Introduction: The relaxin family peptide receptor 2 (RXFP2) is the receptor for Insulin-like peptide-3 (INSL3). The INSL3/RXFP2 signaling is necessary in the first stage of testis descent. Besides, it is proposed that RXFP2 has a role in bone metabolism or in development of cancers (thyroid, prostate). RXFP2 is a rhodopsin-like G protein-coupled receptors (GPCR). Recently, it has been demonstrated that Drosophila RXFP2 had constitutive activity, i.e., a ligand-independent signaling. In addition, RXFP2 exhibits strong structural homology with RXFP1, a constitutively active receptor. In this context, we sought to characterize the constitutive activity of human RXFP2 (hRXFP2).
Methods: HEK293 cells were transiently transfected with hRXFP2 or with the empty vector pcDNA3.1Zeo. Receptor activity was analyzed by measuring intracellular cAMP production under different conditions.
Results: In cells transiently transfected with hRXFP2, we showed that in absence of INSL3 the basal cAMP level was about 13.5±1.2 fold higher than in control cells, after incubation with IBMX, a phosphodiesterase inhibitor. This agonist-independent activity accounted for 60±1.8% of the maximal response to INSL3. We also demonstrated in cDNA-dosing experiments that cAMP production increased linearly with increasing amounts of hRXFP2. Finally, we confirmed the constitutive activity of hRXFP2 showing that hRXFP2 not only increased the basal level of cAMP, but also potentiated forskolin-induced cAMP production. By RT-PCR, we ruled out a local production of INSL3 that can skew the results of a ligand-independent activation. All these results were also confirmed in another cellular model: the COS-7 cell line. Currently, we are validating the constitutive activity of RXFP2 in a cellular model with endogenous expression.
Conclusion: For the first time, we showed that expression of hRXFP2 results in strong agonist-independent increase in intracellular cAMP. Moreover, hRXFP2 stimulates forskolin-induced cAMP production, a feature of constitutively active Gs protein-coupled receptors. The fact that hRXFP2 is highly constitutively active raises a series of questions concerning the physiological importance of this activity which remains to be defined.
18 - 21 May 2019
European Society of Endocrinology