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1521-0081/66/3/570–597$25.00 http://dx.doi.org/10.1124/pr.113.008425PHARMACOLOGICAL REVIEWS Pharmacol Rev 66:570–597, July 2014Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: FINN OLAV LEVY

Pharmacology and Signaling of MAS-RelatedG Protein–Coupled Receptors

Hans Jürgen Solinski, Thomas Gudermann, and Andreas Breit

Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, Munich, Germany

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571II. Genes Encoding MAS-Related G Protein–Coupled Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572

A. Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572B. Phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572C. Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574D. Evolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575

III. Pharmacology and Physiology of MAS-Related G Protein–Coupled Receptors . . . . . . . . . . . . . . . . . 577A. Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577B. MAS-Related G Protein–Coupled Receptors A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577

1. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5772. Signaling Cascades and Physiologic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578

C. MAS-Related G Protein–Coupled Receptors B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579D. MAS-Related G Protein–Coupled Receptors C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579

1. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5792. Signaling Cascades and Physiologic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581

a. Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581b. Pain-enhancing effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581c. Analgesic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583d. Pruritogenic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583

E. MAS-Related G Protein–Coupled Receptors D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5841. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5842. Signaling Cascades and Physiologic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584

F. MAS-Related G Protein–Coupled Receptors E to -H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585G. MAS-Related G Protein–Coupled Receptors X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585

1. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585a. MAS-related G protein–coupled receptors X1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585b. MAS-related G protein–coupled receptors X2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587c. MAS-related G protein–coupled receptors X3 and -4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587

2. Signaling Cascades and Physiologic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587a. Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587b. Somatosensory functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588c. Primary sensory neuron plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588d. Mast cell biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591e. Putative role of MAS-related G protein–coupled receptors X2 in sleep and

blood pressure regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591IV. Agonist-Promoted Internalization and Desensitization of MAS-Related

G Protein–Coupled Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591

This work was supported by a grant from the “Deutsche Forschungsgemeinschaft” [Grant BR 3346/3-1].Address correspondence to: Dr. Andreas Breit, Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-

Universität München, Goethestrasse 33, 80336 Munich, Germany. E-mail: [email protected]. and A.B. are co-senior authors.dx.doi.org/10.1124/pr.113.008425.

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V. Functional Interactions of MAS-Related G Protein–Coupled Receptors withOther Receptor Families. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592A. Heteromultimerization among MAS-Related G Protein–Coupled

Receptors and Receptor Subtypes of Other GPCR Families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592B. Inhibition of Tolerance to Morphine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593

VI. Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594

Abstract——Signaling by heptahelical G protein–coupled receptors (GPCR) regulates many vital bodyfunctions. Consequently, dysfunction of GPCR sig-naling leads to pathologic states, and approximately30% of all modern clinical drugs target GPCR. Onedecade ago, an entire new GPCR family was discov-ered, which was recently named MAS-related Gprotein–coupled receptors (MRGPR) by the HUGOGene Nomenclature Committee. The MRGPR familyconsists of ∼40 members that are grouped into ninedistinct subfamilies (MRGPRA to -H and -X) and arepredominantly expressed in primary sensory neuronsand mast cells. All members are formally still con-sidered "orphan" by the Committee on Receptor No-menclature and Drug Classification of the InternationalUnion of Basic and Clinical Pharmacology. However,several distinct peptides and amino acids are dis-cussed as potential ligands, including b-alanine,

angiotensin-(1–7), alamandine, GABA, cortistatin-14,and cleavage products of proenkephalin, pro-opiomelanocortin, prodynorphin, or proneuropeptide-FF-A. The full spectrum of biologic roles of all MRGPRis still ill-defined, but there is evidence pointing to a roleof distinct MRGPR subtypes in nociception, pruritus,sleep, cell proliferation, circulation, and mast celldegranulation. This review article summarizesfindings published in the last 10 years on thephylogenetic relationships, pharmacology, sig-naling, physiology, and agonist-promoted regula-tion of all MRGPR subfamilies. Furthermore, wehighlight interactions between MRGPR and otherhormonal systems, paying particular attentionto receptor multimerization and morphine toler-ance. Finally, we discuss the challenges the fieldfaces presently and emphasize future directions ofresearch.

I. Introduction

G protein–coupled receptors (GPCR), also knownas seven-transmembrane domain receptors, constitutea large protein family that senses a plethora of distinctphysical and chemical stimuli (biogenic amines, aminoacids, ions, lipids, nucleotides, peptides, proteins, light,odorants, and pheromones) outside the cell. UponGPCR activation, a host of intracellular signaling pro-teins (enzymes, transcription factors, ion channels) areengaged, affecting vital cellular and organismal func-tions, such as cell proliferation, differentiation, de-velopment, survival, circulation, metabolism, neuronalsignal transmission, and sensory perception. Approxi-mately 800 putative GPCR have been identified inhumans and more than 300 of them are nonodorantreceptors (Fredriksson et al., 2003). Interestingly, en-dogenous or natural exogenous ligands have not been

identified for ;100 of the GPCR identified so far,suggesting that many biologic functions of GPCR maynot have been discovered yet (Davenport et al., 2013).It is reasonable to assume that these "orphan" GPCRhave an immense therapeutic potential, because ;30%of all clinically relevant drugs already target GPCRdirectly or modulate their cognate signaling pathways(Overington et al., 2006). Hence, in recent years, con-siderable efforts have been undertaken to identifyendogenous ligands of "orphan" GPCR by high-throughput screening strategies. As a result of one ofthese efforts, Lembo and coworkers (2002) reported agroup of human "orphan" receptors that are activatedby proenkephalin (PENK) cleavage products of thebovine adrenal medulla (BAM) peptide family. Theauthors called these receptors sensory neuron–specificG protein–coupled receptors (SNSR) because of theirrestricted expression pattern in primary sensory

ABBREVIATIONS: BAM, bovine adrenal medulla; CAP, capsaicin; CCR2, chemokine receptors 2; CFA, complete Freund’s adjuvant; CGRP,calcitonin gene-related peptide; CHO, Chinese hamster ovary; D-APV, D-(2)-2-amino-5-phosphonopentanoic acid; DOP, d-OR; DRG, dorsalroot ganglia; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; GPCR, G protein–coupled receptors; HC-030031,1,2,3,6-tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7H-purine-7-acetamide, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; HEK, human embryonic kidney; MK-801, (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine; MOP, m-OR; Mrg, MAS-related gene; MRGPR, MAS-related G protein–coupled receptors; MSH, melanocytestimulating hormone; NC-IUPHAR, Committee on Receptor Nomenclature and Drug Classification of the International Union of Basic andClinical Pharmacology; NFAT, nuclear factor of activated T cells; NMDA, N-methyl-D-aspartate; nNOS, neuronal nitric oxide synthetase;NPAF, neuropeptide AF; NPFF, neuropeptide FF; OR, opioid receptors; PAR, protease-activated receptors; PENK, proenkephalin; PIP2,phosphatidylinositol-3,4-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; PTX, pertussis toxin; SNSR, sensory neuron–specificreceptors; TrkA, neurotrophic tyrosin kinase receptor 1; TRP, transient receptor potential cation channel; TRPA, TRP ankyrin; TRPM, TRPmelastatin 8; TRPV, TRP vanilloid 1; YM58483, N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide.

MAS-Related G Protein–Coupled Receptors 571

neurons and postulated a role for SNSR in painperception. Shortly before, Dong and colleagues(2001) compared cDNA libraries from wild-type miceand from mice lacking the transcription factor neuro-genin 1. Mice without functional neurogenin 1 fail todevelop the neurotrophic tyrosine kinase receptor 1(TrkA)–positive subclass of dorsal root ganglia (DRG)and trigeminal ganglia neurons that detect painfulstimuli, so called TrkA+ nociceptors (Ma et al., 1999).Thus, the authors postulated that genes found in thelibrary of wild-type mice, but not in neurogenin 1–deficient mice, should be specific for TrkA+ nociceptors.By this approach, the authors discovered an entire newfamily of selectively expressed GPCR and named theseproteins MAS-related genes (Mrg), because of theirhomology to the oncogenic GPCR MAS1. The Mrgfamily in rodents and humans comprises ;40 membersthat can be divided into several subfamilies. It soonturned out that one of these subfamilies, the MrgXfamily, is identical to the SNSR proteins (Lembo et al.,2002), such that the same receptor proteins were giventwo different names. Even more confusing, the ratMrgC/SNSR was named after its proposed cognateligand BAM peptide-activated receptor with nonopioidactivity, and MRG is also used as an abbreviation formortality factor on chromosome 4–related genes[mortality factor on chromosome 4/MRG functions inaging are reviewed elsewhere (Chen et al., 2010a)],a group of proteins distinct from the GPCR superfam-ily. Thus, inconsistencies in the nomenclature de-veloped over the last decade may have contributed toconfusions in the field. To overcome this problem, theHUGO Gene Nomenclature Committee now refers toMrg/SNSR proteins as MAS-related G protein–coupledreceptors (MRGPR) (www.genename.org). So far, a to-tal of 38 genes encoding MRGPR proteins has beenlisted in National Center for Biotechnology Informa-tion databases (orthologous proteins from distinctmammalian species are counted as one member, seeFig. 1 and Table 1). Therefore, to the best of ourknowledge, MRGPR represent the nonodorant GPCRfamily with the largest number of members known sofar. At present, MRGPR are arranged in nine sub-families, designated by capital letters, whereas individ-ual subtypes are indicated by numbers. For instance,the MRGPR subtype 1 of subfamily X, originally termedMrgX1 (according to Dong et al., 2001) or SNSR4(according to Lembo et al., 2002), is now officially calledMRGPRX1. This nomenclature is also recognized byCommittee on Receptor Nomenclature and Drug Clas-sification of the International Union of Basic and Clin-ical Pharmacology (NC-IUPHAR; Davenport et al.,2013) and is recommended for use to improve scientificcommunication.Since their first description, considerable efforts

have been undertaken to characterize MRGPR interms of ligand binding profile, regulation, signaling

cascades, and physiologic effects. Significant progresshas been made recently and, thus, several MRGPRmembers emerged as most appealing pharmacologicaltargets for analgesic, antipruritogenic, and antihyper-tensive therapies. However, at the same time, advan-ces in our understanding of the physiology andpathophysiology of MRGPR are hampered by a complexphylogeny, redundancy in ligand binding, and multi-faceted functions. This review is an attempt to summa-rize our present knowledge about the MRGPR familyand to point out current caveats. We present an over-view of MRGPR genetics, including phylogeny and evo-lution, and of pharmacologic and physiologic details ofeach MRGPR subfamily. Finally, we will highlightagonist-induced MRGPR regulation and interactionswith other GPCR families identified so far.

II. Genes Encoding MAS-Related GProtein–Coupled Receptors

A. Preface

MRGPR-encoding genes have been detected inmammals, including rodents, Canis lupus, Bos taurus,and primates. No MRGPR genes have been identifiedin lower vertebrates so far, probably because of lessadvanced gene sequencing and annotation processes oflower vertebrate genomes. Only one MRGPR memberwas annotated in the genome of birds: Solely theMRGPRH was found in Gallus gallus. The GPCRsuperfamily is divided based on sequence homologyinto class A to F. According to the existence of severalconserved motifs, for instance the NPxxY motif intransmembrane domain seven, the MRGPR family isassigned to the rhodopsin-like class A (Dong et al.,2001; Lembo et al., 2002; Fredriksson et al., 2003;Katritch et al., 2013). The rhodopsin-like GPCR class Acan be further subdivided into four groups designateda to d. MRGPR belong to the d-group of class A, whichalso comprises glycohormone, purinergic and the verylarge family of olfactory receptors, with glycohormonereceptors being the receptor family most closely relatedto the MRGPR family (Dong et al., 2001; Lembo et al.,2002; Fredriksson et al., 2003; Katritch et al., 2013).

B. Phylogeny

The mammalian family of MRGPR can be subdividedinto nine separate subfamilies (A–H and X) because ofsequence similarities (see Fig. 1 and Table 1). Sub-families A, B, C, and H exist only in rodents, whereassubfamily X is specific to primates including humans,macaque, and rhesus monkey (Dong et al., 2001;Lembo et al., 2002; Zylka et al., 2003; Zhang et al.,2005; Burstein et al., 2006). In contrast, subfamilies Dto G are conserved in different mammalian species,including rodents and primates (Dong et al., 2001;Lembo et al., 2002; Zylka et al., 2003).

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http://www.genename.org

The MRGPRA subfamily composition differs consid-erably among rodents. Although only one gene is foundin rats, mice harbor 18 protein-encoding MRGPRAgenes as well as several pseudogenes (Dong et al.,2001; Zylka et al., 2003). The MRGPRB subfamilycomprises seven protein-encoding genes in rats, nine inmice, and several pseudogenes in both species (Donget al., 2001; Zylka et al., 2003). According to Zylka et al.(2003), a further subdivision of MRGPRB genes intothe subgroups B2, B4, and B8 is procurable and reflectsthe basic set of MRGPRB in other rodent species, suchas gerbil. The MRGPRC subfamily consists of only onegene in rats and one protein-encoding gene in additionto 13 pseudogenes in mice (Han et al., 2002; Zylka et al.,2003). The MRGPRD subfamily comprises only oneprotein-encoding gene per species (Dong et al., 2001;Lembo et al., 2002; Zylka et al., 2003). Just likeMRGPRD, MRGPRE to -H subtypes exhibit only one

protein-encoding gene per species (Zylka et al., 2003). TheMRGPRX subfamily was originally reported to consist offour distinct genes that are all located on chromosome11p15.1 in humans (Dong et al., 2001). Three of the fourreceptors discovered earlier were identified independentlyby other researchers, and three additional receptors wereisolated from the same laboratory (Lembo et al., 2002),resulting in a total of seven distinct MRGPRX receptors.However, three of the original putative MRGPRXmembers (SNSR2, -3, and -5) have not yet been listedby NC-IUPHAR, most likely because these receptors areup to 98% identical to the listed MRGPRX and, thus,probably represent polymorphisms but not distinct sub-types. Hence, four distinct MRGPRX proteins (MRGPRX1to -4) are currently listed by NC-IUPHAR and will bereviewed herein. Table 2 provides an overview of theidentities and relationships of all MRGPRX memberspublished and officially listed so far.

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


C. ExpressionThe majority of MRGPR is expressed in isolectin B4-

positive small-diameter somatosensory afferents, whichrepresent about half of the nociceptors per DRG (Snider

and McMahon, 1998). All MRGPRA, MRGPRC to -H,and MRGPRX members, as well as the MRGPRB4 and-5 subtype, can be subsumed to be expressed in isolectinB4-positive DRG neurons (Dong et al., 2001; Lembo

TABLE 1Phylogeny of MAS-related G protein–coupled receptors

All 38 MRGPR from 4 different species are listed by their designated names, aliases, and GeneID. The ascribed RefSeqrecords have been reviewed by National Center for Biotechnology Information (NCBI) staff and are annotated to thereference genome (mouse: annotation release 103, rat: build 5.1, human: annotation release 105, rhesus monkey: build1.2). A straight line below the GeneID indicates that the RefSeq record has not yet been subject to individual review byNCBI staff. A GeneID in parentheses indicates that the underlying information was provided by an NCBI collaborator, anNCBI RefSeq record is yet missing, and accordingly no annotation to the reference genome exists. Notice that if MRGPRAand -B subfamily members are not numbered consecutively, numbers in between designate pseudogenes.

Subfamily Number Aliases GeneID

MRGPRA 1 MrgA1 (M) 233221*— MrgA10, AdeninR, MRGPRX3 (R) 252960†2a - (M) 668727*2b MrgA2 (M) 235712*3 MrgA3 (M) 233222*4 MrgA4 (M) 235854*5 MrgA5 (M) 404235*6 MrgA6 (M) 381886*7 MrgA7 (M) 404236*8 MrgA8 (M) (404237)*9 MrgA9 (M) 668725*

10 MrgA10 (M) (404243)*11 MrgA11 (M) (404244)*12 MrgA12 (M) (404245)*13 MrgA13 (M) (404246)*14 MrgA14 (M) (404247)*15 MrgA15 (M) (404248)*16 MrgA16, Gm486 (M) (404249)*19 MrgA19 (M) (404250)*

MRGPRB 1 MrgB1 (M, R), MRGPRX2 (R) 233231*/404640†2 MrgB2 (M, R), MRGPRX2-like (R) 243979*/404645†3 MrgB3 (M, R) 404238*/(404656)†4 MrgB4 (M, R) 233230*/404658†5 MrgB5 (M, R) 404239*/404644†6 MrgB6 (R) 502338†8 MrgB8, MrgB13 (M, R) 404240*/404643†

10 MrgB10, MRGPRX2 (M) 243978*11 MrgB11 (M) (404251)*13 MrgB13 (M) 620137*

MRGPRC — Mrgprc11, MRGPRX1 (M, R),MrgC11 (M), SNSR1 (R)

404242*/282547†

MRGPRD — MrgD, TGR7 (M, R, H, Rh) 211578*/293648†/116512‡/709555x

MRGPRE — MrgE (M, R, H, Rh), GPR167 (H) 244238*/404660†/116534‡/706822x

MRGPRF — RTA, MrgF, GPR140, GPR168 (M, R, H, Rh) 211577*/266762†/116535‡/721546x

MRGPRG — MrgG (M, R, H, Rh), Gm1098 (M), GPR169 (H) 381974*/49929†386746‡/721253x

MRGPRH — GPR90, MrgH (M, R) 80978*/404641†MRGPRX 1 MrgX1 (H, Rh), SNSR4 (H) 259249‡/692101x

2 MrgX2 (H, Rh), MrgX2-2 (Rh) 117194‡/692078x3 MrgX3 (H, Rh), SNSR1 (H) 117195‡/692077x4 MrgX4 (H, Rh), SNSR6 (H) 117196‡/692123x

M, mouse; R, rat; H, human; Rh, rhesus monkey.GeneIDs: *mouse; †rat; ‡human; xrhesus monkey.

TABLE 2The MAS-related G protein–coupled receptors subfamily X

An overview of MRGPRX subfamily members, their identity, and their interrelation according to different authors isgiven.

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

MrgX1 SNSR4 MrgX1-1 (-2 exists additionally) MRGPRX1 259249MrgX2 — MrgX2 MRGPRX2 117194MrgX3 SNSR1 MrgX3-1 (-2 exists additionally) MRGPRX3 117195MrgX4 SNSR6 MrgX4-1 MRGPRX4 117196

— SNSR5 MrgX4-2 — AF474991— SNSR2 MrgX6 — AF474988— SNSR3 MrgX7 — AF474989

574 Solinski et al.

et al., 2002; Robas et al., 2003; Zylka et al., 2003; Zhanget al., 2005; Tatemoto et al., 2006; Cox et al., 2008;Liu et al., 2009). None of the remaining MRGPRBmembers have been detected in primary sensoryneurons so far. However, high amounts of MRGPRB3and -8 transcripts, as well as lower copy numbers of theremaining three rat MRGPRB members, were mea-sured in rat peritoneal mast cells, whereas transcriptsof MRGPRA or -C were not present in these cells(Tatemoto et al., 2006). Recently, MRGPRX1 and -2expression was also shown in human mast cells(Tatemoto et al., 2006; Subramanian et al., 2011b;Solinski et al., 2013). Thus, human MRGPRX mirrorthe expression pattern of rodent MRGPRB and -A/C,lending support to the current theory of MRGPR evo-lution, implying that MRGPRX1 and -2 genes inheritedpromoter elements from ancestral MRGPRB and -A/Cgenes (for details see section II.D).The initial discovery of the MRGPR family pointed to

a selective expression of MRGPR in primary sensoryneurons derived from the TrkA+ population (Donget al., 2001). Perinatally, this results in a largelyoverlapping MRGPRA to -D expression in the samepopulation of neurons (Dong et al., 2001; Zylka et al.,2003; Liu et al., 2008). After birth, TrkA expressionceases in roughly half of the formerly TrkA+ neurons(Molliver et al., 1997). Instead of TrkA, these cells startto express c-ret, the receptor of the glial-derivedneurotrophic factor (Molliver et al., 1997). In adult-hood, expression of all MRGPR is maintained in c-ret+,but not in the remaining TrkA+ neurons (Dong et al.,2001; Zylka et al., 2003; Liu et al., 2008). Interestingly,despite the largely overlapping expression pattern ofall MRGPR in perinatal primary sensory neurons, inadulthood most MRGPR subtypes are not coexpressedin the same c-ret+ population and exhibit a compart-mentalized expression pattern in distinct subpopula-tions (Dong et al., 2001; Zylka et al., 2003; Liu et al.,2008). Notably, these expression patterns are notidentical in mice and rats, suggesting variations inthe physiologic roles of distinct MRGPR in rodents.The analysis of transcriptional pathways responsiblefor expression of different MRGPR in distinct DRGpopulations in mice revealed that expression ofMRGPRA to -D initially depends on the activity ofthe runt-related transcription factor 1, explaining theoverlapping expression pattern in embryonic neurons(Liu et al., 2008). During postnatal development, runt-related transcription factor 1 suppresses MRGPRA to –C,but not MRGPRD expression, through its inhibitoryC-terminal domain, leading to the segregation ofmurine MRGPRD and MRGPRA to -C expression inadolescence. Furthermore, the authors found thatthe transcription factor Smad4 is indispensable forMRGPRB4 expression, but does not affect expressionof other MRGPR subtypes (Liu et al., 2008). Thedistinct expression pattern of MRGPR in adulthood led

to the assumption that neuronal subpopulationscharacterized by their MRGPR expression patternmight have distinct functions. In line with this hypoth-esis, MRGPRB4- or MRGPRD-positive primary sensoryneurons in mice obtain distinct somatosensory inputfrom different skin areas (Zylka et al., 2005; Liu et al.,2007), whereas MRGPRA3 expression specifies neuronsthat induce itch without transducing nociceptive cues(Han et al., 2013).

In addition to the highly selective expression ofseveral MRGPR in small-diameter primary sensoryneurons and the aforementioned expression of someMRGPR in mast cells, MRGPRD to -H and MRGPRX2expression was also found in other tissues. SignificantmRNA levels of MRGPRD were detected in urinarybladder, testis, uterus, and arteries (Shinohara et al.,2004). MRGPRE transcripts were also monitored inmedium- and large-diameter neurons of human DRGsections (Zhang et al., 2005) and in other areas of thecentral nervous system, including cerebral cortex,hippocampus, spinal cord, and cerebellum, as well asin human, mouse, and rat placenta (Zhang et al., 2005;Milasta et al., 2006). MRGPRE and MRGPRF are alsoexpressed in enteric neurons (Avula et al., 2011),belonging to one of the main divisions of the autonomicnervous system that regulates gastrointestinal func-tions. In accordance with these findings, transcripts ofMRGPRF, formally known as rat thoracic aorta re-ceptor, were detected in small and large intestine (Rosset al., 1990). More precisely, MRGPRE and MRGPRFproteins were detected in both myenteric and sub-mucosal neurons, the latter showing coexpression ofboth receptors in 30–50% of neurons analyzed (Avulaet al., 2011). The same study reported that intestinalschistosomiasis and trinitrobenzene sulfonic acid–induced ileitis resulted in decreased expression of bothreceptors, whereas other neuronal marker proteins,e.g., calretinin or neuronal nitric oxide synthetase(nNOS), were not affected by either inflammatoryinsult. MRGPRH transcripts were also detectable inthe heart (Wittenberger et al., 2001), and expression ofhuman MRGPRX2 was described in the adrenal glandsand in several brain areas (Robas et al., 2003; Kamoharaet al., 2005).

D. Evolution

The MRGPR subfamilies D to G constitute evolu-tionarily old genes that are conserved between rodentsand primates. Accordingly, although all MRGPR areencoded on only one chromosome in each speciesanalyzed, the subfamilies D to G lie clustered togethera considerable distance apart from the chromosomalregion encoding subfamilies A to C in rodents orsubfamily X in primates (Zylka et al., 2003). Thephylogenetic relationship between the latter MRGPRsubfamilies poses the question of how and why theentire MRGPR family developed in such a complex


manner during mammalian evolution. A current model(see Fig. 2) rests on the assumption that an ancestralprogenitor gene cluster of one MRGPRA/C/X gene andone MRGPRB gene existed before rodent speciation(Zylka et al., 2003). An unequal crossing over event ledto local MRGPRB gene duplication in the rodentlineage, whereas MRGRPB genes were completely lostin the primate lineage. In rodents, the MRGPRA/C/Xprogenitor developed into two separate gene subfami-lies because of a further round of local gene duplica-tion. Specifically in mice, the de novo insertion of theL1 retrotransposon initiated several additional localgene duplication events of MRGPRA and -C genes.However, additional MRGPRC genes lost their tran-scriptional regulatory elements and are now pseudo-genes. This assumption is supported by the observationthat all MRGPR genes are found on only one chro-mosome, e.g., chromosome 1q in rats or chromosome7B in mice. Notably, such an evolutionary model would

also explain the highly specific expression of rodentMRGPRA and -C and of human MRGPRX in primarysensory neurons, assuming that those promoter ele-ments crucial for the restrictive expression patternalready existed in the progenitor gene and were pre-served during gene duplication events (Dong et al.,2001; Lembo et al., 2002; Zylka et al., 2003; Zhanget al., 2005). Likewise, additional expression ofMRGPRX1 and -2 in humanmast cells could be the resultof conserved ancestral promoter elements responsible forMRGPRB expression in rat mast cells (Tatemoto et al.,2006). Thus, MRGPRX1 and -2 inherited their re-strictive expression in primary sensory neurons and mastcells by a combination of gene expression regulatoryelements of rodent MRGPRA/C and certain rodentMRGPRB genes.

The development of single MRGPRX genes inprimate evolution was also initiated by local geneduplication (note that all human MRGPRX genes are

Fig. 2. Schematic model displaying the putative divergent evolution of the MRGPRA/B/C/X gene cluster in primates and rodents. Based on the dataprovided by Zylka et al. (2003), a theoretical model explaining the evolutionary events leading form a putative ancestral MRGPR gene cluster to theprimate and rodent lineage and further on to species-specific distinctions in the rodent lineage is given.

576 Solinski et al.

located on chromosome 11p15.1) (Dong et al., 2001;Choi and Lahn, 2003; Zylka et al., 2003). Diversion ofdistinct MRGPRX genes was driven by significantpositive selection pressure (Choi and Lahn, 2003;Fatakia et al., 2011), resulting in amino acid sub-stitutions in those parts of transmembrane domainsforming the ligand binding cavity, in extracellulardomains, and in the very C-terminal tail (Choi andLahn, 2003; Fatakia et al., 2011). From a structuralpoint of view, the latter changes may have resulted indiverging receptor proteins with regard to ligandspecificity or affinity (extracellular domains, trans-membrane domains) and the regulation of signaling(C-terminal tail). From an evolutionary point of view,the strong positive selection pressure indicates thatnovel MRGPRX proteins are crucial for primate-specific physiology and represent important dif-ferences between primates and rodents. Indeed,primate-specific genes are clearly over represented inthe fraction of human disease-causing genes (Hao et al.,2010). Interestingly, a continuing positive selectionpressure on MRGPRX genes may account for frequentpolymorphisms of MRGPRX genes in the human pop-ulation and, thus, account for differences in publishedMRGPRX/SNSR subtypes.

III. Pharmacology and Physiology ofMAS-Related G Protein–Coupled Receptors

A. Preface

To date, no MRGPR subtype has officially beendeclared "deorphanized" by NC-IUPHAR (Davenportet al., 2013). This recommendation would require atleast two independent demonstrations of receptor-ligand pairing published in refereed papers. To"deorphanize" a receptor, NC-IUPHAR suggests usingradioligand binding and functional assays in in vitrosystems and native tissues in conjunction with ana-tomic data indicating that the proposed ligand ispresent to activate the receptor in a given tissue.Additionally, genetic approaches that alter expressionlevels of the receptor or the ligand might be beneficial.So far, at least one of the criteria mentioned is lackingfor any given MRGPR. These criteria are particularlyhard to meet for human MRGPRX subtypes: because oftheir specific expression in primates and their re-stricted expression pattern in primary sensory neuronsand mast cells, it appears difficult to obtain data fromnative tissues and to implement genetic approachesthat would alter protein expression levels. However,knowledge of MRGPR biology and pharmacology isexpanding, a fact that is being appreciated byNC-IUPHAR (Davenport et al., 2013). Interestingly, acertain degree of pharmacologic overlap of nonorthologousMRGPR members exists, although MRGPR phylogenyis complex, including subfamilies that are specific torodents and others that are primate specific. Thus, we

will summarize the available pharmacologic data ofMRGPR members ordered by subfamily and reviewthe current knowledge about signaling cascades andphysiologic effects elicited by a given receptor-ligandpair.

B. MAS-Related G Protein–Coupled Receptors A

1. Pharmacology. Endogenous or exogenous ligandshave been assigned to only 4 of 19 MRGPRA members,i.e., rat MRGPRA and three murine MRGPRA (seeTable 3). The murine MRGPRA members 1 and 4 werethe first MRGPR to be analyzed in a functional assay.After recombinant expression in human embryonickidney (HEK)293 cells, both receptors responded toseveral peptides of the RFamide family (Dong et al.,2001) first discovered in mollusks and soon shownto modulate nociception in vertebrates (Price andGreenberg, 1977; Yang et al., 1985). The mammalianneuropeptide FF (NPFF) increased intracellularcalcium concentrations via MRGPRA1 with a potencyof ;200 nM, whereas the mammalian neuropeptideAF (NPAF) likewise activated the MRGPRA4 sub-type with a potency of ;60 nM. These findings weresubsequently confirmed by others (Han et al., 2002;Liu et al., 2009). Both peptides are also known toactivate NPFF receptor 1 or 2 with potencies andaffinities in the nanomolar range (Mollereau et al.,2002). Thus, if these neuropeptides represent theendogenous agonists of murine MRGPRA1 or -4,they would not be selective for the latter receptors.In addition to endogenously occurring neuropep-tides, murine MRGPRA1 was found to be a target ofthe human version of the b-salusin peptide, employ-ing fluorescence imaging plate reader technologyand MRGPRA1 overexpressing HEK293 or Chinesehamster ovary (CHO) cells (Wang et al., 2006b). Asthe orthologous murine peptide failed to activateMRGPRA1, it was concluded that b-salusin is onlya surrogate ligand of MRGPRA1.

The antimalaria drug chloroquine was recentlyshown to induce intracellular calcium transients incells expressing MRGPRA3 (Liu et al., 2009). Thisresponse was characterized by an EC50 value of;27 mMafter heterologous expression in HEK293 cells andwas also noted in dissociated primary sensory neurons.A cluster knockout mouse deficient of several MRGPR(including MRGPRA1-4, -A10, -A12, -A14, -A16, -A19,-B4, -B5, and -C) failed to respond to chloroquine asdid wild-type neurons after small interfering RNA–mediated MRGPRA3 knockdown, indicating thatMRGPRA3 are indeed targeted by chloroquine (Liuet al., 2009). Notably, chloroquine also induced calciumsignals after recombinant expression of rat MRGPRA inprimary sensory neurons derived from the MRGPRcluster knockout mouse (Liu et al., 2009).

The purine adenine might serve as an endogenousagonist of rat MRGPRA, because in silico modeling


revealed a putative adenine binding pocket in the ratMRGPRA protein (Heo et al., 2007b). This assumptionwas corroborated by the observation that adeninebound to rat MRGPRA with a KD value of 24 nM,when the receptor was overexpressed in CHO cells(Bender et al., 2002). In this cellular model, adenineinhibited forskolin-induced adenylyl cyclase activityand induced guanosine 59-O-(3-[35S]thio)triphosphatebinding to the plasma membrane with potencies of ;3and ;60 nM, respectively. However, inhibition ofcAMP production by rat MRGPRA points to Gi/o andnot to Gq/11 as downstream effectors in CHO cells. Thisobservation contrasts with the functional properties ofmurine MRGPRA1 that was found to exclusivelyengage classic Gq/11 signaling cascades (Han et al.,2002; Wang et al., 2006b).2. Signaling Cascades and Physiologic Effects.

Physiologic effects and detailed signaling cascades ofMRGPRA members have so far been described forthe murine chloroquine-sensitive subfamily memberMRGPRA3 (see Fig. 3). Intradermal application ofchloroquine induced a profound itching behavior at theinjection site (Liu et al., 2009). This procedure did notlead to alloknesis, an aberrant sensation of itch whennonitching skin near an itchy skin patch is lightlytouched (Akiyama et al., 2012). Chloroquine-inducedcalcium signals in primary sensory neurons werecompletely inhibited by removal of extracellular cal-cium by EGTA and by ;90% after application ofruthenium red, a blocker of transient receptor poten-tial (TRP) cation channels (Liu et al., 2009). By use ofcalcium imaging of cultured primary sensory neuronsand MRGPRA3 transfected NG108 cells, intracellularsignaling cascades were analyzed that might be re-sponsible for the sensation of itch (Wilson et al., 2011).Most interestingly, chloroquine induced an increase inintracellular calcium through TRP ankyrin 1 (TRPA1)channels, because chloroquine-induced itching was notobserved in TRPA1-deficient mice (Wilson et al., 2011).The TRP vanilloid 1 (TRPV1) channel, an establishedsensor of different painful stimuli such as heat andprotons (Caterina et al., 1997), is also expressed in

chloroquine-responsive neurons (Liu et al., 2009;Wilson et al., 2011). In contrast to TRPA1, TRPV1 isnot required for MRGPRA3-dependent neuronal exci-tation, and chloroquine-induced calcium signals werenot detected in cells coexpressing MRGPRA3 andTRPV1 (Wilson et al., 2011). Functional couplingbetween MRGPRA3 and TRPA1 was assigned to Gbgprotein complexes because of the lack of calciumsignals after incubation with the Gbg inhibitor gallein.The authors further showed that functional interac-tions between TRPA1 and MRGPRA3 are required forthe physiologic effects of the latter. Than and co-workers (2013) confirmed that direct activation ofMRGPRA3 by chloroquine excites murine primarysensory neurons because of TRPA1 activation, becausethe response of these neurons was abolished by theTRPA1 inhibitor HC-030031 [1,2,3,6-tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7H-purine-7-acetamide, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide]. However, the authors revealed thatMRGPRA3 was also able to induce calcium signalsin TRPA1-negative neurons. By use of YM58483 (N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide)asanonsubtypeselective canonical TRP and Pyr3 as a TRPC3 blocker,TRPCs and, in particular, the TRPC3 subtype wereidentified as the downstream effectors mediatingMRGPRA3-induced neuronal excitation in the ab-sence of TRPA1 (Than et al., 2013). In addition, thiswork showed profound sensitization of TRPV1 chan-nels via chloroquine-induced protein kinase C (PKC)activation and inhibition of the cold sensor TRPmelastatin 8 (TRPM8) via direct binding of the Gaq

subunit to the ion channel, because a Gaq chimeraunable to activate phospholipase C (PLC)b stillblocked TRPM8 activity in cells lacking functionalGaq proteins (McKemy et al., 2002; Peier et al.,2002). Thus, although acute signaling of MRGPRA3via TRPA1 solely leads to itch (Liu et al., 2009;Wilson et al., 2011), chloroquine also activates cel-lular signaling cascades that might affect other

TABLE 3Pharmacology of MAS-related G protein–coupled receptors A

MRGPRA subfamily members that have been assigned to a ligand are listed. All data were generated afterrecombinant MRGPR expression.

Receptor Ligand EC50 Readout System Reference

nM

mMRGPRA1 FMRFamide 20 calcium HEK293-Ga15 Dong et al., 2001NPFF 200 calcium HEK293-Ga15 Dong et al., 2001hb-Salusin 300 calcium HEK293 Wang et al., 2006bchloroquine 300,000 calcium HEK293 Liu et al., 2009

mMRGPRA4 NPAF 60 calcium HEK293-Ga15 Dong et al., 2001mMRGPRA3 chloroquine 27,000 calcium HEK293 Liu et al., 2009rMRGPRA chloroquine N.D. calcium mDRG neurons Liu et al., 2009

adenine 3 cAMP inhibition CHO Bender et al., 200260 GTPgS CHO Bender et al., 2002

GTPgS, guanosine 59-3-O-(thio)triphosphate; N.D., not determined; r, rat; m, mouse.

578 Solinski et al.

physiologic processes, such as nociception or tem-perature sensation.

C. MAS-Related G Protein–Coupled Receptors B

None of the rodent MRGPRB members has beenassigned to any ligand so far. However, in a reportermouse that carries a gene for placental alkalinephosphatase under the control of the MRGPRB4promoter, a rare subset of primary sensory neuronsthat only innervates the hairy skin showed highpromoter activity (Liu et al., 2007). These neuronswere nonpeptidergic, TRPV1 negative, displayeda broad arborization of their cutaneous termini, andare often associated with hair follicles. Most interest-ingly, MRGPRB4 positive neurons project to the spinallamina IIo, a spinal pain processing center that is partof the substantia gelatinosa (Rexed, 1952). Thesefindings suggested that MRGPRB4 mark C-fiber tactileafferents that are supposed to detect gentle touchand stroking. Indeed, activation of MRGPRB4-positive

primary sensory neurons, but not activation of theMRGPRB4 itself, by gentle stroking was demonstrated byan elegant in vivo calcium imaging approach. Pharmaco-genetic activation of MRGPRB4 expressing neuronsin freely moving mice resulted in conditioned placepreference, suggesting that activation of these neuronsis positively reinforcing and might be induced byinterindividual social interactions such as allogroom-ing (Vrontou et al., 2013).

D. MAS-Related G Protein–Coupled Receptors C

1. Pharmacology. The rodent MRGPRC subfamilyconsists of only one protein-encoding gene in rats andmice, and both receptor subtypes have been suggestedto bind several ligands (see Table 4). After heterologousexpression in different cell systems, MRGPRC from ratand mouse were activated to elicit calcium signals bya set of different but structurally related peptides (seeFig. 4A) with potencies in the low to medium nano-molar range (Grazzini et al., 2004; Han et al., 2005;

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


Heo et al., 2007a; Solinski et al., 2010, 2013). Amongthese peptides are g2-melanocyte stimulating hor-mone (g2-MSH), BAM peptides, dynorphin-14, andproneuropeptide-FF-A peptides. Notably, these pep-tides originate from PENK (BAM), pro-opiomelanocortin(g2-MSH), prodynorphin (dynorphin-14), and NPPFA(NPFF or NPAF) (see Fig. 4A). Such a wide range inthe binding profile of a distinct GPCR is quite uniqueand suggests that many different physiologic pathwaysrely on MRGPRC signaling. Activation of MRGPRC byBAM peptides was validated in primary sensoryneurons of mice (Liu et al., 2009; Wilson et al., 2011)and rats (Honan and McNaughton, 2007). However,their similarities notwithstanding, several pharmaco-logical differences between murine and rat MRGPRChave also been observed (see Fig. 4B): NPFF peptidesdisplayed lower potency to activate the rat MRGPRCcompared with the murine counterpart, and dynorphin-14 did not activate rat MRGPRC at all (Han et al.,2002; Grazzini et al., 2004; Solinski et al., 2013).Regarding efficacy, g2-MSH and BAM peptides arethe most efficacious activators of both MRGPRC, withg2-MSH clearly surmounting BAM peptides (Solinskiet al., 2013). Using in silico modeling and site-directedmutagenesis, Heo and coworkers (2007a) revealedthat Tyr110, Asp161, and Asp179 of murine MRGPRCare crucially involved in agonist binding by interact-ing with a common C-terminal end of all peptides,which harbors the consensus sequence RF(Y)amide orRF(Y)G. Notably, all three ligand-binding amino acidsare conserved in rat MRGPRC.Several peptides that are now recognized as

MRGPRC agonists were previously shown to activatedistinct receptors from other GPCR families, e.g.,g2-MSH also binds to melanocortin 3 receptors and

some BAM peptides to opioid receptors (OR). Interest-ingly, ligand sharing with receptor subtypes of otherGPCR families is not restricted to MRGPRC, butappears to be a common feature of many MRGPR assummarized in Table 9 (Bowery et al., 1980; Quirionand Weiss, 1983; Roselli-Rehfuss et al., 1993; de Leceaet al., 1996; Fukusumi et al., 1997; Kaupmann et al.,1997; Oosterom et al., 1999; Han et al., 2002; Lemboet al., 2002; Mollereau et al., 2002; Robas et al., 2003;Santos et al., 2003; Grazzini et al., 2004; Shinoharaet al., 2004; Gembardt et al., 2008; Lautner et al.,2013). Although, ligand sharing between MRGPRCand other GPCR subtypes is of physiologic interest, itis also a major concern when considering the specificityof distinct MRGPRC-activating peptides in vivo. In-terestingly, after N-terminal truncation of g2-MSH(g2-MSH6–12) or of BAM1–22 (BAM8–22) activity towardMRGPRC but not toward melanocortin 3 receptor orOR is preserved (Lembo et al., 2002; Grazzini et al.,2004). Accordingly, g2-MSH6–12 or BAM8–22 areregarded as specific MRGPRC ligands and can be usedin in vivo studies to analyze the physiologic effects ofMRGPRC in mice or rats. When considering g2-MSH orBAM derivatives as putative endogenous MRGPRCligands, it is also important to investigate whetherthese peptides are actually released from precursorsunder physiologic or pathophysiological conditions.g2-MSH and BAM1–22 have both been shown to bepresent in several tissues, including spinal cord andbrain (Mizuno et al., 1980; Pelletier et al., 1981; Holltet al., 1982; DeBold et al., 1988a,b; Cai et al., 2007a).Thus, peptides that bind other GPCRs and MRGPRCcould serve as endogenous ligands of the latter. Thespecific MRGPRC agonist BAM8–22 was also identi-fied in vivo after microdialysis of exogenous BAM1–25

TABLE 4Pharmacology of MAS-related G protein–coupled receptors C

MRGPRC subfamily members are listed by the ligands to which they have been assigned. Data were generated afterrecombinant MRGPRC expression, despite when endogenous expression is indicated by “(e)”.

Receptor Ligand EC50 System Reference

nM

mMRGPRC 11 HEK293 (GFP-tag) Han et al., 2002g2-MSH 340 HEK293 Heo et al., 2007a

290 HEK293 Solinski et al., 201353 HEK293 (GFP-tag) Han et al., 2002

BAM8–22 292 HEK293 Heo et al., 2007a330 HEK293 Solinski et al., 2013

Dynorphin-14 22 HEK293 (GFP-tag) Han et al., 2002310 HEK293 Solinski et al., 2013

NPFF 54 HEK293 (GFP-tag) Han et al., 2002358 HEK293 Heo et al., 2007a

NPAF 282 HEK293 (GFP-tag) Han et al., 2002SLIGRLamide 10,000 CHO Liu et al., 2011

50,000 DRG neurons (e) Liu et al., 2011rMRGPRC g2-MSH 44 HEK293-Gaqi5 Grazzini et al., 2004

440 HEK293 Solinski et al., 2013Tyr6-g2-MSH6–12 15 HEK293-Gaqi5 Grazzini et al., 2004BAM8–22 120 HEK293-Gaqi5 Grazzini et al., 2004

2600 HEK293 Solinski et al., 2013

m, mouse; r, rat.

580 Solinski et al.

into the striatum of anesthetized rats, using a combi-nation of solid-phase preconcentration capillary elec-trophoresis and imaging matrix-assisted laser desorption/ionization mass spectrometry (Zhang et al., 1999).However, although the proteolytic machinery to generateBAM8–22 in vivo is present in the brain, it has not yetbeen shown that BAM8–22 or g2-MSH6–12 are producedendogenously in tissues adjacent to primary sensoryneurons. Thus, it remains unclear whether and underwhich circumstances specific MRGPRC ligands exist invivo.Very recently, it was proposed that the "SLIGRL"-

peptide activates murine MRGPRC (Liu et al., 2011)."SLIGRL" is not a classic neuropeptide released fromcommon precursor proteins but part of the tetheredligand domain of murine protease-activated receptors 2(PAR2) [for further reading see these comprehensivereviews (Steinhoff et al., 2005; Ramachandran andHollenberg, 2008)]. PAR are GPCR that are activatedby serine proteases such as thrombin or trypsin. Toactivate PAR, proteases cleave N-terminal peptidesfrom the PAR protein, thereby exposing a “cryptic”PAR-activating sequence at the N terminus. Shortpeptides that are derived from theses cryptic sequenceshave also been shown to activate PAR without previouscleavage, e.g., the "SLIGRL"-amide was established asa PAR2-specific ligand. However, it may also be possible

that peptides such as the "SLIGRL"-peptide are re-leased from PAR through consecutive cleavage by twoor more proteases and in turn serve as ligand ofdistinct GPCR. By use of CHO cells recombinantlyexpressing MRGPRC, small interfering RNA–

mediated MRGPRC knockdown in wild-type primarysensory neurons, and primary sensory neurons fromMRGPRC-deficient mice, "SLIGRL"-induced calciumsignals in the presence, but not in the absence of theMRGPRC were identified, indicating that the "SLIGRL"-peptide is indeed a ligand of this receptor. Analysis ofstructure-activity relationships revealed that amida-tion of the "SLIGRL" C terminus is important for itsfunction as a ligand, which fits well to the proposedRF(Y)G/amide consensus sequence of other MRGPRCagonists. Notably, "SLIGRL"-induced itch, which wasbelieved to be mediated by PAR2 (Shimada et al.,2006), was not changed in PAR2-deficient mice (Liuet al., 2011). In MRGPRC overexpressing CHO cells,a high EC50 value of ;10 mM for "SLIGRL"-inducedcalcium signals was measured (Liu et al., 2011).However, because PAR2 and MRGPRC are both ex-pressed in primary sensory neurons, it is appealing tospeculate that the "SLIGRL" peptide might physiolog-ically exist in concentrations high enough to stimulateMRGPRC.

2. Signaling Cascades and Physiologic Effects.a. Preface. Rat and murine MRGPRC elicit their

effects primarily by the activation of G proteins. Bothreceptors were shown to induce intracellular calciumrelease via activation of PLCb (Han et al., 2002;Grazzini et al., 2004). For murine MRGPRC couplingto Gq/11 proteins seems to be essential, becauseMRGPRC-dependent calcium signaling was absent inmurine embryonic fibroblasts derived from Gq/11-deficientembryos (Han et al., 2002). In accordance with theirselective expression in small-diameter primarysensory neurons, MRGPRC signaling in mice andrats has been implicated in somatosensory detectorfunctions in the skin. Indeed, published data on thein vivo effects of MRGPRC agonists detected pain-enhancing, pruritogenic, but also analgesic effects(summarized in Fig. 5).

b. Pain-enhancing effects. Intraplantar injectionsof the MRGPRC-specific peptides BAM8–22 or Tyr6-g2-MSH6–12 into juvenile or adult rats dose dependentlyresulted in acute nocifensive behavior, thermal hyper-algesia, and mechanical allodynia (Grazzini et al.,2004; Ndong et al., 2009). Likewise, intrathecal in-jection of MRGPRC agonists into juvenile or adult ratsand Kunming mice also induced acute pain-like be-havior and thermal hyperalgesia (Grazzini et al.,2004; Chang et al., 2009; Wei et al., 2010). MRGPRCmay also be responsible for inflammatory pain,because complete Freund’s adjuvant (CFA)–inducedthermal hyperalgesia was alleviated by RNAi-mediatedMRGPRC knockdown in rats (Ndong et al., 2009).

Fig. 4. Schematic presentation and comparison of the pharmacology ofBAM8–22-sensitive MRGPR. (A) Amino acid sequences and precursorsof the classic peptidergic MRGPRC agonists are given. Dashed linesindicate that the synthesis of the peptide in vivo has not been detectedyet. The C-terminal consensus, which is proposed to be crucial forMRGPRC binding, is highlighted in red. (B) The most potent agonists ofmurine (m) or rat (r) MRGRPC and human (h) MRGPRX1 are provided.The ligands are drawn to scale by linearly modifying the font size inaccord with their published potencies (Han et al., 2002; Lembo et al.,2002; Grazzini et al., 2004; Liu et al., 2009, 2011; Solinski et al., 2013).For simplicity, ligands that exhibit potencies more than 10 times lowerthan g2-MSH (in the case of MRGPRC) or BAM8–22 (in the case ofMRGPRX1) are not shown.


Notably, pain-enhancing effects of MRGPRC can only beassessed at low agonist doses (e.g., up to 20 nmol of Tyr6-g2-MSH6–12) and in a rigid time window of a maximumof 20 minutes postinjection (Wei et al., 2010). This lowdose effect may be due to the activation of an analgesicoff-target at higher peptide concentrations, most likelythe Kyotorphin receptor, which masked MRGPRC-induced pain, whereas rapid peptide degradation maybe responsible for the short-term response (Grazziniet al., 2004; Wei et al., 2010).Several publications identified heat-sensitive TRPV1

ion channels as major downstream targets of MRGPRCresponsible for its pain-enhancing effects (Honan andMcNaughton, 2007; Hager et al., 2008; Ndong et al.,

2009; Wilson et al., 2011). In rat primary sensoryneurons, BAM8–22 sensitized TRPV1 for its agonistcapsaicin (CAP) via a PKC-dependent pathway (Honanand McNaughton, 2007). The PKC isoforms d, «, and zwere excluded to be part of this signaling cascade,because these isoforms did not translocate to theplasma membrane after MRGPRC stimulation. TRPV1sensitization was also made responsible for an in-creased CAP- or heat-induced calcitonin gene-relatedpeptide (CGRP) release from rat or mouse paw skinafter preincubation with BAM1–22 in conjunction withnaloxone, an opioid receptor antagonist that blockseffects of BAM1–22 on opioid receptors but still allowsBAM1–22 to act on MRGPRC (Hager et al., 2008).

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

582 Solinski et al.

Interestingly, BAM8–22 or g2-MSH alone failed toinduce CGRP release, and, surprisingly, a combinationof BAM1–22 and naloxone that sensitized TRPV1 forCAP still induced CGRP release from skin prepara-tions derived from TRPV1-deficient mice. In rats,MRGPRC-mediated thermal hyperalgesia was abro-gated by a specific TRPV1 inhibitor (Ndong et al.,2009), and coexpression of TRPV1 with murineMRGPRC in NG108 cells resulted in augmented calciumsignals by BAM8–22 (Wilson et al., 2011). Thus, TRPV1has been defined as a common target for cellular sig-naling induced by MRGPRC, which might be of highphysiologic significance because coexpression of TRPV1with MRGPRC in rodent primary sensory neurons wasfrequently observed (Lembo et al., 2002; Hager et al.,2008; Liu et al., 2009).In addition to TRPV1, Chang and coworkers (2009)

identified the spinal N-methyl-D-aspartate (NMDA)receptor and the nNOS as players in MRGPRC-mediated thermal hyperalgesia, because the NMDAreceptor antagonists D-APV [D-(2 )-2-amino-5-phosphonopentanoic acid] and MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine] and the nNOS inhibitor NG-nitro-L-argininemethyl ester were found to dose-dependently inhibitthe pronociceptive effects of Tyr6-g2-MSH6–12 inmice (Chang et al., 2009). Because nNOS inhibitorsalso diminished NMDA-induced pain-like behavior(Kitto et al., 1992), it remained unclear whether bothplayers are directly modulated by MRGPRC in thesame cell.

c. Analgesic effects. In contrast to pain-enhancing effects of MRGPRC, several studies haveestablished analgesic effects of MRGPRC-specific ago-nists (Hong et al., 2004; Zeng et al., 2004; Chen et al.,2006, 2008, 2012; Cai et al., 2007a; Guan et al., 2010;Jiang et al., 2013). Acute nocifensive behavior afterformalin or NMDA injection was found to be diminisheddose-dependently by BAM1–22 plus naloxone and byBAM8–22 or Tyr6-g2-MSH6–12 applied alone (Honget al., 2004; Zeng et al., 2004; Chen et al., 2006, 2008). Inparallel, spinal c-Fos immune reactivity, an establishedmarker of nociception (Bullitt, 1989), was reduced byMRGPRC agonists. Thermal hyperalgesia after intra-plantar formalin or CFA injection, quantified either bytail flick or Hargreaves’s tests, were found to bediminished by MRGPRC agonists (Hong et al., 2004;Guan et al., 2010; Jiang et al., 2013). Interestingly,CFA-induced thermal hyperalgesia was augmented inthe MRGPR cluster knockout mice (see section III.B.1)that are deficient for MRGPRC (Guan et al., 2010).Mechanical hyperalgesia, a hallmark of CFA-inducedinflammatory and spinal nerve ligation– or chronicconstriction injury–induced neuropathic pain, wasalso reduced by application of MRGPRC agonists(Cai et al., 2007a; Guan et al., 2010; Chen et al.,2012; Jiang et al., 2013). MRGPRC upregulation and

increased abundance of its agonist BAM1–22 havebeen observed during the process of CFA-inducedpain chronification (Cai et al., 2007a; Jiang et al.,2013). Consequently, acute intrathecal application ofa BAM1–22-neutralizing antibody decreased CFA-induced mechanical hyperalgesia, thus highlightingongoing MRGPRC signaling during inflammatorypain (Cai et al., 2007a). After CFA injection, enhancedMRGPRC signaling inhibited the induction of CGRPin primary sensory and of nNOS in spinal projectionneurons (Jiang et al., 2013), which is believed to exertlong lasting effects on spinal sensitization [for detailssee (Latremoliere and Woolf, 2009)]. This was attrib-uted to the engagement of m-OR (MOP), as evidencedby the lack of inhibition after preincubation with theMOP inhibitor CTAP.

d. Pruritogenic effects. Concomitant to the obser-vation that chloroquine-induced scratching behavior ismediated largely by murine MRGPRA3 signaling,intradermal injections of BAM8–22 into the nape ofthe neck of mice also elicited a profound scratchingbehavior (Liu et al., 2009). This behavioral result wasconfirmed by several other studies, the proposedmurine MRGPRC agonist "SLIGRL" also elicited itch,and agonist-induced scratching was diminished to;25% in the MRGPR cluster knockout mouse (seesection III.B.1) that lacks MRGPRC (Liu et al., 2009,2011; Wilson et al., 2011; Akiyama et al., 2012; Hanet al., 2013). However, intradermal injections into thenape of the neck as a secure test for itch werequestioned because pain-inducing algogens and pruri-togens elicit nearly indistinguishable reactions afterthis application procedure (Shimada and LaMotte,2008). Instead the “cheek model of itch” was proposed,which is based on intradermal cheek injections andenables discrimination of itch and pain. Cheek injec-tions of pain-inducing algogens are followed by intensewiping, whereas pruritogens elicit a vigorous scratch-ing response. Therefore it is important to note thatBAM8–22 injected into the cheek of mice elicited onlyscratching without any wiping reaction (Wilson et al.,2011). The notion that murine MRGPRC cause itchwithout pain is further accompanied by the findingthat ablation of MRGPRA3-positive primary sensoryneurons that almost all express MRGPRC (Zylka et al.,2003; Liu et al., 2009) completely abolished BAM8–22-or "SLIGRL"-induced scratching (Han et al., 2013). Thelatter study is of particular interest because it showedthat MRGPRA3/MRGPRC-positive neurons are involvedin itch, but not pain perception in mice. Noteworthy,activation of MRGPRC by BAM8–22, but not by"SLIGRL" induced alloknesis in addition to acuteitch, which were both independent of histaminesignaling (Akiyama et al., 2012).

Wilson and coworkers (2011) analyzed downstreameffectors involved in MRGPRC-induced itching. Al-though the percentage of primary sensory neurons that


elicited BAM8–22-induced calcium signals was dimin-ished in TRPV1-deficient cells, action potentials afterBAM8–22 application were not altered. In contrast,TRPA1 deficiency resulted in the reduction of bothresponses, calcium signals and action potentials. Ac-cordingly, BAM8–22-induced scratching responses wereabsent only in TRPA1-deficient mice, whereas TRPV1deficiency was dispensable.

E. MAS-Related G Protein–Coupled Receptors D

1. Pharmacology. The MRGPRD subfamily consistsof only one receptor subtype per species, which isconserved in rodents and primates, and several ligandshave been implicated in MRGPRD pharmacology (seeTable 5). b-[3H]Alanine is able to bind to the humanMRGPRD after heterologous expression in CHO cells(Shinohara et al., 2004). Measuring calcium signals inthese cells, an EC50 value of ;15 mM was determinedthat is three to 4-fold below b-alanine concentrationsobserved in rat sciatic nerve or cat brain samples(Tallan et al., 1954; Marks et al., 1970). In line withthis observation for human MRGPRD, rat or mouseMRGPRD also responded to b-alanine after heterolo-gous expression in CHO cells with calcium signals andEC50 values between 3 and 44 mM (Shinohara et al.,2004; Zhang et al., 2007; Ajit et al., 2010). b-Alanineinduced signaling in MRGPRD-enriched fractions ofprimary sensory neurons from rat (Crozier et al., 2007)and calcium signals or action potentials in murineprimary sensory neurons (Liu et al., 2012). Thus,b-alanine has been established as a ligand of MRGPRDand could be responsible for their endogenous effects.Interestingly, two other amino acids, GABA andb-aminoisobutyric acid, have also been shown to induce

calcium signals via human MRGPRD, although withlower potency compared with b-alanine (Shinoharaet al., 2004; Ajit et al., 2010; Uno et al., 2012).However, because b-alanine and GABA also bind toother proteins, including GPCR and ion channels(Bowery, 1993; Macdonald and Olsen, 1994; Tiedjeet al., 2010), in vivo effects of both amino acids wouldhave to be interpreted with caution regarding themolecular receptor entity.

In addition to amino acids, two peptides produced bythe renin-angiotensin system have also been identifiedas MRGPRD ligands. Precisely, angiotensin-(1–7) andalamandine [Ala1-angiotensin-(1–7)] were proposed toactivate MRGPRD (Gembardt et al., 2008; Lautneret al., 2013). Angiotensin-(1–7) is also established tobind the MAS1 receptor in vivo (Santos et al., 2003)and is therefore not specific for MRGPRD. Furtherscreening approaches revealed novel exogenous com-pounds that specifically activate or inhibit MRGPRDactivity (Zhang et al., 2007; Ajit et al., 2010; Uno et al.,2012). These specific ligands will be very useful toolsfor the analysis of the in vivo effects of MRGPRD in thefuture.

2. Signaling Cascades and Physiologic Effects.Several cellular studies agree that MRGPRD couple toGq/11 and pertussis toxin (PTX)–sensitive Gi/o proteins,because MRGPRD induced PTX-insensitive calciumreleases and PTX-sensitive inhibition of forskolin-induced adenylyl cyclase activation (Shinohara et al.,2004; Ajit et al., 2010). Gi/o coupling links MRGPRD toKCNQ2/3 channels, and inhibition of KCNQ2/3 activityis mainly responsible for the inhibition of noninactivatingpotassium currents (M-currents) induced by MRGPRD in

TABLE 5Pharmacology of MAS-related G protein–coupled receptors D

MRGPRD subfamily members are listed by the ligands to which they have been assigned. All data were generatedafter recombinant MRGPRD expression.

Receptor Ligand EC50 Readout System Reference

nM

hMRGPRD b-Alanine 15 calcium CHO Shinohara et al., 20044 calcium CHO Ajit et al., 2010

23 calcium HEK293 Uno et al., 2012220 GTPgS HEK293 Uno et al., 2012350 IP HEK293 Uno et al., 2012

GABA 191 calcium CHO Shinohara et al., 200453 calcium CHO Ajit et al., 2010

4000 calcium HEK293 Uno et al., 2012BABA 53 calcium HEK293 Uno et al., 2012

820 GTPgS HEK293 Uno et al., 2012380 IP HEK293 Uno et al., 2012

Angiotensin-(1–7) N. D. arachidonic acid COS Gembardt et al., 2008Alamandine N. D. nitric oxide CHO Lautner et al., 2013

mMRGPRD b-Alanine 44 calcium CHO Shinohara et al., 2004rMRGPRD b-Alanine 14 calcium CHO Shinohara et al., 2004

4 calcium CHO Ajit et al., 20101 calcium CHO Zhang et al., 2007

GABA 191 calcium CHO Shinohara et al., 200450 calcium CHO Ajit et al., 2010

L-Carnosine 34 calcium CHO Shinohara et al., 2004

BABA, b-aminoisobutyric acid; GTPgS, guanosine 59-3-O-(thio)triphosphate; h, human; IP, phosphoinositol pro-duction; m, mouse; N.D., not determined; r, rat.

584 Solinski et al.

primary sensory neurons (Crozier et al., 2007). Thissignaling cascade was implicated to contribute toMRGPRD-dependent enhanced neuronal activity(Crozier et al., 2007). Likewise, decreased neuronalactivity was detected in MRGPRD-deficient mice (Rauet al., 2009).Just like other MRGPR members, MRGPRD also

specifically mark a discrete subset of primary sensoryneurons. Genetically engineered axonal tracers mapthe stratum granulosum of the skin as the only pe-ripheral target of MRGPRD-positive neurons (Zylkaet al., 2005). MRGPRD neurons form synapses in thespinal lamina II (Zylka et al., 2005) and convey theirsignal monosynaptically to almost all known classes ofspinal lamina II neurons, as evidenced by an optoge-netical circuit mapping approach (Wang and Zylka,2009). MRGPRD-positive neurons were classified asnonpeptidergic C-fiber nociceptors that coexpress theionotropic ATP receptor P2X3, tetrodotoxin-insensitiveNaV channels, MOP, and partly the TRPV1 ion channel(Shinohara et al., 2004; Zhang et al., 2005; Zylka et al.,2005; Dussor et al., 2008). The functional analysis ofthese cells for sensory transduction revealed a complexpicture. MRGPRD-deficient mice exhibit deficits inthe detection of mechanical, heat, and cold stimuli,whereas ablation of MRGPRD-positive neurons onlyimpacted acute mechanical pain and mechanicalhyperalgesia in a CFA model of inflammatory pain,whereas heat and cold transduction was completelypreserved (Cavanaugh et al., 2009; Rau et al., 2009).In accordance with the latter finding, ablation ofMRGPRD-positive primary sensory neurons reducedthe firing rate and abundance of spinal dorsal hornneurons responding to mechanical but not heat stimuli(Zhang et al., 2013). Finally, stimulation of MRGPRD-positive neurons with b-alanine only induced calciumsignals and action potentials in ;40% of geneticallylabeled MRGPRD-expressing cells (Liu et al., 2012).This response elicited itch in mice, because itch wascompletely abrogated by MRGPRD deficiency. In vivoelectrophysiological recordings revealed that b-alanine–activated neurons are heat- and mechanosensitive,whereas the remaining heat-insensitive subset ofMRGRPD neurons that did not respond to b-alaninewas solely mechanosensitive. Thus, the latter pop-ulation might be responsible for deficits in mechanicalpain that was observed after ablation of MRGPRD-positive neurons (Cavanaugh et al., 2009). Interestingly,intradermal application of b-alanine during a pilothuman study that comprised 11 individuals also resultedin itch, pricking/stinging, and burning (Liu et al., 2012),indicating that MRGPRD similarly affect itching in hu-mans and rodents.Transcripts of MRGPRD were also detected in other

tissues than in primary sensory neurons, includingarteries (Shinohara et al., 2004). Moreover, the angio-tensinogen cleavage product alamandine circulates in

human blood and produces physiologic effects thatresemble those produced by angiotensin-(1–7), includ-ing relaxation of aortic rings. These actions ofalamandine were not mediated via MAS1 or angioten-sin II type 2 receptors (AT2R) but required MRGPRDactivation (Lautner et al., 2013). Surprisingly, theabovementioned MRGPRD agonist b-alanine did notrelax aortic rings, but blocked the effects of alaman-dine, suggesting that both MRGPRD agonists directMRGPRD-induced signaling to different pathways.Effects of MRGPRD in primary sensory neurons andthe vasculature are summarized in Fig. 6.

F. MAS-Related G Protein–Coupled Receptors E to -H

The subfamilies MRGPRE to -H consist of only onereceptor per species and are conserved in rodents andprimates, except for MRGPRH that only exists inrodents. So far, no member of these MRGPR has everbeen assigned to any ligand, which severely hamperedtheir functional characterization. A MRGPRE-deficientmouse strain, which exhibited normal acute painresponses in a hot plate assay but showed a trendtoward decreased nocifensive behavior in both phasesof the formalin test as well as statistical significantdeficits in the induction, but not maintenance, ofmechanical allodynia after chronic constriction injury,was also established (Cox et al., 2008).

G. MAS-Related G Protein–Coupled Receptors X

1. Pharmacology.a. MAS-related G protein–coupled receptors X1.

The human MRGPRX1 was the first primate-specificMRGPR to be assigned to a ligand, and several ad-ditional agonistic and antagonistic compounds havebeen identified thereafter (see Table 6). MRGPRX1have been shown to bind BAM peptides with highaffinity (KD value of BAM8–22 ;10 nM) after expres-sion in HEK293 cells (Lembo et al., 2002). Likewise,BAM peptides elicited intracellular calcium release inthe same cells (Lembo et al., 2002). In accord withrodent MRGPRC, agonistic activity of BAM peptideswas completely preserved in an N-terminal truncatedform of BAM1–22 (BAM8–22). EC50 values of eitherBAM8–22 or BAM1–22 and MRGPRX1 in calciumassays varied between ;14 and ;800 nM (Lemboet al., 2002; Burstein et al., 2006; Tatemoto et al., 2006;Shemesh et al., 2008; Malik et al., 2009; Solinski et al.,2013).

Despite sharing BAM peptides as agonists, humanMRGPRX1 and rodent MRGPRC also displayed phar-macological differences (see Fig. 4B). For instance,proneuropeptide-FF-A cleavage products or dynorphin-14, which are partial agonists of MRGPRC, did notactivate MRGPRX1 at all, and importantly, g2-MSH,the most potent and efficacious agonist of rodentMRGPRC, only faintly activated MRGPRX1 whenchimeric G proteins were coexpressed (Lembo et al.,


2002; Solinski et al., 2013). Moreover, cyclic dimers ofthe C-terminal part of g2-MSH were introduced toantagonize BAM8–22-induced MRGPRX1 activation,but do not bind rodent MRGPRC (Schmidt et al., 2009).Therefore, the common RF(Y)G/amide consensus ofMRGPRC agonists does not apply to MRGPRX1. Asa consequence MRGPRC exhibit a high promiscuitytoward many ligands, whereas MRGPRX1 are muchmore restrictive and solely bind BAM peptides, afeature conserved to a certain degree in the MRGPRX1of rhesus monkeys (Burstein et al., 2006).

In addition to potential endogenous agonists, exog-enous agonists of MRGPRX1, e.g., tetracyclic benzimi-dazoles, have been proposed (Malik et al., 2009).Furthermore, 2,4-diaminopyrimidine derivates and 2,3-disubstituted azabicyclooctanes antagonize BAM8–22-induced signaling via MRGPRX1 (Kunapuli et al.,2006; Bayrakdarian et al., 2011). Interestingly, theMRGPRA3 ligand chloroquine also activated MRGPRX1,although affinity was 1000-fold and efficacy ;2.5-foldreduced compared with BAM8–22 (Liu et al., 2009).Hence, in accordance with its evolutionary relation

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

TABLE 6Pharmacology of human MAS-related G protein–coupled receptors X1

The potency of agonists is given as the EC50 value to induce calcium signaling in the given cellular system and thepotency of antagonists is given as the IC50 value to inhibit BAM8–22-induced calcium signaling in the given cellularsystem. All data were generated after recombinant MRGPRX1 expression.

EC50 IC50 System Reference

nM

AgonistBAM8–22 14 HEK293-Gaqi5 Lembo et al., 2002

25 HEK293 Burstein et al., 200640 HEK293 Tatemoto et al., 2006

8–50 CHO Kunapuli et al., 2006150 HEK293 Solinski et al., 2013

BAM1–22 16 HEK293-Gaqi5 Lembo et al., 2002800 HEK293 Burstein et al., 200680 CHO-Ga16 Shemesh et al., 2008

800 HEK293 Malik et al., 2009Chloroquine 300,000 HEK293 Liu et al., 2009Benzimidazole derivates 320 HEK293 Malik et al., 2009

AntagonistCyclic g2-MSH6–12 derivates 12 HEK293-Gaqi5 Schmidt et al., 2009Diaminopyrimidine derivates 14 HEK293-Gaqi5 Bayrakdarian et al., 2011Azabicyclooctane derivates 100 CHO Kunapuli et al., 2006

586 Solinski et al.

MRGPRX1 share some pharmacological character-istics with rodent MRGPRC and MRGPRA subfamilymembers but also display unique pharmacologicalfeatures.

b. MAS-related G protein–coupled receptors X2.The MRGPRX2 subtype is clearly distinguishable fromMRGPRX1 regarding its pharmacology (see Table 7),because MRGPRX2 do not bind BAM peptides(Burstein et al., 2006). However, several other pep-tidergic ligands have been proposed in different heter-ologous or endogenous expression systems (Robaset al., 2003; Kamohara et al., 2005; Burstein et al.,2006; Tatemoto et al., 2006; Shemesh et al., 2008;Malik et al., 2009; Kashem et al., 2011; Liu et al., 2011;Subramanian et al., 2011a,b, 2013). The best charac-terized MRGPRX2 ligand is the cortistatin-14 peptidethat activated MRGPRX2 with potencies in themedium to high nanomolar range (Robas et al., 2003;Kamohara et al., 2005; Burstein et al., 2006; Shemeshet al., 2008; Malik et al., 2009). Notably, similar toMRGPRX1, the ligand binding profile of MRGPRX2 isconserved in rhesus monkeys, because cortistatin-14also activates MRGPRX2 from this species (Bursteinet al., 2006; Malik et al., 2009).MRGPRX2 show a broader expression pattern than

other MRGPRX, because MRGPRX2 expression wasdetected in primary sensory neurons, several brainareas, mast cells, and the adrenal medulla (Kamoharaet al., 2005). In line with this observation, proadreno-medullin peptides that endogenously occur as sideproducts during adrenomedullin synthesis in theadrenal medulla have also been shown to activateMRGPRX2 (Kamohara et al., 2005). MRGPRX2 were

also shown to be involved in mast cell activation bya set of structurally similar, endogenous (Tatemotoet al., 2006; Subramanian et al., 2011a, 2013) orexogenous (Kashem et al., 2011; Subramanian et al.,2011b) basic secretagogues (see section III.G.2.d).Finally, in concordance with murine MRGPRC, thehuman PAR2-derived "SLIGKV"-peptide was shown toactivate MRGPRX2 after expression in CHO cells ata concentration of 20 mM (Liu et al., 2011).

c. MAS-related G protein–coupled receptors X3and -4. So far no study reported activation of MRGPRX3and -4 by a given ligand. Therefore, data about signalinginduced by MRGPRX3 and -4 or their biologic role arenot available at present. For MRGPRX3 it has beenshown that expression of this receptor subtype undercontrol of the b-actin promoter in rats resulted inincreased proliferation of lens fiber cells and keratino-cytes in basal and suprabasal layers of the skin (Kaishoet al., 2005). With regard to the MRGPRX4, a screeningapproach using human colorectal cancer cells revealedthat the MRGPRX4 protein is one of 15 mutational hotspots in these cancer cells (Gylfe et al., 2013). Thus,MRGPRX3 and -4 may behave as oncogenes in certaincancers.

2. Signaling Cascades and Physiologic Effects.a. Preface. MRGPRX1 and -2 function via acti-

vation of G proteins, as indicated by their ability toinduce guanosine 59-O-(3-[35S]thio)triphosphate incor-poration into plasma membrane fractions (Kamoharaet al., 2005; Burstein et al., 2006; Tatemoto et al.,2006). Both receptors further induced the release ofcalcium from internal stores via PLCb-mediated pro-duction of inositol-1,4,5-trisphosphate (Lembo et al.,

TABLE 7Pharmacology of human MAS-related G protein–coupled receptors X2

Potency of the ligand is given as the EC50 value. The lowest concentration (LC) used to elicit a significant effect when measuring the respective readout is given. Data weregenerated after recombinant MRGPRX2 expression, despite when endogenous expression is indicated by “(e)”.

Ligand EC50 LC Readout System Reference

nM

Cortistatin-14 25 calcium HEK293-Ga15 Robas et al., 200350 calcium CHO Kamohara et al., 200565 cAMP inhibition CHO Kamohara et al., 200530 GTPgS CHO Kamohara et al., 2005

2000 calcium HEK293 Burstein et al., 2006610 calcium CHO-Ga16 Shemesh et al., 2008500 calcium HEK293 Malik et al., 2009

PAMP1-12 60 calcium CHO Kamohara et al., 200540 cAMP inhibition CHO Kamohara et al., 200520 GTPgS CHO Kamohara et al., 2005

Substance P 8000 calcium HEK293 Tatemoto et al., 2006Benzimidazole derivates 400 calcium HEK293 Malik et al., 2009SLIGKVamide N.D. calcium CHO Liu et al., 2011PMX-53 100 calcium LAD2 (e), RBL-2H3, human

mast cells (e)Subramanian et al., 2011b

10 degranulationE7 1000 calcium HMC-1 (e), LAD2 (e), human

mast cells (e)Kashem et al., 2011

10 degranulationSubstance P 100 degranulation human mast cells (e) Tatemoto et al., 2006LL-37 100 calcium LAD2 (e), HMC-1, RBL-2H3,

human mast cells (e)Subramanian et al., 2011a

1000 degranulationb-Defensins 100 calcium LAD2 (e), RBL-2H3, HEK293, murine

mast cells, human mast cells (e)Subramanian et al., 2013

100 degranulation

GTPgS, guanosine 59-3-O-(thio)triphosphate; N.D., not determined.


2002; Robas et al., 2003; Breit et al., 2006; Solinskiet al., 2012). Different groups agree that activation ofGq/11 proteins is involved in this process (Lembo et al.,2002; Robas et al., 2003; Kamohara et al., 2005; Breitet al., 2006; Burstein et al., 2006). However, apart fromits Gq/11 coupling, additional coupling to PTX-sensitiveGi/o proteins was described for MRGPRX1 (Chen andIkeda, 2004; Gales et al., 2005; Burstein et al., 2006) or-2 (Kamohara et al., 2005; Subramanian et al., 2013).According to their expression pattern, MRGPRX1

and -2 may be involved in somatosensory detectorfunctions and plasticity of primary sensory neurons(see sections III.G.2.b and III.G.2.c) and in mast cellbiology (see section III.G.2.d). However, despite specificexpression, data highlighting MRGPRX2-mediatedfunctions in primary sensory neurons are currentlyelusive. Moreover, MRGPRX2 may also induce hypo-tension and slow-wave sleep (see section III.G.2.e).Figures 7 and 8 give an overview of effects induced byMRGPRX1 or MRGPRX2, respectively.

b. Somatosensory functions. To shape an organ-ism’s somatosensory performance, a GPCR needs totransfer stimuli detected in the skin into enhancedor reduced primary sensory neuron activity. Indeed,MRGPRX1 have been shown to modulate the activityof several distinct ion channels, thereby shapingneuronal activity. After heterologous expression in ratDRG, hippocampal, and superior cervical ganglionneurons, MRGPRX1 inhibited voltage-gated calciumchannels, at least partly via PTX-sensitive Gi/o proteins,leading to decreased synaptic transmission (Chen andIkeda, 2004). On the other hand, MRGPRX1 alsoinhibited M-type potassium channels via Gq/11 proteinsin the same cells, thereby relieving the neuron froma background potassium conductance that limits excit-ability (Chen and Ikeda, 2004). In line with this notion,human MRGPRX1 induced action potentials afterheterologous expression in murine DRG neurons (Liuet al., 2009). The pain-enhancing TRPV1 might beinvolved in MRGPRX1-induced sensory neuron excita-tion, inasmuch as MRGPRX1 were shown to sensitizeand directly activate this pain sensor (Solinski et al.,2012). Interestingly, MRGPRX1 engaged TRPV1 via twodistinct signaling pathways: sensitization took place viaPKC-mediated phosphorylation of serine residues 502and 800 of the channel, whereas direct TRPV1 activa-tion relied on diacylglycerol binding to channel regionsencompassing tyrosine 511 and phosphatidylinositol-3,4-bisphosphate (PIP2) degradation. This multifacetedmodulation of TRPV1 activity is unique among theGPCR superfamily (Chuang et al., 2001; Prescott andJulius, 2003; Woo et al., 2008; Kim et al., 2009; Solinskiet al., 2012) and, thus, points to an important role of theTRPV1-MRGPRX1 regulatory axis in somatosensation.The fact that MRGPRX1 were shown to either

dampen or increase neuronal activity raises thequestion about the mechanisms responsible for these

opposing effects. In this context it should be mentionedthat distinct effects of MRGPRX1 on neuronal activitywere observed in different cell types and that in-hibition of neuronal activity apparently depends on Gi/o

and increased activity on Gq/11 signaling. Because Gprotein coupling of a given GPCR might differ indistinct cell types (Hermans, 2003; Kukkonen, 2004),distinct G protein coupling of the MRGPRX1 in dif-ferent cells might be the most straightforward expla-nation for its opposing effects on neuronal activity.Because of the lack of endogenous model systems,effects of MRGPRX1 on neuronal activity have so faronly been analyzed after overexpression of theMRGPRX1 protein also affecting its G protein couplingprofile (Kenakin, 1997, 2006). Thus, at this point it isnot clear whether MRGPRX1 can act analgetically bydecreasing neuronal activity under certain conditionsand algetically by increasing neuronal activity indifferent circumstances. Alternatively, the receptorsmay even induce both effects at the same time, suchthat the net effect in a given cell would depend onwhich of the two signaling pathways dominates theother. It will be an important task for the future toanalyze whether and how MRGPRX1 regulate theactivity of primary sensory neurons of primates and,thus, nociception in vivo. These experiments are dif-ficult to perform, because functional primary sensoryneurons from humans are not available and rodents donot harbor MRGPRX1-encoding genes. However, ro-dent MRGPRC and human MRGPRX1 both bindBAM8–22, which has been shown to induce pain-enhancing, analgesic, and pruritogenic effects in miceor rats (see section III.D.2). Interestingly, in a seminalstudy in which BAM8–22 was applied to the skin ofhealthy human volunteers using cowhage spicules,acute itching accompanied by nociceptive sensations(pricking, stinging, and burning) was reported (Sikandet al., 2011), thus showing that BAM8–22 actssimilarly in rodents and humans and that MRGPRX1function as somatosensory receptors with nociceptiveand pruriceptive actions. However, because applicationby cowhage spicules is more likely to elicit itchcompared with pain (Sikand et al., 2009), more workis needed to determine unequivocally if MRGPRX1 ex-ert acute functions in pruriceptive and/or nociceptiveprimary sensory neurons in vivo.

c. Primary sensory neuron plasticity.Small-diameter primary sensory neurons respond tovarious acute or chronic stimuli, thereby showing highplastic changes in connectivity, morphology, or re-ceptor expression pattern [see Woolf and Ma (2007) forfurther reading]. Interestingly, pathways that wereimplicated in adaptive responses of primary sensoryneurons are well established players of proliferativesignaling in dividing cells. One example is the extra-cellular signal-regulated kinase (ERK)-1/2 pathwaythat was implicated in adaptive responses of the

588 Solinski et al.

nociceptive system to chronic inflammation (Ko et al.,2005; Wang et al., 2006a; Utreras et al., 2009; Su et al.,2010; Romero et al., 2012).Several indications point to the engagement of

nuclear signaling pathways by MRGPRX, leading totranscriptional modulation. Overexpression of MRGPRX1or -4 increased proliferation of 3T3 fibroblasts,highlighting considerable constitutive activity of theseMRGPRX after recombinant expression (Burstein et al.,2006). We recently found that after expression inprimary sensory neuron–derived F11 cells, MRGPRX1

induced activation of ERK-1/2 and serum responseelement–dependent transcription as well as ERK-1/2–dependent expression of the immediate early genesc-Fos and early growth response protein 1 (Solinskiet al., 2013). Because of the published pathophysiologicimportance of this pathway in rodents and humans (Koet al., 2005; Wang et al., 2006a; Utreras et al., 2009; Suet al., 2010; Romero et al., 2012), MRGPRX1 mayrepresent interesting targets in chronic inflammatorypain if this pathway is also activated by MRGPRX1 invivo as well.

Fig. 7. Schematic presentation of signaling cascades, downstream targets, and physiologic effects proposed for human MRGPRX1 in primary sensoryDRG/trigeminal ganglia (TG) neurons and mast cells. (A) Application of BAM8–22 into human skin elicits histamine-independent pruriceptive andnociceptive sensations. Recombinant human MRGPRX1 inhibit voltage-dependent calcium channels via Gi/o proteins and M-current-conductingKCNQ2/3 channels via Gq/11 proteins. TRPV1 activity is enhanced by recombinant MRGPRX1 through PKC, diacylglycerol (DAG), and PIP2. (B) Effectsof sustained MRGPRX1 activation in recombinant cell systems include ERK-1/2 and NFAT activation, which induces c-Fos, early growth responseprotein 1 (EGR-1), and CCR2 expression. In a human mast cell line endogenously expressing MRGPRX1, BAM8–22 enhanced the release of the CCR2agonist chemokine ligand 2 (CCL2).


It is interesting to mention that MRGPRX1 not onlysignal to the nucleus via classic proliferative pathwaysbut also induced transcriptional alterations in primarysensory neurons via the calcineurin-dependent tran-scription factor nuclear factor of activated T cells(NFAT) (Solinski et al., 2013). Activation of NFATwas already implicated in several plastic changes ofprimary sensory neurons, for instance axonal growth(Graef et al., 2003; Nguyen and Di Giovanni, 2008) andactivity-dependent transcription (Groth et al., 2007;Jackson et al., 2007; Jung and Miller, 2008). Moreover,

BAM8–22-induced NFAT activation led to increasedexpression of chemokine receptors 2 (CCR2) in primarysensory neurons after recombinant MRGPRX1 expres-sion (Solinski et al., 2013). CCR2 were consistentlyfound to be upregulated in primary sensory neuronsunder neuropathic conditions and to cause pain chron-ification (Abbadie et al., 2003; Sun et al., 2006; Bhangooet al., 2007, 2009; White et al., 2007; Jung et al., 2009;Fu et al., 2010; Serrano et al., 2010; Wang et al., 2010).Thus, MRGPRX1 may also be interesting targets inchronic neuropathic pain.

Fig. 8. Schematic presentation of signaling cascades, down-stream targets, and physiologic effects proposed for human MRGPRX2 in mast cells andpotential effects in chromaffin cells and hippocampal neurons. (A) Several agonists have been implicated in mast cell degranulation via activation ofMRGPRX2 including the C5a receptor antagonist PMX-53, the C3a receptor super-agonist E7, substance P (SP), the cathelicidin LL-37, andb-defensins. PTX-sensitive Gi/o proteins via PKC and PLCb-activating G proteins, presumably Gq/11, elicit IgE-independent degranulation of connectivetissue type mast cells. (B) MRGPRX2 have been proposed to cause hypotensive and slow-wave sleep inducing effects. Hypotension might be mediatedvia adrenomedullary PAMP1–20-induced inhibition of (nor)epinephrine release. Sleep phase modulation is conveyed by cortistatin-14 (COR-14) andpotentially engages MRGPRX2-induced reduction of hippocampus excitability.

590 Solinski et al.

d. Mast cell biology. Mast cells are long-livedmononuclear cells that reside in tissues near externalsurfaces, e.g., in skin or mucosa, and thereby areamong the first cells of the immune system to respondto pathogens or allergens (Galli et al., 2011). Mast cellsfulfill their tasks in innate and adaptive immuneresponses by secretion of a plethora of mediators(degranulation), invaluable for host defense (Galliet al., 2011). Mast cell degranulation is caused byIgE-dependent and -independent processes (Metcalfeet al., 1997; Ferry et al., 2002). A very heterogeneousgroup of agents, either released endogenously byimmune cells, neurons, and epithelial cells, or appliedexogenously, mediates IgE-independent mast cell de-granulation and is referred to as secretagogues (Sharmaet al., 2002). However, underlying signaling pathwaysare diverse and a matter of debate.Endogenously expressed MRGPRX2 were proposed

as receptors for different secretagogues in culturedhuman primary mast cells and in the differentiatedhuman mast cell line LAD2 (Tatemoto et al., 2006;Kashem et al., 2011; Subramanian et al., 2011a,b,2013). Early during infection, a first line of mast cellactivation is thought to arise from complement-mediated anaphylatoxin production (e.g., C3a or C5a)(Kashem et al., 2011; Subramanian et al., 2011b).Although MRGPRX2 are not involved in mast cellactivation provoked by endogenous anaphylatoxins,mast cell degranulation induced by a synthetic C5areceptor antagonist (PMX-53) or a C3a receptor super-agonist (E7) required MRGPRX2 expression (Kashemet al., 2011; Subramanian et al., 2011b). Later onduring infection, epithelial cells can secrete b-defensinsor cathelicidins, both inducing mast cell degranulationvia MRGPRX2 (Subramanian et al., 2011a, 2013).Hence, MRGPRX2 can integrate paracrine input fromvarious cell types by detecting alterations of the localmilieu and inducing mast cell degranulation.Degranulation of mast cells after MRGPRX2 activa-

tion depends on PTX-sensitive G proteins, irrespectiveof the MRGPRX2 ligand under investigation (Tatemotoet al., 2006; Kashem et al., 2011; Subramanian et al.,2011a,b, 2013). However, MRGPRX2 activation byLL-37 or human b-defensin-3 involves two differentsignaling cascades that act synergistically on degran-ulation, but only one of which depends on Gi/o-inducedPKC activation (Subramanian et al., 2011a, 2013). Thesecond PTX-insensitive pathway involved calcium in-flux and release, as suggested by the inhibitory actionsof the calcium release–activated calcium channelblocker, lathanium (La3+), and the inositol 1,4,5-trisphosphate receptor and canonical TRP blocker,2-aminoethoxydiphenyl borate (Subramanian et al.,2011a, 2013).Because some basic secretagogues can be secreted by

primary sensory neurons, MRGPRX2 may also en-hance neuron-to-mast cell signaling, thereby shaping

neuroimmune interactions. In congruence with thisconcept, MRGPRX1 were implicated in the secretion ofchemokine ligand 2 from LAD2 mast cells afterstimulation with BAM8–22 (Solinski et al., 2013).Given that MRGPRX1 induced the expression of thechemokine ligand 2 receptor CCR2 in primary sensoryneurons (Solinski et al., 2013), it is appealing tohypothesize that MRGPRX1 could additionally en-hance mast-cell-to-neuron signaling. Because mastcells contribute to chronic pain (Zuo et al., 2003),inhibition of such a paracrine signaling circuitwould be an interesting approach for future analgesictherapy.

e. Putative role of MAS-related G protein–coupledreceptors X2 in sleep and blood pressure regulation.PAMP1-20 is a potent hypotensive peptide, functioningmainly via inhibition of norepinephrine and epineph-rine release from sympathetic neurons or adrenalchromaffin cells, respectively (Shimosawa et al., 1995;Kobayashi et al., 2001). On the protein level, PAMP1-20 is mainly distributed in adrenal medulla and atrium(Washimine et al., 1994). Because MRGPRX2 proteinshave been detected in adrenal chromaffin cells andbecause PAMP1-20 activated MRGPRX2 in heterolo-gous expression systems, Kamohara and coworkers(2005) proposed that epinephrine-dependent hypoten-sive functions of PAMP1-20 might be mediated byMRGPRX2 signaling.

Cortistatin-14 activates all five somatostatin receptor(SSTR1-5) subtypes, but some effects of cortistatin-14appear to be clearly independent of these receptors andare not caused by somatostatin (de Lecea, 2008). Amongthese effects is the induction of slow-wave sleep thatmight be partly mediated by inhibition of hippocampalneurons. Because MRGPRX2 are expressed in hippo-campal neurons of layer CA2-4 and because MRGPRX2responded specifically to cortistatin-14, Robas and co-workers (2003) put forward the proposal that MRGPRX2may be involved in cortistatin-14–mediated slow-wavesleep induction.

IV. Agonist-Promoted Internalization andDesensitization of MAS-RelatedG Protein–Coupled Receptors

GPCR signaling is commonly regulated by agonist-induced receptor phosphorylation that leads to uncou-pling from cognate G proteins (desensitization) andsubsequent receptor internalization (endocytosis), mostlyvia association with b-arrestins (Ferguson, 2001). Inaddition, b-arrestins have been shown to induceG protein-independent signaling pathways, furtherincreasing the repertoire of GPCR-mediated signaling(Shenoy and Lefkowitz, 2003). Only a few GPCR havebeen shown to resist agonist-promoted processes thatnegatively regulate receptor activity, including k-OR,


b3-adrenoceptors, and SSTR4 (Nantel et al., 1993; Chuet al., 1997; Csaba and Dournaud, 2001).Detailed analysis of the subcellular distribution of

human MRGPRD fused to green fluorescent protein(GFP) in CHO cells after a 30-minute period ofstimulation with saturating concentrations of b-alanine(Shinohara et al., 2004) revealed translocation of thereceptor protein from the plasma membrane to punctateintracellular vesicles, indicative of ligand-inducedinternalization. Dose-dependent internalization ofc-myc–tagged rat MRGPRD by b-alanine after recombi-nant expression in HEK293 cells was also identified,using either a qualitative immunofluorescence or aquantitative enzyme-linked immunosorbent assay ap-proach (Milasta et al., 2006). Thus, MRGPRD, the onlyMRGPR conserved in rodents and primates and as-signed to an agonist, apparently belongs to the group ofendocytosis-prone GPCR.After recombinant expression of murine MRGPRA1

or MRGPRA4 in HEK293 cells, Dong and coworkers(2001) observed a prominent decline in ligand-inducedcalcium signals after repetitive short term stimulationwith FLRFamide or NPAF, indicating agonist-promoted desensitization. Desensitization was revers-ible by ligand wash-out over ;20 minutes and mostlikely occurred because of phosphorylation and endo-cytosis of the respective receptor protein. In fact, inHEK293 cells murine MRGPRA1 fused to GFP wasinternalized to punctate intracellular vesicles after 30minutes of agonist stimulation (Han et al., 2002).Likewise, MRGPRC-GFP fusion proteins also in-

ternalized after 30 minutes of stimulation by saturat-ing concentrations of g2-MSH in HEK293 cells (Hanet al., 2002). This work was corroborated by the findingthat 1) other full agonists of murine MRGPRC in-cluding BAM8–22 are able to induce the same extent ofMRGPRC endocytosis; 2) endocytosis is not confined toHEK293 cells, but also detectable in several DRGneuron-derived cell lines; 3) b-arrestins are indispens-able for proper ligand-induced MRGPRC endocytosis inCos7 cells (Solinski et al., 2010). Notably, rat MRGPRCwas also found to be sensitive to BAM8–22- org2-MSH–induced endocytosis in the same cell models(Solinski et al., 2010). In sharp contrast to rodentBAM8–22-sensitive MRGPRC, human MRGPRX1resisted BAM8–22-induced desensitization and endo-cytosis after recombinant expression in differentcellular systems, including primary sensory neuron-derived F11 and ND-C cells (Solinski et al., 2010).Thus, identical ligands regulateMRGPRC andMRGPRX1endocytosis in diametrically opposed ways. Moreover,similar to MRGPRX1, ligand-induced phosphorylationand internalization of the MRGPRX2 was not detectable(Subramanian et al., 2011a). Thus, absence of agonist-promoted receptor desensitization appears as a commonfeature among MRGPRX, defining the MRGPRX sub-family as the first among all GPCR families to harbor

more than one endocytosis resistant member. WhetherMRGPRX3 and -4 also resist agonist-mediated de-sensitization still needs to be confirmed in the future.Notably, serine residues in the C-terminal tail of a givenGPCR are profoundly involved in agonist-induced re-ceptor regulation (Ferguson, 2001) and, as alreadymentioned, C-terminal tails of MRGPRX developedunder strong positive selection pressure during evo-lution. It is tempting to speculate that considerablephysiologic benefits have been earned by enrichingthe lack of receptor desensitization and endocytosisamong MRGPRX. A summary of the available litera-ture analyzing ligand-induced regulation of MRGPRis given in Table 8.

V. Functional Interactions of MAS-RelatedG Protein–Coupled Receptors with Other

Receptor Families

A. Heteromultimerization among MAS-RelatedG Protein–Coupled Receptors and Receptor Subtypesof Other GPCR Families

Heteromultimerization among GPCR has been pro-posed to alter ligand binding, G protein coupling,protein trafficking, and agonist-promoted desensitiza-tion of the receptors involved (Bulenger et al., 2005).Thus, direct protein-protein interactions due to re-ceptor multimerization represent an important mech-anism by which distinct hormone systems interact witheach other (Jordan et al., 2001; Torvinen et al., 2005;Parenty et al., 2008). Because MRGPRX1 and ORshare a common ligand, the BAM1–22 peptide, arecoexpressed in primary sensory neurons, and bothaffect pain modulation, functional interactions be-tween MRGPRX1 and the d-OR (DOP) due to multi-merization have been analyzed. Bioluminescenceresonance energy transfer experiments revealed thatMRGPRX1 interacted with human DOP in livingHEK293 cells stably overexpressing each receptor(Breit et al., 2006). Individual activation of eitherDOP (with Leu-Enkephalin) or MRGPRX1 (withBAM8–22) allowed both receptors to modulate theirrespective signaling pathways. In contrast, the DOP/MRGPRX1 bivalent agonist BAM1–22, which activatedeach receptor expressed individually, fully activatedthe MRGPRX1 but did not promote DOP-mediatedsignaling within the heteromultimer. Similarly, concom-itant activation of the DOP/MRGPRX1 heteromultimerby selective DOP and MRGPRX1 agonists (Leu-Enkephalin and BAM8–22) promoted MRGPRX1 butnot DOP signaling. Furthermore, coexpression of theendocytosis resistant MRGPRX1 inhibited agonist-promoted internalization of the endocytosis-proneDOP. Overall, these data suggest that MRGPRX1signaling within the DOP/MRGPRX1 heteromultimeracts in a dominant-negative fashion on DOP signaling

592 Solinski et al.

and thus, could affect the fine tuning of pain sensationregulated by DOP.MRGPR have not only been shown to interact with

members of other GPCR families, but also among eachother. The rat MRGPRD and -E subtypes form multi-meric complexes when expressed in HEK293 cells orperipheral nociceptive neurons, as previously indicatedby coimmunoprecipitation and time-resolved fluores-cence resonance energy transfer (Milasta et al., 2006).These interactions increased the potency of b-alanineto phosphorylate ERK-1/2, maintained the capacity ofb-alanine to elevate intracellular calcium concentra-tions, and inhibited b-alanine–induced internalizationof MRGPRD. Given the large number of MRGPRfamily members and assuming that heteromultimericinteractions also exist between other subtypes, onewould predict an immense variety of putative bindingsites for MRGPR-selective ligands, expanding theimpact of this receptor family on many physiologicpathways even further.

B. Inhibition of Tolerance to Morphine

As mentioned above, BAM-sensitive, humanMRGPRX1 functionally interact with the opioid systembased on receptor multimerization. Interestingly,multimerization-independent interactions between

BAM-sensitive MRGPRC and opioid receptors havealso been described in vivo. A major problem of chronicpain treatment with morphine is the development oftolerance characterized by declining analgesic effectsafter repeated dosing. The mechanisms that cause theunderlying adaptation processes are manifold andnot completely understood [for further reading seeWilliams et al. (2013)]. In rats, tolerance to morphine canbe generated by its daily application over six to sevendays. Interestingly, after intrathecal application ofMRGPRC-specific agonists in rats, morphine regained;50% of its analgesic actions in formerly morphine-tolerant animals on the consecutive day (Jiang et al.,2006; Cai et al., 2007b; Chen et al., 2010b). MRGPRCagonists were not only able to restore analgesic efficacyafter tolerance to morphine had already been estab-lished, intrathecally applied BAM8–22 or Tyr6-g2-MSH6–12 on every second day during induction ofmorphine tolerance dose-dependently inhibited the de-velopment of morphine tolerance (Cai et al., 2007b; Chenet al., 2010b). Compensatory upregulation of pain-enhancing molecular players in the DRG and spinalcord including PKCg-dependent upregulation of nNOSor CGRP is involved in the development of morphinetolerance (Menard et al., 1996; Kolesnikov et al., 1997;Granados-Soto et al., 2000). Because MRGPRC agonists

TABLE 8Ligand-promoted desensitization and endocytosis of MAS-related G protein–coupled receptors

MRGPR that have been analyzed for their ligand-induced regulation are listed in conjunction with the respective ligand and expression systemused.

Receptor Ligand Desensitization Endocytosis System Reference

mMRGPRA1 FLRFamide YES YES HEK293 Dong et al., 2001Han et al., 2002

mMRGPRA4 NPAF YES N.D. HEK293 Dong et al., 2001mMRGPRC g2-MSH YES YES HEK293, F11, ND-C, Cos7 Han et al., 2002

BAM8–22 Solinski et al., 2010Dyn-14

rMRGPRC g2-MSH BAM8–22 N.D. YES HEK293, F11, ND-C Solinski et al., 2010hMRGPRD b-Alanine N.D. YES CHO Shinohara et al., 2004rMRGPRD b-Alanine N.D. YES HEK293 Milasta et al., 2006hMRGPRX1 BAM8–-22 NO NO HEK293, F11, ND-C, Cos7 Solinski et al., 2010hMRGPRX2 Cortistatin-14 NO NO HEK293 Subramanian et al., 2011a

LL-37 LAD2, HMC-1

h, human; m, mouse; N.D., not determined; r, rat.

TABLE 9Ligand sharing between MAS-related G protein–coupled receptors and members of other GPCR families

Ligands that have been implicated to bind and activate both, MRGPR subtypes and other GPCR, are given.

Ligand MRGPR Reference Other GPCR Reference

BAM1–22 r/mMRGPRC hMRGPRX1 Lembo et al., 2002 DOP, MOP, KOP Quirion and Weiss,1983Han et al., 2002Grazzini et al., 2004

NPFF mMRGPRC Han et al., 2002 NPFF1R, NPFF2R Mollereau et al., 2002g2-MSH r/mMRGPRC Han et al., 2002 MC3R, MC4R Roselli-Rehfuss et al., 1993

Grazzini et al., 2004 Oosterom et al., 1999GABA h/rMRGPRD Shinohara et al., 2004 GABAB Bowery et al., 1980

Kaupmann et al., 1997Angiotensin-(1–7) hMRGPRD Gembardt, 2008 MAS1, AT2R Santos et al., 2003

Walters, 2005Cortistatin-14 hMRGPRX2 Robas, 2003 SSTR1-5 de Lecea et al., 1996

Fukusumi et al., 1997

h, human; KOP, k-OR; m, mouse; MC3R, melanocortin 3 receptor; r, rat.


blocked enhanced PKCg activity in the spinal cord anddiminished nNOS and CGRP immune reactivity in DRGand spinal cord slices, this signaling cascade mightinhibit morphine tolerance by MRGPRC (Chen et al.,2010b).

VI. Conclusions and Perspectives

Over the last decade, our knowledge about MRGPRhas been significantly extended: 1) the murineMRGPRA3 subtype has been identified as the itch-mediating chloroquine receptor; 2) rodent MRGPRChave also been shown to be involved in itching andMRGPRC have been made responsible for analgesicand pain-enhancing effects; 3) MRGPRD enhanceneuronal excitability and induce vasorelaxation; and4) human MRGPRX also modulate excitability ofprimary sensory neurons and gene expression in thesecells. However, because of the lack of suitable modelsystems, these data are solely based on experimentsobtained after heterologous expression of the MRGPRX1protein. In mast cells, endogenously expressing theMRGPRX1 and -2 subtype effects on degranulation andmediator release have been found. Adaptive evolu-tionary development and resistance to agonist-promoted desensitization/endocytosis further highlightthe extraordinary features of the primate-specificMRGPRX subfamily. Notably, MRGPRX can be consid-ered as valuable drug targets with low or even no off-target effects because of their restricted expression inprimary sensory neurons and mast cells.However, our knowledge about the MRGPR family is

still superficial and central questions need to beaddressed: 1) to what extent do MRGPR serve asmarkers for primary sensory neurons that executespecific functions; 2) what is the difference betweendistinct rodent species in this regard; 3) what is thephysiologic role of distinct MRGPR in these specializedneurons? Consider, in particular, that some subtypes ofthe MRGPRA and -X subfamilies are not yet analyzed,mainly because not a single ligand for these receptorsis available. Similarly, progress in the work withMRGPRE to -H receptors is significantly hindered bythe lack of agonists. Moreover, it will be essential todefine the signaling events responsible for the effects ofMRGPRC agonists on nociception. Interestingly, hu-man MRGPRX1 have been shown to enhance or inhibitsignaling pathways that supposedly modify neuronalactivity, suggesting that MRGPRX1 might also exertproalgetic and analgesic effects in humans. In thiscontext it will be important to analyze to which extentdata obtained from BAM8–22-sensitive rodent MRGPRCcan be extended to BAM8–22-sensitive humanMRGPRX1.Convincing evidence that rodents can in fact be usedas a native model systems for human BAM8–22-sensitive MRGPRX1 would propel the "deorphaniza-tion" process of these receptors forward and would

simplify the development of drugs tested in establishedanimal models but intended to target humanMRGPRX1. In parallel, it would be of interest todevelop primate- or even human-based DRG-derivedcell models that endogenously express MRGPRX1.Considering mast cells, a step forward has been madeat this point, because the human mast cell line LAD2has been shown to endogenously express MRGPRX1and -2. Furthermore, the problem of ligand-sharingbetween MRGPR and subtypes of other GPCR families,observed for many endogenous ligands (see Table 9), isa major drawback in the field. Thus, screening effortsyielding MRGPR subtype–specific agonists and antag-onists would significantly help attain a better un-derstanding of the physiologic pathways engaged byMRGPR in vivo.

Acknowledgments

The authors thank Dr. Alexander Dietrich (Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, Munich, Germany) for critical reading of themanuscript.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Solinski,Gudermann, Breit.

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