Abstract:
G-protein-coupled receptors (GPCRs), which make up the largest protein superfamily
in the human body, are essential for cellular signaling. They are essential
pharmacological targets for the development of drugs. The relaxin family peptide
receptor 3, commonly known as relaxin-3 receptor 1 (RXFP3), belongs to the class A
GPCR group. It is prominently expressed in brain regions such as the hypothalamus,
amygdala, and the brainstem. The signaling pathways activated by the RXFP3 receptor
upon binding with its ligands are inhibition of cyclic adenosine monophosphate
(cAMP) accumulation, ERK1/2, MAPK, and beta-arrest in recruitment. However,
additional information is needed to determine the distinct preferences of different
RXFP3 ligands regarding their recruitment of beta-arrest in or activation of G-proteins.
One of the upstream effectors activated by the RXFP3 receptor is Gαi/o, which is the
primary focus of this work. The objective is to establish a GloSensorTM cAMP signaling
assay to determine the functional selectivity of RXFP3 receptor activation by relaxin-3
agonists and its inhibition of forskolin-induced cAMP assays. In this method, a
luciferase enzyme connected to a cAMP-responsive component is used. The luciferase
enzyme is triggered when cAMP levels rise, producing light. This luminescence
provides a reliable measure of Gαi/o activation via RXFP3.In comparison to existing
cAMP measurement assays like Enzyme-Linked Immunosorbent Assay (ELISA), the
GloSensorTM cAMP assay, being a luminescence-based technique has several benefits,
such as being cost-effective and efficient. In the initial stages of the study, we isolated
and validated a pCAGGS mammalian expression plasmid construct containing the
GloSensor coding sequence. Then, the study focused on HEK-RXFP3 cell line-based
forskolin-induced cAMP assays. Adenylate cyclase activation and cAMP signaling in
cells were investigated using the well-known stimulant forskolin. The assay detected
the highest luminescence signal in response to forskolin and 3-Isobutyl-1
methylxanthine (IBMX), after transfecting HEK-RXFP3 cells with the GloSensor
plasmid, whereas the lowest signal was seen at the highest relaxin-3 concentration. The
resulting dose-response curve showed fluctuation of cAMP that was released in
response to relaxin-3 ligands. It is interesting to note that lower relaxin-3 concentrations
showed the greatest cAMP accumulation. During the optimization phase, experimental
parameters, including PEI concentration (1mg/ml) and cell count per well, were fine
tuned. The results revealed that 5 µl (25 µg/ml) of branched PEI yielded the most
robust signal compared to other concentrations. Additionally, relaxin-3 demonstrated
the ability to reduce cAMP synthesis by activating Gαi/o inhibitory pathways through
RXFP3 binding, highlighting the receptor's modulation of both stimulatory and
inhibitory pathways.
