Introduction
The Mas-related gene D (MRGPRD) receptor is a seven transmembrane
G-protein coupled receptor (GPCR) belonging to the mas-related gene
family (Dong et al ., 2001), also known as the sensory neuron
specific receptor family (Lembo et al ., 2002), comprised of
approximately 50 GPCRs related to the Angiotensin-(1-7) receptor Mas 1.
There are only four functional MRGPR genes in humans, compared with
three large families and six single copy genes in mice, but MRGPRD has
clearly defined orthologs in the mouse and rat (Ajit et al .,
2010). MRGPRD receptors are predominantly expressed in small diameter
Griffonia simplicifolia lectin IB4+ nociceptive
sensory neurons in the Dorsal Root Ganglion (DRG) (Dong et al .,
2001) and neurons located in the stratum granulosum of skin (Liuet al ., 2008; Rau et al ., 2009). These small diameter
IB4+ neurons are a subpopulation of neurons that
include cutaneous nociceptors which are associated with the perception
of painful stimuli and have been implicated with conditions such as
neuropathic pain (Dong et al ., 2001; Zykla et al ., 2004;
Rau et al. , 2009; Wang et al. , 2019). MRGPRD knockout mice
have impaired ability to sense mechanical and thermal stimuli (Rauet al. , 2009), whereas ablating MRGPRD containing neurones in an
adult mouse model similarly impacted the ability to sense mechanical
pain without affecting heat and cold transduction (Cavanagh et
al. , 2009; Rau et al. , 2009). In addition to a role in pain
sensation, MRGPRD has also been implicated in the perception of itch,
the transduction of pruritogenic stimuli in animal models, and the
sensation of itch in man following intradermal injection with the amino
acid β-alanine (Liu et al., 2009; Liu et al ., 2012). The
ability of MRGPRD to enhance neuronal activity is thought to be a
consequence of inhibition of the ‘M’-current, carried by KCNQ2/3
potassium channels, which is involved in pain perception (Crozieret al. , 2007). This very specific expression and response pattern
in nociceptive-related afferents and the availability of animal models
makes MRGPRD an interesting target of study in drug discovery. Current
treatment options for neuropathic pain, which include opioids, have
significant limitations in their therapeutic use including CNS and other
side effects (Brooks and Kessler, 2017). Pharmacological agents acting
against MRGPRD may therefore provide an alternative as they would likely
have fewer side effects due to its restricted expression in sensory
neurons (Zhang et al. , 2005).
MRGPRD receptors have been shown to couple to both Gαi/oand Gαq proteins (Shinohara et al., 2004; Crozieret al ., 2007; Ajit et al ., 2010; Uno et al., 2012).
Ajit and colleagues (2010) reported a complete loss in MRGPRD
signalling, following activation with the agonist β-alanine, after
inhibition of PLC and a reduction of approximately 50% when blocking
Gαi/o, indicating that MRGPRD signalling was
predominantly coupled via Gαq G proteins. In contrast
Crozier et al., (2007), reported the inhibition of M currents,
mediated by KCNQ2/3 potassium channels in DRG neurons via the activation
of endogenous MRGPRD receptors, were inhibited by exposure to the
Gαi/o G protein
inhibitor pertussis toxin. Interestingly, in this same study, responses
via recombinant MRGPRD receptors expressed in CHO cells were only
partially inhibited by PTX pre-treatment. More recently MRGPRD has been
implicated as a second receptor for Angiotensin-(1-7) (alamandine), the
effects of which are reported to be mediated via Gαs,
involving adenylyl cyclase, cAMP and protein kinase A (PKA) (Lautner,et al. , 2013; Tetzner et al ., 2016). This indicates
plasticity in the coupling of MRGPRD receptors and these differences are
likely to be dependent on cell/tissue type, assay technique or the
ligand used. It will therefore be important to carefully characterise
the signalling of MRGPRD when working within and comparing between
different cell and tissue systems.
β-alanine was first described as a MRGPRD agonist by Shinohara and
colleagues (2003), who showed that it raised intracellular
Ca2+ levels and supressed cAMP production in CHO cells
expressing mouse, rat or human MRGPRD receptors;
[35S]GTPγS binding was also investigated, with
specific binding being observed in whole cell isolates. β-alanine is a
small amino acid that is structurally related to both γ-aminobutyric
acid (GABA) and glycine and has been shown to bind weakly to GABA and
Glycine receptors (Shinohara et al ., 2003). Both GABA and Glycine
also activate MRGPRD receptors transfected into CHO cells but with lower
potency than β-alanine (β-alanine >> GABA
> Glycine; Ajit et al ., 2010). The heptapeptide
alamandine also appears to be an agonist for MRGPRD; as described by
Lautner et al (2013), who observed that both β-alanine and the
putative MRGPRD antagonist PD123,319, competed for fluorescently
labelled alamandine binding when the receptor was expressed in CHO
cells. Several synthetic small molecule antagonists have also been
described; these include thioridazine hydrochloride (Ajit et al.,2010), PD123,319 (Lautner et al., 2013) and MU-6840 (Uno et
al., 2012). Thioridazine hydrochloride and the related molecules
chlorpromazine and R(-)-propylnorapomorphine are dopaminergic
antagonists also found to inhibit β-alanine activation of MRGPRD (Ajitet al., 2010). PD123,319, an antagonist of the
AT2 angiotensin receptor, was shown to compete with the
binding of alamandine to MRGPRD transfected CHO cells (Lautner et
al., 2013). However, PD123,319 also abolished the alamandine response
in AT2 receptor knockout mice, indicating an interaction
with multiple targets. MU-6840 was identified as a MRGPRD antagonist in
recombinant CHO cells by Uno et al (2012) using a high-throughput
Ca2+ flux Fluorometric Imaging Plate Reader (FLIPR)
assay in addition to having effects in [35S]GTPγS
binding, inositol phosphate accumulation and spheroid proliferation
assays.
High throughput screens of MRGPRD have been reported, principally using
FLIPR-based Ca2+ flux assays utilising recombinant
cell lines (Zhang et al., 2007; Ajit et al., 2010; Unoet al., 2012). Most groups have screened for antagonists using
β-alanine as the reference agonist whilst Ajit et al (2010)
simultaneously screened for agonists and antagonists. Many HTS assays
have the potential to generate false positives, for a
Ca2+ flux assay this includes the discovery of
auto-fluorescent compounds, calcium ionophores and chemicals that
permeabilise the cell membrane (Zhang et al., 2007). Many of
these false positives can be discounted by testing them against a
non-transfected cell line and/or in orthogonal, lower throughput assays.
For MRGPRD these orthogonal assays include
[35S]GTPγS binding (Uno et al., 2012) and
cAMP assays (Zhang et al., 2007) the latter of which have been
used because MRGPRD also couples through Gαi/o proteins.
However, these assays, like most, have limitations; radioactive tracers
have safety implications, and the associated assays are relatively low
throughput, whilst assays to monitor the inhibition of cAMP accumulation
require the addition of a stimulant such as forskolin to induce a
response, which is then inhibited via receptor activation when coupling
through Gαi/o proteins. This increased complexity
increases the chances of error and potential sources of interference
from screening compounds. An orthogonal assay technology that removes
this requirement for complexity and the use of downstream labels of GPCR
activation is dynamic mass redistribution (DMR), also known as
Corning® Epic® (Fang et al.,2006).
DMR uses wave-guide resonant grating to measure changes in the
wavelength of refracted light, as a consequence of cellular mass
changes, within 120-200nm of a gold sensor surface measured in pm (Liet al., 2012; Jiang and Eichelberger, 2015). This is of use
because the activation of specific cellular pathways, particularly those
mediated via GPCRs, results in rearrangement and redistribution of the
intracellular architecture, including receptor dimerization,
internalisation, cytoskeletal rearrangements and changes in cell
adhesion and shape (Coffman et al., 2011). This technology has
not previously been reported for studying MRGPRD receptor pharmacology
and the work described herein assesses its suitability in characterising
agonists and antagonists in comparison with the more traditional
FLIPR-based Ca2+ flux assay.