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.