Discussion
To our knowledge this is the first reported use of a label free
technology to characterise the pharmacology of recombinantly expressed
MRGPRD receptors. DMR is now a well established technique for studying
GPCR function with examples including Muscarinic M1 (Peters et
al., 2010), Muscarinic M2 (Peters et al., 2010, Lee et
al., 2008), Muscarinic M3 (Dodgson et al., 2009), Dopamine D3
(Lee et al., 2008), corticotropin-releasing factor (CRF1) (Peterset al., 2010) and formyl peptide 1 (Christensen et al.,2017) receptors. GPCRs can couple through and activate multiple
signaling pathways, which can be ligand or cell specific and may result
in differences in pharmacological profile when using different assay
formats, ligands and cell types. Biophysical technologies such as DMR
provide an opportunity to measure a phenotypic, cellular, response in
real time in the absence of labels, which can be prone to compound
interference. Scott and Peters (2010), compared both cellular impedance
(cellkey) and optically based (BIND and DMR) label free systems and
noted that all three systems produced consistant responses that were
dependent on the type of G-protein coupling of the receptor
investigated. In general, GPCRs coupled via Gi pathways
produced an increase in response, stimulation of Gqcaused a transient decrease followed by a larger increase, while
Gs activation produced a decrease in the response. They
also found that with optical biosensors both Gi and
Gq responses were rapid in onset and peaked within
around 5 minutes of agonist addition before declining, whilst
Gs-coupled responses were slower and more sustained.
Here we observed that the putative MRGPRD agonist, β-alanine produced a
dose-dependent transient increase in the DMR response with a peak at
approximately 4 minutes before declining, which is consistent with the
observations of both Scott and Peters (2010) and Lee et al.(2008) for Gi-coupled receptor responses. The lack of
response to β-alanine in untransfected control CHO cell lines confirms
the specificity of the activation of MRGPRD receptors. In addition, we
wanted to use an orthogonal method for confirming the activity observed
in the DMR assay and so monitored intracelullar Ca2+flux using a FLIPR, a technique that has previously been used to study
many GPCRs including MRGPRD receptors (Ajit et al., 2010; Zhanget al., 2007). We observed a rightward shift in
pEC50 of approximately one order of magnitude when
comparing DMR with FLIPR MRGPRD responses for all three agonists tested.
One explanation for this discprenency may be differing G-protein
coupling between the two assays, in addition to differences in the
kinetics of the two readouts. MRGPRD receptors have been reported to be
able to couple via both Gi and Gq G
protiens (Shinohara et al., 2004; Crozier et al., 2007;
Ajit et al., 2010; Uno et al., 2012), and we found that
the Gq inhibitor, YM-254890 abolished the response to
β-alanine in Ca2+ assays whereas incubating with the
Gi inhibitor PTX only resulted in an approximate 50%
loss in response, corresponding with the findings of Ajit el al ,
(2010). However, in the DMR assay, YM-254890 caused only minimal
inhibition of the agonist response, with the most marked reduction in
response due to PTX. These data indicate that in the DMR assay and, by
extension, the gross cellular re-arrangements of mass that take place in
response to MRGPRD receptor activation, were principally coupled via
activation of Gi while in contrast the increase in
intracellular Ca2+ was primarily via
Gq. This difference in signalling is a likely
explanation for the shift in agonist potencies observed between the two
techniques and we speculate that MRGPRD when interacting with either
Gi or Gq G proteins may adopt different
conformers that alter the affinity of β-alanine. Dodgson et al.,(2009) compared DMR and FLIPR assays when investigating the muscarinic
M3 receptor and observed similar shifts in agonist potency between the
techniques leading them to suggest that the signal coupling, when
measured proximal to the receptor, tends to be amplified leading to an
increase in apparent agonist potency compared to DMR which measures a
global change in cellular phenotype. They also comment on the time
period being measured; in the FLIPR, responses are recorded within the
first minute following activation, while in the DMR assay the peak
measurement is taken after 4 minutes. Lee et al., (2014) also
found discrepancies in the potency of antagonists of the urotensin
receptor, which were less potent in the DMR assay compared to
Ca2+ mobilisation.
The MRGPRD agonists GABA and glycine both produced a response with a
similar DMR profile to β-alanine, but with reduced potency, that was
consistent across both assay formats and consistent with the rank order
of agonist potency described in the literature (Ajit et al.,2010). The differential coupling pathways for GABA and glycine in the
two assays were also broadly consistent with those observed with
β-alanine, although the weak DMR response to glycine was not fully
inhibited by PTX while the β-alanine and GABA responses were not
entirely abolished by co-administration of the Gi and
Gq inhibitors, which may suggest the presence of
additional signalling pathways.
Thioridazine hydrochloride, described by Ajit et al., (2010) as
an MRGPRD antagonist, caused a response in DMR which was unaffected by
the addition of PTX and which was also observed in the control cell
line, indicating that its activity is not related to MRGPRD in our assay
system; additionally, it only inhibited the β-alanine induced
Ca2+-response in the FLIPR assay at the highest
concentrations tested. We also show that thioridazine hydrochloride is
cytotoxic at these higher concentrations (Fig 5E), bringing into
question the specificity of this compound as an MRGPRD antagonist.
Another putative MRGPRD inhibitor, PD123,319, first described as an
AT2R angiotensin receptor antagonist and shown to
inhibit the agonist effects of alamandine in CHO cells expressing MRGPRD
(Lautner et al., 2013), failed to demonstrate any significant
inhibition of the agonists used in either of the assay formats studied
here. We also tested the effects of alamandine in both FLIPR and DMR
assays but observed only minimal responses (supplementary data) and so
could not characterise its sensitivity to antagonism. It is, therefore,
possible that the agonist activity of alamandine (Lautner et al.,2013) is tissue or cell specific, similarly the effects of PD123,319,
whereas we find no evidence, in a similar recombinant system, that
PD123,319 affects β-alanine, GABA or glycine MRGPRD responses. Finally,
we observed that MU-6840 (Uno et al., 2012) dose-dependently
inhibited the response to all three agonists. However, analysis of the
FLIPR Ca2+-data indicated that inhibition by MU-6840
although competitive may be cooperative as suggested by a Schild slope
of 2, which is different to the results obtained in the DMR assay where
the slope was not significantly different from 1
(pA2=6.25). This divergent pharmacology observed between
two different assay techniques may, therefore, provide evidence for two
distinct populations of receptor due to differential G protein coupling.
In conclusion, this study presents for the first time, a novel label
free assay for studying MRGPRD receptors. Comparing the pharmacology of
agonists and antagonists between DMR and a well characterised
Ca2+ mobilisation assay has provided interesting
insights into how MRGPRD receptors can differentially couple through
separate G proteins in the same cell line. In addition, we find good
evidence for MU-6840, but neither thioridazine hydrochloride nor
PD123,319, to be a MRGPRD antagonist with an indication of cooperativity
in its mechanism when coupled via Gq. This data argues
for a holistic, unbiased approach to studying MRGPRD receptor
pharmacology by employing multiple techniques that facilitate dissection
of divergent on-target pharmacology and potentially off target effects,
which will be particularly important in defining the activity of
molecules identified in drug discovery programmes.