Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules

Abstract

Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism.

International Union of Basic and Clinical Pharmacology. XCV. Recent Advances in the Understanding of the Pharmacology and Biological Roles of Relaxin Family Peptide Receptors 1–4, the Receptors for Relaxin Family Peptides

“…Studies using chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped C-terminally to the final cysteine residue of the module demonstrate that RXFP2 likely uses a similar mechanism to drive receptor activation. Importantly the chimeric receptors were still able to bind ligand normally and were also able to signal, albeit with altered activity compared with wild-type receptors (Bruell et al, 2013). Furthermore, when the TM domains of the RXFP1 chimera with an RXFP2 LDLa (RXFP211) were swapped to match the LDLa module (RXFP212) the activity of the receptor approached wild type, suggesting a specific interaction between the LDLa module and the receptor TM domains.…”

“…Modeling studies suggest that A-chain residues in relaxin and INSL3 interact with this potential binding site (Hartley et al, 2009), but this has yet to be fully characterized. The current model of activation of RXFP1 and RXFP2 suggests that ligand binding to the LRRs and TM ECLs directs the LDLa to interact with the TM domain of a receptor homodimer partner to drive receptor activation (Kong et al, 2010;Bruell et al, 2013). However this model still requires experimental verification using site-directed mutagenesis or other methods that prevent dimer formation.…”

Investigation of Interactions at the Extracellular Loops of the Relaxin Family Peptide Receptor 1 (RXFP1)

“…Mutant receptor expressing cells were then selected for semistable expression by Zeocin selection followed by FACS using a fluorescently tagged FLAG antibody 1 week later as previously described (24). HEK293T cells stably expressing WT RXFP1 were used as a positive control in the FACS sort, and only the top 10% of cells with fluorescence levels significantly higher than the background fluorescence in nontransfected HEK293T control cells were collected.…”

“…action demonstrated the possibility of the LDLa module also having access to the TM domain of receptors (44). Functional characterization of LDLa-less, chimeric, and mutant receptors has demonstrated a critical functional role for the LDLa module (10,24). Because of the inability of concentrating EL1/EL2-GB1 beyond ϳ200 M, 15 N-LDLa was titrated to only equimolar with EL1/EL2-GB1.…”

Activation of Relaxin Family Receptor 1 from Different Mammalian Species by Relaxin Peptide and Small-Molecule Agonist ML290

“…Saturation-binding studies using Eu-labeled human H2 RLN (Eu-H2 RLN) were performed on whole cells as described previously ( 12 ). Cells stably expressing RXFP1 ( 13 ) or with a semi-stable transfection of R1-RXFP1 selected using FACS ( 14 ) were used in this experiment. Increasing concentrations of Eu-H2 RLN (0.1–50 nM) were utilized and non-specific binding was determined in presence of 1 μM of unlabeled H2 RLN.…”