin-(1–7)
NPFF
Angioprotectin
FLRF
NPFF
Salusin-b
Chloroquine
NPAF
ACTH
Adenine
Adenine
Adenine
DRVYIHP
FLFQPQRF-amide
PEVYIHP
FLRF-amide
FLFQPQRF-amide
AIFIFIRWLLKLGHHGRAPP
see below
AGEGLNSQFWSLAAPQRF-amide
39 amino acids
see below
see below
see below
g1-MSH
g2-MSH
FLRF
NPFF
SLGRL
BAM22
BAM8–22
Antho-RFamide
BAM22,
BAM8–22,
g1-MSH
g2-MSH
b-Alanine
GABA
Alamandine
g1-MSH
BAM22,
BAM8–22
g1-MSH
Compound 48/80
BAM22,
BAM8–22
Chloroquine
MCD
YVMGHFRWDRF-amide
YVMGHFRWDRFG
FLRF-amide
FLFQPQRF-amide
SLGRL
YGGFMRRVGRPEWWMDYQKRYG
VGRPEWWMDYQKRYG
pEGFR-amide
YGGFMRRVGRPEWWMDYQKRYG
VGRPEWWMDYQKRYG
YVMGHFRWDRF-amide
YVMGHFRWDRFG
see below
see below
ARVYIHP
YVMGHFRWDRF-amide
YGGFMRRVGRPEWWMDYQKRYG
VGRPEWWMDYQKRYG
YVMGHFRWDRF-amide
see below
YGGFMRRVGRPEWWMDYQKRYG
VGRPEWWMDYQKRYG
see below
MCICKNGKPLPGFIGKICRKICMMEETHamide
38 amino acids
RPKPEEFFGLM-amide
70 amino acids
HSDAVFTDNYTRLRKQMAVKKYLNSILN
PCKNFFWKTFSSCK
FRKKWNKWALSR
see below
AGCKNFFWKTFTSC
. 40 amino acids
35–40 amino acids
PACAP
Substance P
Platelet factor-4
VIP
Cortistatin-14
PAMP-12
Compound 48/80
Somatostatin
b-Defensins
Cathelicidines
Chloroquine
Adenine
b-Alanine
N.D., kD: 0.08nM
0.4
N.D.
0.02–0.4
0.2–2.1
0.30
0.028
0.06
0.2
0.01
0.01
0.02
0.017
0.011
0.157
0.054
10.1
0.026
0.053
0.016
0.155
0.12
0.063
0.037
14–44
165- . 300
0.46
0.013
0.028
0.6
70
0.016
0.014–0.04
0.3
1.25
1.64
2.4–8.0
8.96
2.63
0.03–0.06
3.75
0.06
0.03–5.70
N.D.
65
GABA
Dong et al., 2001; Santos
et al., 2003; Jankowski
et al., 2011
Dong et al., 2001; Han
et al., 2002; Wang et al., 2006
Liu et al., 2009
Dong et al., 2001
von Kügelgen et al., 2008
Thimm et al., 2013
Bender et al., 2002; Gorzalka
et al., 2005
Han et al., 2002; Liu et al., 2011;
He et al., 2014
Grazzini et al., 2004;
He et al., 2014
Shinohara et al., 2004;
Lautner et al., 2013
Lembo et al., 2002
Lembo et al., 2002; Tatemoto
et al., 2006; Hager et al.,
2008; Liu et al., 2009;
Kashem et al., 2011
Robas et al., 2003; Kamohara
et al., 2005; Nothacker
et al., 2005; Tatemoto
et al., 2006; Subramanian
et al., 2013
Compound 48/80
N.D., not determined; NPAF, neuropeptide AF; NPFF, neuropeptide FF.
forkhead box protein O1 (Verano-Braga et al., 2012). It
is not yet clear how these early signaling events induced
by Ang-(1–7) are linked to the NO and phospholipase A2
pathway. In the phosphoproteomic study, we also saw an
Ang-(1–7)–induced dephosphorylation of mitogen activated
protein kinases in contrast to some cell types of the kidney,
in which an activation was observed (Zimpelmann and
Burns, 2009; Liu et al., 2012b). Some kidney cells also
showed an increase in cAMP and activation of protein
kinase A after Ang-(1–7) challenge, suggesting Gs activation (Magaldi et al., 2003; Liu et al., 2012b). Nevertheless,
in these studies there is only indirect evidence that Mas is
involved in the Ang-(1–7) effects based on the fact that they
could be inhibited with A-779.
Mas-Related Genes
On the other hand, there is no evidence for an effect
of Ang-(1–7) and AVE0991 on the stimulation of other
Mas-mediated signaling pathways such as Ca2+ influx/
release or inositol 1,4,5-trisphosphate accumulation
(Dias-Peixoto et al., 2008; Shemesh et al., 2008; Gomes
et al., 2012; Zhang et al., 2012) (Fig. 4), which are particularly pronounced in strongly Mas-overexpressing cells.
These Ang-(1–7) independent signaling pathways are
most likely the ones induced by the constitutive activity
of the receptor and are responsible for its transforming
activity. Some studies have found ligands of Mas that
modulate these pathways, especially at high concentrations (. 1 mM), such as the agonists MBP7 (Bikkavilli
et al., 2006), AR234960 (Zhang et al., 2012), and CGEN856S (P61 in Shemesh et al., 2008) with serine instead of
cysteine (Savergnini et al., 2010), and the inverse agonist,
AR244555 (Zhang et al., 2012). Interestingly, CGEN-856S
has been shown to activate both pathways, Ca2+ and
AKT—but Ca2+ only in Ga16 transfected cells with a
diverted signaling (Shemesh et al., 2008; Savergnini et al.,
2013). Thus, the still limited evidence about Mas signaling
suggests that Mas behaves like other GPCRs, for which it
has also been shown that different ligands activate different signaling pathways, a phenomenon called functional
selectivity or biased signaling (Urban et al., 2007; Kenakin
and Christopoulos, 2013) (Fig. 4).
B. hMRGPRX1 (hSNSR4)
hMRGPRX1 stably expressed on mast cells was shown
to be activated by BAM22, a product of proenkephalin A
1091
metabolism, and by compound 48/80, a classic mast cell
activator (Kashem et al., 2011) (Table 3). This was
consistent with the findings of Lembo et al. (2002), who
had cloned hMRGPRX1 as SNSR4 and had discovered
that it can be activated by peptides derived from
proenkephalin A at an EC50 of 16 nM. The signaling of
this receptor was not abolished by pertussis toxin and,
thus, was transmitted by Gq proteins. When they
analyzed the binding domain on BAM22, they found that
the first 7 amino acids are not relevant for hMRGPRX1
binding, in contrast to its binding to opioid receptors. The
peptide lacking this Met-enkephalin motif, BAM8–22,
had the same affinity to hMRGPRX1 as BAM22 itself.
BAM8–22 also activated rat neurons stably expressing
hMRGPRX1 (Chen and Ikeda, 2004). In these cells,
hMRGPRX1 inhibited the activity of high-voltage–
activated Ca2+ and M-type K+ channels. Whether this
is also true in naturally hMRGPRX1 expressing cells
remains elusive. First small-molecule antagonists for
hMRGPRX1 have also already been developed (Kunapuli
et al., 2006; Bayrakdarian et al., 2011).
C. rMrgprX1 (rSNSR1, rMrgC), mMrgprX1 (mMrgC11)
Peptides derived from proenkephalin A and
proopiomelanocortin (POMC) have been shown to bind
cells expressing rMrgprX1 (Grazzini et al., 2004) and
mMrgprX1 (Han et al., 2002) with EC50 values of 11 to
150 nM (Table 3). In particular, the POMC derivatives,
g1- and g2-melanocyte-stimulating hormone (MSH),
show highest affinity and selectivity for these receptors.
Fig. 4. Signaling of Mas. The most important known signaling pathways of Mas involve phospholipase A (PLA) to generate arachidonic acid (AA) and
phosphoinositide 3 kinase (PI3K) and AKT to activate eNOS by phosphorylation at serine 1177 and dephosphorylation at threonine 495. Ang-(1–7),
AVE0991, CGEN-856S, and angioprotectin have been shown to activate these pathways and A-779 antagonizes these actions. Ang-(1–7) also activates
forkhead box protein O1 by dephosphorylation at serine 236. The ligands for the PLA and PI3K pathways (other than CGEN-856S), however, do not
activate a phospholipase C (PLC)/Ca2+ signaling pathway observed in Mas-overexpressing cells, which in some studies were additionally transfected
with promiscuous G proteins. This pathway is activated by the ligands neuropeptide FF (NPFF), AR234960, MPF7, and CGEN-856S and blocked by
AR244555. Thus, different Mas ligands activate distinct signaling pathways, a phenomenon called biased agonism.
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Bader et al.
g2-MSH elicits Ca2+ release but no change in cAMP
metabolism in mMrgprX1 expressing cells, supporting
Gq coupling of this receptor (Han et al., 2002). Because
rMrgprX1 and mMrgprX1 also bind the proenkephalin
A metabolite BAM22, they may be functional orthologs
to the human MRGPRX1 receptor (see above). However,
one study unraveled a difference between the human
and the rodent MrgprX1s: Solinski et al. (2010) showed
that hMRGPRX1 is not internalized after agonist binding,
in contrast to mMrgprX1 and rMrgprX1.
Another functionally important ligand of mMrgprX1
is the peptide SLIGRL, which is released from the
protease activated receptor 2 upon trypsin digestion
(Liu et al., 2011), and which is responsible for the itch
response induced by intradermal trypsin injections.
He et al. (2014) recently showed that the hMRGPRX1
antagonist, 2,3-disubstituted azabicyclo-octane (Kunapuli
et al., 2006), also blocks rMrgprX1 and mMrgprX1. They
also describe a first synthetic MrgprX1 agonist, JHU58
(He et al., 2014).
D. mMrgprA1, mMrgprA4, mMrgprX1 (mMrgC11)
Already the first description of Mrgprs had discovered
that peptides terminating with the amino acids RF/Y-amide
or RF/YG are ligands for some members of this family,
including Mas itself (Dong et al., 2001). Such peptides
include the already mentioned BAM and MSH derivatives. In addition, the tetrapeptide FLRF and neuropeptide AF (NPAF) (Table 3) were shown to bind to
mMrgprA1 and mMrgprA4, respectively, with EC50
values of lower than 100 nM (Dong et al., 2001). The
authors could also demonstrate that these receptors
desensitize after activation with the preferred ligand.
Moreover, mMrgprX1, in addition to its affinity for
proenkephalin A and POMC metabolites, is also activated
by RF-amide peptides with comparable EC50 as the
MrgprA family members (Han et al., 2002). These
peptides elicit Ca2+ influx in cells expressing mMrgprA1
or mMrgprX1 only depending on Gq proteins.
A high-throughput approach testing 16,000 compounds
identified two other agonists for MrgprA1, which, however, are not further chemically defined (Zhang et al.,
2007). Furthermore, salusin-b, a peptide of yet not well
defined functions, has been characterized as agonist for
mMrgprA1, but no MRGPR ortholog in humans seems
to react on this substance, questioning the physiologic
importance of this finding (Wang et al., 2006).
The specific activation of mMrgprA1 by FLRF and
its normally very restricted expression in DRGs was
exploited for the generation of transgenic mice in
which Ca2+ signaling in all astrocytes can be activated
by FLRF (Table 3). These animals were created by
breeding mice expressing the tetracycline repressor
under the control of the astrocyte-specific glial-fibrillary
acidic protein promoter, with animals carrying the
mMrgprA1 coding region under the control of the tet
operator (Fiacco et al., 2007) (Table 2). When they are
given doxycycline, the mice ectopically express mMrgprA1
only on astrocytes and have been used to study the role of
this cell type in synaptic transmission by applying the
agonist, FLRF (Fiacco et al., 2007; Agulhon et al., 2010;
Lacar et al., 2012; Xie et al., 2012; Cao et al., 2013b;
Devaraju et al., 2013; Wang et al., 2013b).
E. mMrgprA3, rMrgprA, hMRGPRX1
There is yet no endogenous ligand known for mMrgprA3.
However, it has been shown to be activated by the
antimalaria compound, chloroquine (Table 3), and to be
responsible for its pruritogenic actions (Liu et al., 2009,
2011; Wilson et al., 2011). When activated, mMrgprA3
opens transient receptor potential (TRP) A1 channels
via Gbg release (Wilson et al., 2011). In the rat and in
humans, rMrgprA and hMRGPRX1, respectively, seem
to be the responsible receptors for chloroquine sensing
(Liu et al., 2009). EC50 values are in the high micromolar
range, which is sufficient to explain the itch racking
chloroquine-treated patients, because the drug accumulates in the skin (Liu et al., 2009).
F. hMRGPRX2, rMrgprB3
hMRGPRX2 on mast cells was shown to be activated
by compound 48/80 (Kashem et al., 2011) (Table 3).
Moreover, an artificial C3a receptor agonist, E7, also
stimulates hMRGPRX2 (Kashem et al., 2011). The same
group showed that antimicrobial peptides of two classes,
b-defensins (e.g., hBD2 and hBD3) and cathelicidines
(e.g., LL-37), are ligands at the hMRGPRX2 receptor on
human mast cells and activate Gq- and Gi-dependent
signaling pathways (Subramanian et al., 2011a, 2013).
On rat mast cells, the rMrgprB3 receptor probably
confers this function, but it is not linked to Gq proteins,
and there are no receptors for these peptides detectable
in functional assays using the mouse defensin, mCRAMP,
on mouse mast cells (Subramanian et al., 2013). Another
group has confirmed in human and rat mast cells that
hMRGPRX2 and rMrgprB3 (but not rMrgprA, B2, B6, B8,
and B9), respectively, are transmitting the degranulating
action of numerous basic substances including mast cell
degranulating peptide, substance P, pituitary adenylate
cyclase–activating peptide (PACAP), and platelet activating
factor 4, with EC50 values in the low micromolar range
(Tatemoto et al., 2006).
Furthermore, cortistatin-14 (EC50: 25 nM) and -17
(EC50: 99 nM) have been shown to be agonistic ligands
of hMRGPRX2 (Robas et al., 2003) (Table 3), but their
tissue distribution does not overlap much and there is
no hMRGPRX2 homolog in rodents, which challenges
the physiologic relevance of this finding (Kamohara
et al., 2005; Siehler et al., 2008; van Hagen et al.,
2008). Cortistatin increases Ca2+, but not cAMP, in
hMRGPRX2-expressing cells, arguing in favor of Gq
coupling of hMRGPRX2 (Robas et al., 2003). However,
the same publication lists several other peptides with
only slightly higher EC50s (.100 nM). Furthermore, the
Mas-Related Genes
proadrenomedullin C-terminal peptides, PAMP-12 and
-20, showed comparable affinities to hMRGPRX2, and
activated Gi and Gq proteins in hMRGPRX2-expressing
cells (Kamohara et al., 2005; Nothacker et al., 2005).
These data again show that Mrgprs are quite promiscuous
and that ligands bind these receptors with considerably
lower affinity than the ligands of most other GPCRs.
Nevertheless, the expression of short hairpin RNA against
hMRGPRX2 leads to an attenuation of the effects of
cortistatins and b-defensins in a mast cell line (Subramanian
et al., 2013), which suggests a physiologic role of this
receptor in mast cell degranulation by these agonists.
G. hMRGPRX3 (hSNSR1), hMRGPRX4 (hSNSR6)
For hMRGPRX3 (hSNSR1) and hMRGPRX4 (hSNSR6),
no ligand has been described yet.
H. rMrgprA (rMrgprX3), mMrgprA9, mMrgprA10
The only rat MrgprA receptor (also called rMrgprX3)
has been shown to bind adenine (Table 3) with a
dissociation constant of 24 nM and to inhibit cAMP
generation by Gi protein coupling (Bender et al., 2002).
In mouse, two members of the MrgprA subfamily,
mMrgprA9 and A10, have also been described to bind
adenine, albeit with somehow lower affinities (Kd: 113
and 286 nM, respectively), and to inhibit cAMP generation
(von Kügelgen et al., 2008; Knospe et al., 2013; Thimm
et al., 2013). The same group has discovered an adenine
binding homolog in Chinese hamster, but there has not
yet been any adenine receptor of the Mrgpr family found
in humans. The binding of adenine to its receptors is very
specific, because even small molecular alterations markedly
reduce affinity of the ligand for rMrgprA and mMrgprA10
(Gorzalka et al., 2005; von Kügelgen et al., 2008; Borrmann
et al., 2009). A first small-molecule antagonist for rMrgprA
has also been developed (Kishore et al., 2013).
I. MrgprD, MrgprE
The first ligand described for MrgprD of human, rat,
mouse, and monkey was b-alanine (Shinohara et al.,
2004) (Table 3). This amino acid analog is produced in
the liver from uracil or from dietary carnosine by the
enzyme carnosinase (Stellingwerff et al., 2012). MrgprD
activation is transmitted by both Gq and Gi proteins
onto changes of intracellular Ca2+ and cAMP, respectively (Shinohara et al., 2004). The EC50 of b-alanine for
a rise in intracellular Ca2+ in MrgprD-expressing cells
was found to be between 14 and 44 mM, depending on
the species (Shinohara et al., 2004). b-Alanine binding
leads to rapid internalization of MrgprD (Shinohara
et al., 2004), which is inhibited when MrgprE is coexpressed
in transfected cells, because these two receptors most
likely form heterodimers (Milasta et al., 2006). When
KCNQ2/3 potassium channels are coexpressed with
MrgprD, as in most cells expressing the receptor naturally,
a potent inhibition of the channels is initiated by b-alanine
(Crozier et al., 2007). Also, a coupling to calcium-activated
1093
chloride channels via Gq proteins, phospholipase C, and
inositol-3 phosphate–induced Ca2+ release has been described, albeit in Xenopus oocytes (Zhuo et al., 2014).
A recent study, which systematically searched for
agonists in the Library of 640 Pharmacologically Active
Compounds (LOPAC) using a fluorescence image plate
reader assay confirmed the G protein coupling and
found even lower EC50 values for b-alanine [4 mM (Ajit
et al., 2010)]. In addition, this study showed that
GABA as well as 5,7-dihydroxytryptamine and HA-966
are potent agonists of MrgprD (Ajit et al., 2010). Another
high-throughput study using a fluorescence image plate
reader and 8000 compounds identified two further
agonists for MrgprD, which, however are not further
chemically defined (Zhang et al., 2007). Recently,
b-aminobutyric acid and diethylstilbestrol were added
to the list of agonists with EC50 values for Ca2+ release
of 53 and 10 mM, respectively (Uno et al., 2012). This
study also demonstrated the very high constitutive
activity of MrgprD: as Ajit et al. (2010) had observed,
cells overexpressing MrgprD at too-high levels could no
longer be stimulated by any agonist (Uno et al., 2012).
Uno et al. (2012) also describe an antagonist for MrgprD:
the chemical compound Mu-6840 inhibits constitutive
and b-alanine-induced activity of MrgprD in transfected
cells and can therefore be described as the first inverse
agonist of an Mrgpr, albeit not a natural one.
However, small molecules are not the only agonists
of MrgprD. Gembardt et al. (2008) discovered weak
effects of angiotensin peptides [Ang III and Ang-(1–7)]
on MrgprD-expressing cells. More recently, we described
a novel component of this peptide family, alamandine,
Ala1-Ang-(1–7), as potent agonist of MrgprD (Lautner
et al., 2013). Peptides seem to interact differently with
the receptor as b-alanine, because b-alanine does not
inhibit the vasodilatory actions of alamandine (Lautner
et al., 2013). However, D-Pro7-Ang-(1–7) was shown to be
an inhibitor of alamandine binding to MrgprD in addition
to its blocking effect on Ang-(1–7) binding to Mas
(Lautner et al., 2013).
J. MrgprF (RTA), MAS1L (mrg)
The first two Mrgprs discovered after Mas were MrgprF
(originally named RTA and expressed in all mammals)
and MAS1L (originally named mrg and only found in
primates) (Ross et al., 1990; Monnot et al., 1991). In both
papers describing the discoveries, no ligands could be
identified, but Ang II could be excluded (Ross et al., 1990;
Monnot et al., 1991). Nevertheless, angiotensins stimulated
MAS1L-expressing Xenopus oocytes, indicating stimulatory
cross-talk between MAS1L and angiotensin receptors
opposite to what was shown for Mas itself (Kostenis
et al., 2005). For MrgprF, this information is not available.
More recently, Gembardt et al. (2008) showed that Ang III
and Ang-(1–7) and also, slightly, Ang II can stimulate
MAS1L-expressing cells. They did not, however, exclude
cross-talk to potentially coexpressed angiotensin receptors.
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Bader et al.
K. MrgprG, MrgprH
For MrgprG and MrgprH receptors, no ligand has
been described yet.
L. Receptor Hetero-Oligomerization
GPCRs are known to form hetero-oligomers with
functions distinct from monomers or homo-oligomers
(Fuxe et al., 2008). Also, Mrgprs seem to influence the
signaling of other GPCRs by such interactions. Accordingly,
it has been shown that hMRGPRX1 can form dimers with
the d-opioid receptor, and binding of hMRGPRX1 ligands
to this heterodimer inhibits d-opioid signaling (Breit
et al., 2006). rMrgprX1 activation modulates m-opioid
receptor coupling in rat DRG neurons, in which they are
coexpressed (Wang et al., 2013a), and activation of
rMrgprA inhibits the signaling of coexpressed vasopressin
V2 receptors in the collecting duct of the kidney (Kishore
et al., 2013), but in both cases a direct interaction of the
receptors was not analyzed. Moreover, Mas was found to
bind the Ang II AT1 receptor (Kostenis et al., 2005; Santos
et al., 2007). This may explain the early findings that Ang
II could activate Mas-expressing cells more than control
cells (Jackson et al., 1988) and that permanent transfection of kidney cells with Mas can change the
hypertrophic actions of Ang II into a proliferative response (Wolf and Neilson, 1992) despite Mas not being
a receptor for Ang II. In these cells, however, as well as
in the study of Kostenis et al. (2005), an antagonizing
interaction between the two receptors was shown (Wolf
et al., 1995).
Also different Mrgprs can form hetero-oligomers with
each other when they are coexpressed in one cell: as
already mentioned above, rMrgprE binds to rMrgprD
and inhibits its internalization and increases its signaling (Milasta et al., 2006). Thus, hetero-oligomerization
with other GPCRs may be a common way by which
Mrgprs exert their actions.
V. Physiologic and Pathophysiologic Functions
of Mas-Related G Protein–Coupled Receptors
A. Mas-Related G Protein–Coupled Receptors in
Sensory Neurons
Most Mrgprs (all MrgprA, MrgprB, and MrgprC subfamily members, as well as MrgprD) are nearly exclusively
expressed in specific dorsal root and trigeminal ganglia
neurons. Therefore, it is not surprising that for these
Mrgprs functions, mainly nociception, itch, or pruritus,
and thermosensation have been described.
Mouse MrgprA3 and MrgprX1 are mostly coexpressed
in the same DRG neurons (Fig. 3) (Zylka et al., 2003).
When both genes were ablated (together with 10 other
ones in the same cluster; Fig. 2) the itch response to
chloroquine, SLIGRL, and BAM8–22 was blunted (Liu
et al., 2009, 2011). These primary sensory neurons have
endings in the epidermis of the skin and seem to be
responsible for itch induced by pruritogens in the skin
(Fig. 3). When these neurons were specifically ablated
by injecting diphtheria toxin into transgenic mice, in
which the diphtheria toxin receptor gene was activated
by Cre recombinase expressed under the control of
the mMrgprA3 promoter (Table 2), itch was much less
inducible by most pruritogens. This included substances
known to activate Mrgprs, such as chloroquine and
BAM8–22 (see section IV), and also histamine and
serotonin, at least in juvenile mice (Han et al., 2013). In
older animals, the cells become more selective most
likely by changes in receptor expression (Akiyama et al.,
2012). The itch specificity of these neurons was further
validated by an elegant gain-of-function experiment.
When the expression of TRPV1, which is involved in itch
and thermosensation (Mishra and Hoon, 2013), was
only rescued in mMrgprA3-expressing neurons of a
TRPV1 knockout mouse, these animals reacted only
with itch response on intradermal injections of capsaicin, which in normal animals would also induce pain
(Han et al., 2013). Concordantly, the overexpression of
mMrgprA3 in TRPV1-positive nociceptors induced by
a BRAF-expressing transgene led to a marked increase
in their response to pruritogens (Zhao et al., 2013).
Nevertheless, the main channel activated by mMrgprA3
during pruriception is not TRPV1 but transient receptor
potential ankyrin 1 (TRPA1), because TRPA1 knockout
mice hardly respond to the mMrgprA3 agonist chloroquine (Wilson et al., 2011), whereas in TRPV1 knockout
mice only histamine-induced itch is blunted (Imamachi
et al., 2009). Furthermore, TRPA1 knockout mice are not
responsive anymore to the mMrgprX1 agonist BAM8–22
despite that mMrgprX1 cannot directly open TRPA1
channels. In this case, mMrgprX1 activation seems to
open first TRPV1 channels by phospholipase C activation,
which, in turn, activates TRPA1 channels for effective
signaling (Wilson et al., 2011). These results were
confirmed in a recent experiment using chemical silencing
or pruriceptors, which also showed that TRPA1 mediates
the itch response to the mMrgprA3 and mMrgprX1 agonists chloroquine and SLIGRL, respectively (Table 3), and
that TRPV1+ cells are responsive to histamine (Roberson
et al., 2013). mMrgprX1 is also responsible for an itchinducing cross-talk between mast cells and DRG neurons:
mast cells release neuropeptide FF upon IgE stimulation,
which induces itch by binding to mMrgprX1 (Table 3) on
neighboring nerve endings (Lee et al., 2008).
Interestingly, itch could still be induced by b-alanine
in mice depleted of the mMrgprA-expressing neurons
(Han et al., 2013) (Table 2). The reason may be that the
receptor for b-alanine, mMrgprD, is expressed in other
neurons, and thereby defines as second subclass of
pruriceptors besides the one characterized by mMrgprA3
expression. When these mMrgprD-expressing neurons,
which do not express TRPV1 (Pogorzala et al., 2013),
were ablated by the diphtheria toxin receptor approach (Table 2), it was revealed that they are, indeed, not
involved in the itch response to most pruritogens (however,
Mas-Related Genes
b-alanine was not tested) (Imamachi et al., 2009). Instead
the cells are responsible for sensing of pain induced by
noxious mechanical stimulation (Cavanaugh et al.,
2009) and involved in the response to noxious hot and
cold temperatures (Pogorzala et al., 2013) but not for
the nocifensive responses to formalin (Shields et al.,
2010).
The role of mMrgprD in the nociceptive function of
these neurons is, however, not completely clear. Although,
as expected, b-alanine induced itch is virtually abolished
in mMrgprD knockout mice (Liu et al., 2012c) (Table 2),
these animals show no impairment in mechanical or
thermal nociception (Rau et al., 2009). Nevertheless,
isolated nociceptive neurons were clearly less responsive to mechanical and thermal stimuli in the absence of
functional mMrgprD (Rau et al., 2009). Thus, mMrgprD
seems to sensitize nociceptive neurons, either by the
known presence of b-alanine in the skin (Crush, 1970)
or, more likely, by the notorious constitutive activity
of MrgprD (Uno et al., 2012). However, the lack of
sensitization may be compensated in the sustained
absence of mMrgprD, possibly by other nociceptive
fibers in knockout mice.
For other Mrgprs, their influence on pain perception
is not completely resolved. Activators of mMrgprX1,
rMrgprX1, and hMRGPRX1, such as BAM8–22, have
been shown to induce both hyperalgesia and hypoalgesia
(Grazzini et al., 2004; Ndong et al., 2009; Guan et al.,
2010; Jiang et al., 2013). Recent experiments with the
cluster knockout mice lacking 12 Mrgprs, including
mMrgprX1 (Fig. 2; Table 2), shed some light on this
controversial issue (Guan et al., 2010). These animals
exhibit unaltered nociceptive thresholds at baseline,
showing that normal pain processing is not dependent
on the Mrgprs deleted in the cluster. The same was
true in rats with downregulation of the rMrgprX1
gene by local siRNA injection in the spinal cord
(Ndong et al., 2009). However, the cluster-knockout
mice and the rMrgprX1-siRNA–injected rats exhibited
prolonged pain sensitivity after hind paw inflammation
(Ndong et al., 2009; Guan et al., 2010). The so-called
“windup effect,” the activity-dependent sensitization
of wide dynamic range neurons in the spinal cord, is
involved in this hyperalgesia observed in inflammation.
Intrathecal administration of BAM8–22 inhibited this
effect in wild-type but failed to do so in the cluster-knockout
mice (Guan et al., 2010). Interestingly, BAM8–22 even had
the opposite effect in these mice by inducing hyperalgesia,
which may explain some of the inconsistency in the
published data about this molecule. Accordingly, BAM8–
22 and the MrgprX1 agonist JHU58 inhibited neuropathic
pain induced by nerve ligation (He et al., 2014). This effect
was abolished by the MrgprX1 inhibitor 2,3-disubstituted
azabicyclo-octane (Kunapuli et al., 2006) and absent in
Mrgpr-cluster knockout mice and rMrgprX1-siRNA–
treated rats (He et al., 2014). Furthermore, BAM8–22
inhibits the upregulation of inflammatory mediators,
1095
such as CGRP, in the spinal cord (Jiang et al., 2013),
and it changes the signaling of m-opioid receptors,
enhancing and maintaining their antinociceptive activity even in the presence of morphines, which normally
would induce tolerance (Cai et al., 2007b; Chen et al., 2010;
Wang et al., 2013a). Thus, Mrgprs binding BAM8–22, such
as mMrgprX1, rMrgprX1, and hMRGPRX1, exert different
effects in the skin, where they elicit itch and pain (Liu
et al., 2009; Sikand et al., 2011), and in the spinal cord,
where they exert mainly hypoalgesic but also, albeit less
potently, hyperalgesic actions (Guan et al., 2010). These
actions are more pronounced in inflammatory conditions, probably because the expression of BAM22 (Cai
et al., 2007a) and of rMrgprX1 (Jiang et al., 2013) are
upregulated in DRGs in inflammation, e.g., induced by
intrapaw injection of complete Freund’s adjuvant; however, there are also conflicting data in the same model
(Ndong et al., 2009), and a downregulation of rMrgprX1
has been observed after spinal nerve ligation (Gustafson
et al., 2005).
The role of mMrgprE in pain perception was studied
using a knockout mouse for the protein (Table 2). However, despite the high expression of this Mrgpr in DRGs,
spinal cord, and brain, there were only slight alterations
in nociception after its ablation, namely a delayed
development of allodynia after sciatic nerve injury (Cox
et al., 2008). This mere absence of effect may be due to
the observed, probably compensatory, upregulation of
mMrgprF in mMrgprE-deficient mice.
mMrgprB4 marks a subset of sensory neurons that
have recently been shown to confer the sensation of
gentle touch and induce anxiolytic effects when activated
in mice (Liu et al., 2007; Vrontou et al., 2013). However,
the function of mMrgprB4 itself in these effects, if any,
was not reported, and because there is no agonist known
yet, it cannot easily be tested. Some indirect evidence for
a role of mMrgprB4 in behavior comes from knockout
mice for the acid-sensing ion channel 3, which show
changes in maternal behavior and a drastic dysregulation of the mMrgprB4 gene in the dorsal root and
trigeminal ganglia (Huang et al., 2013).
Taken together, one major function of Mrgprs is the
induction of itch by a specific subset of primary sensory
neurons with endings in the epidermis and cell bodies
in a DRG (Fig. 3). These neurons have recently been shown
to use brain natriuretic peptide as transmitter activating
natriuretic peptide A receptors on secondary pruriceptor
neurons (Mishra and Hoon, 2013). Furthermore, these
neurons also seem to be involved in nociception, but the
respective functions of the Mrgprs in these cells remain to
be clarified.
B. Mas-Related G Protein–Coupled Receptors in
Mast Cells
Some Mrgprs are probably the long-sought low-affinity,
low-selectivity receptors on mast cells that mediate IgEindependent degranulating actions of numerous, mainly
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Bader et al.
basic compounds. As already mentioned above (section
IV.F), hMRGPRX2 and rMrgprB3, which are highly
expressed in mast cells, are activated by mast-cell
degranulating peptide, substance P, PACAP, compound
48/80, b-defensins, cathelicidines, and platelet activating
factor 4 (Table 3) and trigger the release of histamine
(Tatemoto et al., 2006; Kashem et al., 2011; Subramanian
et al., 2011a, 2013). Histamine, in turn, activates
pruriceptive neurons in the skin different from the ones
mentioned above that express Mrgprs. Thus, the final
effect of Mrgprs on mast cells is again the induction of
itch, corroborating the concept that they have evolved as
pruriceptors. Pruritogens in the skin can either activate
Mrgpr family members directly on somatosensory
neurons (e.g., hMRGPRX1 in humans) or other members on mast cells (e.g., hMRGPRX2 in humans), which
via the release of histamine activate another class
of neurons altogether, eliciting the sensation of itch.
Thereby the selectivity of the Mrgprs is not very high,
enabling the response to a large variety of substances
that may be evolutionarily advantageous and allows the
reaction on diverse skin-penetrating noxious stimuli.
C. Mas-Related G Protein–Coupled Receptors
in Tumors
As mentioned in section I, Mas was originally but
erroneously described as an oncogene (Young et al.,
1986, 1988). Other Mrgprs are overexpressed in tumors,
such as hMRGPRD, and the expression of hMRGPRD,
hMRGPRX1, and hMRGPRX4 as well as of rhesus
monkey MRGPRX genes elicit hyperproliferation in
transfected cells, most likely by their strong constitutive
activity (Andrawis et al., 1992; Burstein et al., 2006;
Nishimura et al., 2012). Similar effects are described
from Mrgpr-overexpressing transgenic animals (Table 2).
Transgenic rats overexpressing hMRGPRX3 controlled
by a ubiquitously active promoter develop eye and skin
phenotypes based on hyperproliferation of cells (Kaisho
et al., 2005). When Mas is overexpressed in retina using
the opsin promoter (Table 2), degeneration of photoreceptors is the consequence, which is probably induced
by proliferative signaling pathways activated in these
cells because of the constitutively active Mas protein (Xu
et al., 2000).
This transforming mechanism of Mrgprs has best
been studied for Mas itself in stably transfected cells
without addition of a ligand. Mas was shown to simultaneously couple to Gi and Gq proteins, which, in turn,
activate Rho proteins, such as rac1 (Zohn et al., 1998;
Booden et al., 2002; Chen and Ikeda, 2004; Singh et al.,
2010a). Rac1 stimulates cell cycle progression, and
thereby, proliferation of cells. It is conceivable that this
mechanism is also engaged by other Mrgprs, which induce
hyperproliferation, but there are no studies available.
However, none of the Mrgprs has yet been shown to
be a real oncogene causing cancer, even not Mas (see
section I). In the opposite, Ang-(1–7), the specific agonist
of Mas, has been described to have antitumor activities,
which could even be therapeutically exploited (Gallagher
et al., 2011). The tumorigenic and proproliferative actions
of Mas and maybe also the other Mrgprs may only be
observed above a certain threshold of overexpression in
cells which is normally not reached in vivo.
D. Mas-Related G Protein–Coupled Receptors and
Mas in Cardiovascular Organs
rMrgprA, mMgprD, rMrgprF, and mMrgprH have
been shown to be expressed in cardiovascular organs, but
very little is known about their functions there (Monnot
et al., 1991; Wittenberger et al., 2001; Shinohara et al.,
2004; Milasta et al., 2006) other than the possible
regulation of vasopressin signaling in the kidney by
rMrgprA (Kishore et al., 2013).
In contrast, there is a fair amount of data concerning
cardiovascular actions of Mas. Mas is expressed in many
cardiovascular-related organs and tissues, including
brain (hypothalamus, brain stem) (Becker et al., 2007;
Freund et al., 2012), heart (Santos et al., 2006; DiasPeixoto et al., 2008), kidney (Santos et al., 2003; Pinheiro
et al., 2004), and blood vessels (Sampaio et al., 2007a).
Most of the data related to cardiovascular actions of Mas
were obtained taking advantage of the availability of
Mas-deficient (Mas-KO) mice and with the use of Mas
agonists [Ang-(1–7), AVE 0991 (Wiemer et al., 2002), and
CGEN-856S] and the Mas antagonists, A-779 (Santos
et al., 1994) and D-Pro7 Ang-(1–7) (Santos et al., 2003).
Ang-(1–7) was identified as a Mas agonist in 2003
(Santos et al., 2003), whereas AVE0991 and CGEN-856S
were reported later (Pinheiro et al., 2004; Shemesh et al.,
2008; Savergnini et al., 2010). On the basis of evidence
obtained in the last 10 years, Mas is now considered to be
part of the novel axis of the RAS, ACE2/Ang-(1–7)/Mas
(Xu et al., 2011; Bader, 2013; Passos-Silva et al., 2013;
Santos et al., 2013a). In the following, we will summarize
the numerous cardiovascular and metabolic actions of
this axis, but for more comprehensive descriptions the
reader is referred to other recent reviews (Xia and
Lazartigues, 2010; Rabelo et al., 2011; Xu et al., 2011;
Ferreira et al., 2012; Passos-Silva et al., 2013; Santos
et al., 2013a; Simoes e Silva et al., 2013).
1. Mas in Heart. Mas expression was detected in
cardiomyocytes (Tallant et al., 2005) and cardiac fibroblasts (Iwata et al., 2011) and, more recently, also in the
sinoatrial node, providing the morphologic basis for the
antiarrhythmogenic effect of Ang-(1–7) (Ferreira et al.,
2011).
Recently, using picomolar concentrations of Ang-(1–7),
Souza et al. (2013) were able to unveil a significant Masmediated vasodilator effect of Ang-(1–7) in isolated rat
hearts. This effect was offset in hearts taken from aortacoarcted rats. Intriguingly, the blunted vasodilation in
hypertensive animals was restored by acute or chronic
AT1 blockade with losartan. These observations are in
keeping with the previously described interaction of AT1
Mas-Related Genes
receptor with Mas (Castro et al., 2005; Kostenis et al.,
2005; Canals et al., 2006), which still needs to be
addressed in more detail.
In cardiomyocytes, acute Mas stimulation with Ang(1–7) has no demonstrable effect on Ca2+ transients but
promotes NO release by activating endothelial nitric
oxide synthase (eNOS) and neuronal NOS (Dias-Peixoto
et al., 2008; Costa et al., 2010; Gomes et al., 2010). On
the other hand, chronic Mas stimulation or genetic
deletion of Mas (Mas-KO; Table 2) produces significant
effects on Ca2+ handling proteins (Santos et al., 2006;
Gomes et al., 2012). Transgenic rats expressing an
Ang-(1–7) producing fusion protein in the heart present
an increased Ca2+ transient amplitude, faster Ca2+
uptake, and increased expression of SERCA2 (Gomes
et al., 2012). Accordingly, cardiomyocytes from Mas-KO
mice presented a smaller peak Ca2+ transient and
slower Ca2+ uptakes due to a decreased expression of
SERCA2 (Gomes et al., 2012). These changes were
turned into a decreased heart function in Mas-KO
mice (Santos et al., 2006; Botelho-Santos et al., 2012;
Gava et al., 2012). The changes in the calcium handling
proteins were paralleled by changes in the NO production machinery (Dias-Peixoto et al., 2008). Cardiomyocytes from Mas-KO mice presented normal eNOS
protein levels. However, Mas deficiency resulted in
a 70% increase in caveolin 3 expression and a decrease
in heat shock protein 90 (Dias-Peixoto et al., 2008).
These two alterations may lead to a decrease in eNOS
activity, because caveolin 3 prevents calmodulin interaction with NOS and heat shock protein 90 act as
a scaffold protein for the recruitment of AKT to the
eNOS complex (Wu, 2002; Takahashi and Mendelsohn,
2003).
Most of the data related to Mas and its ligand Ang-(1–7)
in the heart have shown cardioprotective effects of
Ang-(1–7) (Ferreira et al., 2001, 2007; Santos et al., 2004;
Iwata et al., 2005, 2011; Tallant et al., 2005; Grobe et al.,
2006; Iusuf et al., 2008; Mercure et al., 2008; Li et al., 2009;
Giani et al., 2010; Gomes et al., 2010; Pei et al., 2010;
Santiago et al., 2010; Varagic et al., 2010; Marques et al.,
2011, 2012; Qi et al., 2011; Durik et al., 2012; McCollum
et al., 2012; Patel et al., 2012; Zeng et al., 2012; Cunha
et al., 2013; de Almeida et al., 2013) with only one
exception (Zhang et al., 2012).
In transgenic rats with Ang-(1–7) elevation in the
circulation produced by a fusion protein [TGR(A1–7)
3292], a marked attenuation of isoproterenol-induced
cardiac fibrosis was observed (Santos et al., 2004).
Accordingly, He et al. (2004), and later on many other
investigators (Iwata et al., 2005; Tallant et al., 2005;
Grobe et al., 2006; Iusuf et al., 2008; Mercure et al., 2008;
Li et al., 2009; Giani et al., 2010; Gomes et al., 2010; Pei
et al., 2010; Varagic et al., 2010; Iwata et al., 2011; Durik
et al., 2012; McCollum et al., 2012; Patel et al., 2012;
Zeng et al., 2012; de Almeida et al., 2013), described
antiremodeling effects of Ang-(1–7) and of other Mas
1097
agonists (AVE 0991/CGEN-856S) (Ferreira et al., 2007;
Savergnini et al., 2010). These observations are in line
with the deleterious cardiac effects of genetic ablation of
Mas in mice (Santos et al., 2006; Gava et al., 2012).
Interestingly, even acute blockade of Mas with A-779
produced a deterioration of heart function in isolated
mouse hearts (Castro et al., 2005).
An antihypertrophic effect of Ang-(1–7) was also
observed in culture cardiomyocytes treated with Ang II
(Gomes et al., 2010; Flores-Munoz et al., 2012), vasopressin
(Flores-Munoz et al., 2012), and endothelin (Gallagher
et al., 2008).
Contrasting with several reports showing a cardioprotective effect of the Mas/Ang-(1–7) axis, overexpression of Mas in neonatal rat cardiomyocytes by means of
infection with adenovirus encoding human Mas elicited
a significant increase in inositol 1,4,5-trisphosphate
accumulation and cellular hypertrophy. These responses
were due to enhanced Gq-mediated signaling via Mas
(Zhang et al., 2012). These data and others with Ang-(1–7)
(Neves et al., 1995; De Mello et al., 2007) support the
concept mentioned above (section V.C) that differences in
signaling may arise in conditions where Mas or Ang-(1–7)
are increased far above physiologic concentrations.
2. Mas in Blood Vessels. In humans and other species,
Ang-(1–7) is formed in the endothelial layer of blood
vessels (Santos et al., 1992) that also express Mas (Santos
et al., 2000; Sampaio et al., 2007a; Xu et al., 2008). In
addition, Mas is expressed in the vascular smooth muscle
cells (VSMC) of different species (Goncalves et al., 2007).
Studies performed in experimental models of permanent gain- or loss-of-function of Ang-(1–7) and Mas
indicate that Mas is capable of chronically influencing
systemic and local hemodynamics. It has been reported
that Mas-deficient mice present an important increase
in the vascular resistance in many territories, such as
kidney, lung, adrenal gland, mesentery, spleen, and
brown fat tissue (Botelho-Santos et al., 2012). A parallel
increase in total peripheral resistance and a decreased
cardiac index was also observed. On the contrary, in the
transgenic rats, TGR(A1-7)3292, with a life time increase in circulating Ang-(1–7), an opposite change was
reported (Botelho-Santos et al., 2007). These data suggest
the existence of an Ang-(1–7)/Mas tonus in blood vessels
with a previously unsuspected physiologic relevance.
However, because these regional blood flow measurements were made under anesthesia, caution should
be exercised in terms of transposing these finding to
nonanesthetized animals.
One of the most prominent consequences of Mas
deletion is endothelial dysfunction (Rabelo et al., 2008;
Xu et al., 2008). In the FVB/N mouse background, the
endothelial dysfunction is associated with an increase in
blood pressure (Xu et al., 2008), whereas in C57Bl/6
mice, no alteration of blood pressure was observed
(Rabelo et al., 2008). The endothelial dysfunction associated
with Mas deficiency in two genetic backgrounds is in
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Bader et al.
keeping with the improvement of endothelial function
produced by short-term Ang-(1–7) infusion in normotensive rats (Faria-Silva et al., 2005) and with the
improvement of Mas-mediated vascular function in
many species and conditions (Langeveld et al., 2008;
Durand et al., 2010; Stegbauer et al., 2011; Beyer
et al., 2013; Jarajapu et al., 2013; Tassone et al.,
2013).
In addition to its effects on vascular tone, Ang-(1–7)
has antiproliferative effects in VSMCs (Freeman et al.,
1996; Tallant and Clark, 2003). A similar action was
described for AVE0991 (Sheng-Long et al., 2012). This
feature appears not to be restricted to VSMC, considering
that Ang-(1–7) has well documented antiproliferative
effects in other cell types, including cardiac fibroblasts
(McCollum et al., 2012) and tumoral cells (Gallagher and
Tallant, 2004; Ni et al., 2012). The involvement of Mas in
these Ang-(1–7) effects was supported by the use of the
Mas antagonist A-779.
3. Mas in Cardiovascular Centers of the Brain.
As described above, Mas transcripts and protein are
expressed in many brain regions (Young et al., 1986;
Bunnemann et al., 1990; Martin and Hockfield, 1993;
Walther et al., 1998; Becker et al., 2007; Freund et al.,
2012), including those related to cardiovascular control
(Becker et al., 2007; Freund et al., 2012; Jiang et al., 2013).
The presence of Mas in areas, such as the hypothalamus,
nucleus tractus solitarii, rostral and caudal ventrolateral
medulla, provides the basis for several effects produced
by its agonist, Ang-(1–7), in the brain. Modulation of
sympathetic activity (Silva et al., 1993; Fontes et al., 1994;
da Silva et al., 2011; Kar et al., 2011; Li et al., 2013),
increase of vagal tonus (Guimaraes et al., 2012), and
improvement of baroreflex sensitivity (Chaves et al., 2000)
are some of the effects of Ang-(1–7) that can be blocked
by the Mas antagonist A-779. In agreement with these
findings, genetic ablation of Mas produces a decrease in
baroreflex sensitivity and changes in sympathetic activity
(Walther et al., 2000b). In addition to its role in neuronal
activity, in the past few years the protective effect of
Ang-(1–7) and Mas in stroke has received much attention
(Mecca et al., 2011; Regenhardt et al., 2014a,b).
4. Mas in Kidney. Mas is present in the rodent
kidney (Santos et al., 2003; Pinheiro et al., 2004; da
Silveira et al., 2010), but it could not be detected in the
human kidney in one study (Shalamanova et al., 2010).
Its ablation in C57Bl/6 mice leads to reduction in urine
volume and fractional sodium excretion without any significant change in free-water clearance. A significantly
higher inulin clearance and microalbuminuria concomitant with a reduced renal blood flow indicates glomerular
hyperfiltration in the Mas-KO mice (Pinheiro et al.,
2009). Genetic ablation of Mas abolished the specific
binding of [125I]Ang-(1–7) to kidney slices and the
antidiuretic effect of Ang-(1–7) in water-loaded animals,
whereas the binding of Ang II was unaltered (Santos
et al., 2003). The absence of the antidiuretic action of
Ang-(1–7) in Mas-KO mice is in agreement with the
antidiuretic effect of Ang-(1–7) in water-loaded rats
(Santos and Baracho, 1992). Diverging from many reports
describing Mas-mediated anti-inflammatory effects in different tissues (Al-Maghrebi et al., 2009; da Silveira et al.,
2010; El-Hashim et al., 2012; Giani et al., 2012; Jiang
et al., 2012; Santos et al., 2012; Souza and Costa-Neto,
2012; Sukumaran et al., 2012; Chen et al., 2013;
Feltenberger et al., 2013; Moore et al., 2013; Regenhardt
et al., 2013; Wagenaar et al., 2013; Wang et al., 2013c;
Acuna et al., 2014; Meng et al., 2014), in the kidney, a
proinflammatory role for Ang-(1–7) and Mas was reported
by using a model of unilateral ureteral obstruction in mice
(Esteban et al., 2009). In contrast, anti-inflammatory and
beneficial effects of Ang-(1–7) were observed in other
models of kidney injury (Singh et al., 2010b; Moon et al.,
2011; Bernardi et al., 2012; Giani et al., 2012; Harris,
2012; Chou et al., 2013; Santos et al., 2013b; Zhang et al.,
2014). Interestingly, the outcome of adriamycin-induced
nephropathy was similar in Mas-KO and wild-type mice.
However, the beneficial effects of losartan in this model
were suppressed in Mas-KO animals (Silveira et al., 2013).
Further studies are obviously necessary to clarify whether
influences of genetic background, the severity of the kidney disease model, an increased AT1 expression in renal
vessels (Pinheiro et al., 2009), or a still unknown factor
contribute to these contrasting findings (Zimmerman and
Burns, 2012).
E. Mas in Metabolism
Mas is present in several tissues involved in glucose
and lipid metabolism, including pancreas (Bindom and
Lazartigues, 2009; Frantz et al., 2013), liver (Herath
et al., 2007), adipose tissue (Santos et al., 2008; Liu
et al., 2012a), vascular wall (Karpe and Tikoo, 2014),
and skeletal muscle (Prasannarong et al., 2012; Acuna
et al., 2014). In these organs, Mas stimulation with
Ang-(1–7) promotes improvement of insulin sensitivity
(Munoz et al., 2012; Marcus et al., 2013; EcheverriaRodriguez et al., 2014), facilitates insulin signaling
(Munoz et al., 2012; Chhabra et al., 2013; Santos et al.,
2014), increases glucose uptake and decreases production of reactive oxygen species in adipocytes (Liu
et al., 2012a), and increases glucose uptake in skeletal
muscle (Echeverría-Rodriguez et al., 2014). In addition,
our phosphoproteomic study in endothelial cells suggests that Mas stimulation triggers changes in the
phosphorylation status of several known downstream
effectors of insulin signaling, indicating an important
role of Mas and Ang-(1–7) in glucose homeostasis in
human endothelial cells (Verano-Braga et al., 2012).
Accordingly, genetic ablation of Mas in FVB/N mice
leads to a metabolic syndrome–like status comprising
hypertension, increase in blood glucose, elevated plasma
triglycerides and cholesterol levels, and an increase in
abdominal fat (Santos et al., 2008). However, as observed
for blood pressure (Rabelo et al., 2008), ablation of Mas in
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Mas-Related Genes
C57BL/6 mice produced only minor changes in glucose
and lipid metabolism. Only cholesterol levels were
elevated, and this occurred in only old mice (Santos
et al., 2008). In this genetic background, however, Mas
deficiency produced worsening of the lipid profile and
severe hepatic steatosis in apolipoprotein E knockout
mice (Silva et al., 2013).
F. Mas in Behavior
There is also evidence for Mas being important for
the manifestation of certain behaviors. Mas-KO mice
exhibit sex-specific alterations in anxiety-like behavior, with only males showing a proanxious phenotype
in the elevated plus maze (Walther et al., 1998, 2000a).
Electrophysiological experiments revealed a more
pronounced long-term potentiation in the hippocampus
of Mas-KO mice, which is a prerequisite of memory
formation (Walther et al., 1998; Hellner et al., 2005).
Surprisingly however, only insignificant improvements
in the hippocampus-dependent spatial learning were
observed in Mas-KO mice (Walther et al., 1998, 2000a). In
contrast, object recognition memory, another hippocampusdependent behavior, was impaired in these animals
(Lazaroni et al., 2012), confirming an important but yet
elusive role of Mas in hippocampal functions that is
not linked to its electrophysiologic actions. However, it
may be explained by the role of Mas in neuronal NO
generation, which is also involved in memory formation
(Yang et al., 2011).
G. Mas in Reproductive Functions
Despite the fact that Mas exhibits a localized and
well ontogenically controlled expression in testis during
puberty (Metzger et al., 1995; Alenina et al., 2002b), no
drastic alterations in fertility are observed in Mas-KO
mice, which have normal pregnancy rates and an equal
number of male and female offspring (Walther et al.,
1998). However, they exhibit a reduction in testis
weight, an increased number of apoptotic cells during
meiosis, the presence of giant cells and vacuoles in
seminiferous epithelium, and a decreased amount of
daily produced sperm (Leal et al., 2009), pointing to an
important role of Mas in the modulation of spermatogenesis. Accordingly, a strong correlation between Mas
expression in seminiferous tubules and male infertility
was observed in humans (Reis et al., 2010). Moreover,
Ang-(1–7) is released in the corpus cavernosum and can
attenuate oxidative stress and DNA damage, processes
leading to the degeneration of this tissue, and facilitate
electrical-stimulated release of NO (Goncalves et al.,
2007; Kilarkaje et al., 2013). Consequently, the genetic
ablation of Mas results in an increase of the fibrous
tissue content, leading to a compromised erectile
function (Goncalves et al., 2007).
The Ang-(1–7)/Mas axis also seems to play a modulatory role in the female reproductive system, being
involved in folliculogenesis, ovulation, and pregnancy.
Mas expression was detected in human ovaries (Reis
et al., 2011) and is regulated by gonadotropin-releasing
hormone in rat ovary and in granulosa and theca cells
in cattle (Pereira et al., 2009; Tonellotto dos Santos
et al., 2012). It is also involved in ovulation in rabbits
(Viana et al., 2011). Moreover, the Mas antagonist
A-779 inhibited the germinal vesicle breakdown induced by Ang-(1–7) and reduced oocyte maturation
stimulated by luteinizing hormone (Honorato-Sampaio
et al., 2012), further corroborating the role of Mas in
female reproduction. Mas is also present in rat uterus
(Vaz-Silva et al., 2012) and in human placenta, where
it is decreased in pre-eclamptic women (Velloso et al.,
2007). Thus, Mas seems to be a relevant but not essential
modulator of male and female fertility.
VI. Conclusions and Outlook
More than 25 years after the discovery of Mas and
more than 10 years after the first description of the
Mrgpr family, there is still not much known about their
physiologic functions. Only Mas has been intensively
studied, and it is believed to be part of a beneficial axis
of the renin-angiotensin system. For most other Mrgprs,
numerous low-affinity agonists have been described, but
the natural ligands are still uncertain and none has
been approved for therapy. However, as mentioned
above, there may be no agonist, but rather, the receptors
are constitutively active and we have to search for
endogenous inverse agonists that modulate their functions. Alternatively, the receptors may have been evolved
for promiscuous binding to several low-affinity agonists
in response to challenges of noxious plants and parasites
affecting the outer layer of the skin. Thus, the Mrgprs
may have allowed the evolutionary invention of itch in
Tetrapods, which, by initiating scratching upon their
activation, avoids harmful invasion of noxious agents.
Nevertheless, some members of the Mrgpr family definitively have additional functions in nociception and
other physiologic processes, and therefore, several therapeutic applications of Mrgpr agonists or antagonists are
conceivable.
The most advanced Mrgpr in respect to pharmacologic application is Mas itself, exploiting its protective
actions in cardiovascular and metabolic diseases. Several
different strategies have been developed to directly
activate this receptor or to increase the concentration of
its main agonist, Ang-(1–7), and some have already
entered clinical trials (Steckelings et al., 2011; Bader
et al., 2012; Ferreira et al., 2012). However, none is
already approved for therapy.
Nevertheless, the other Mrgprs also hold potential
for pharmacological applications. In particular, they
may be interesting targets for antinociceptive therapies. Furthermore, inhibition of Mrgprs may be a novel
working mode for anti-itch drugs. For example, antagonists of hMRGPRX1 may help block the itch occurring
1100
Bader et al.
under malaria therapy with chloroquine in some patients
(Liu et al., 2009). On the other hand, agonists for the same
receptor, such as JHU58, might be therapeutically
exploited to inhibit inflammatory hyperalgesia and
neuropathic pain (Guan et al., 2010; Guan, 2013; He
et al., 2014). These obviously contradictory actions of
drugs interacting with hMRGPRX1, however, may
create a problem in their clinical application.
In conclusion, we are on the eve of the pharmacologic
exploitation of Mrgprs in the clinic, with MAS and
hMRGPRX1 as first targets. We need further basic
investigations on Mrgprs to reveal their physiologic
and pathophysiologic functions, and thereby, their
therapeutic potential. Such research is worthwhile in
view of the unsolved medical needs that Mrgpr-specific
drugs may alleviate, such as chronic itch and pain as well
as cardiovascular diseases and metabolic syndrome.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: Bader,
Alenina, Andrade-Navarro, Santos.
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