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In vitro pharmacological characterization of RXFP3 Allosterism: an example of probe dependency
Author(s) -
Alvarez-Jaimes Lily,
Sutton Steven W,
Nepomuceno Diane,
Motley S Timothy,
Cik Miroslav,
Stocking Emily,
Shoblock James,
Bonaventure Pascal
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.836.7
Subject(s) - allosteric modulator , receptor , chemistry , allosteric regulation , radioligand , g protein coupled receptor , relaxin , in vitro , biophysics , pharmacology , microbiology and biotechnology , biochemistry , biology
Expression of the relaxin‐3 receptor (RXFP3) in brain areas involved in the modulation of circadiam rhythm, emotion, and neuroendocrine processing suggests this GPCR is a potential target for the development of pharmacotherapies for the treatment of various CNS diseases. In this study, we characterized 3‐[3,5‐ Bis(trifluoromethyl)phenyl]‐1‐(3,4‐dichlorobenzyl)‐1‐[2‐(5‐ methoxy‐1H‐indol‐3‐yl)ethyl]urea (135PAM1), the first selective RXFP3 positive allosteric modulator (PAM). 135PAM1 increased calcium mobilization in the presence of relaxin‐3 NH2 and R3/I5 NH2 with EC50 values of 280 nM [230–350] and 860 nM [630–1160] in cell lines expressing the cloned human RXFP3 receptor. In a cAMP accumulation assay, 135PAM1 inhibited the CRE response to forskolin with an IC50 of 750 nM [540–1000] in the presence of a probe (10 nM) concentration of relaxin‐3 NH2 . 135PAM1 did not compete for binding with the orthosteric radioligand, [ 125 I] R3I5 NH2 , in membranes prepared from cells expressing the cloned human RXFP3 receptor. Selectivity for RXFP3 was also evaluated by testing the ability of 135PAM1 to modulate the CRE response in the presence of relaxin‐3 NH2 and R3/I5 NH2 in cells expressing RXPF4, the highest homologous sequence compare to RXPF3. These results show the compound does not directly activate RXFP3 receptor but increases functional responses to relaxin‐3 NH2 or R3/I5 NH2 . When using the free acid (native) form of relaxin‐3 or R3/I5, 135PAM1 doesn't activate RXFP3 indicating that the compound's effect is probe dependent. Altogether, these data demonstrate that the state of the probe's c‐terminus is crucial to allosteric modulation of RXFP3 by a PAM.