Distinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin

Abstract

Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. It is structurally related to the human relaxin receptor RXFP1 and can be activated by human gene-2 (H2) relaxin as well as its cognate ligand insulin-like peptide 3 (INSL3). Both receptors possess an N-terminal low-density lipoprotein type a (LDLa) module that is necessary for activation and is joined to a leucine-rich repeat domain by a linker. This linker has been shown to be important for H2 relaxin binding and activation of RXFP1 and herein we investigate the role of the equivalent region of RXFP2. We demonstrate that the linker’s highly-conserved N-terminal region is essential for activation of RXFP2 in response to both ligands. In contrast, the linker is necessary for H2 relaxin, but not INSL3, binding. Our results highlight the distinct mechanism by which INSL3 activates RXFP2 whereby ligand binding mediates reorientation of the LDLa module by the linker region to activate the RXFP2 transmembrane domains in conjunction with the INSL3 A-chain. In contrast, relaxin activation of RXFP2 involves a more RXFP1-like mechanism involving binding to the LDLa-linker, reorientation of the LDLa module and activation of the transmembrane domains by the LDLa alone.

Multi-Component Mechanism of H2 Relaxin Binding to RXFP1 through NanoBRET Kinetic Analysis

“…To test the hypothesis that the two binding states of RXFP1 are associated with the binding sites in the LRRs and the linker domain, we conducted binding studies on RXFP2, which does not contain the linker-binding site (Bruell et al., 2017). We therefore first cloned a Nanoluc tag at an equivalent position of FLAG-tagged RXFP2 to Nanoluc-RXFP1, and the resulting Nanoluc-RXFP2 construct was tested for cAMP activity in response to relaxin in the same way (Figure 9A).…”

“…Relaxin potency at Nanoluc-RXFP2 was not significantly different from RXFP2 (Table 1), and similarly, the potency of TamRLX was not significantly different from relaxin at RXFP2 (Figure 9B; EC 50 = 8.46 ± 0.25 versus 8.57 ± 0.26). We previously reported the affinity of relaxin for RXFP2 as being around 10 nM using a saturation binding assay with europium-labeled H2 relaxin (Bruell et al., 2017). Similarly, here using the NanoBRET system with Nanoluc-RXFP2 titrated with TamRLX, we demonstrated that the K d was 10.74 ± 1.64 nM (Figure 9C).

Figure 9Characterization of TamRLX and Nanoluc-RXFP2 and NanoBRET Saturation Binding(A) Dose-response curves of cAMP activity in HEK293T cells expressing RXFP2 when stimulated with relaxin or TamRLX.

…”

“…However, more recent work has demonstrated that the linker domain has an important role in binding (Sethi et al., 2016). Relaxin can also bind to and activate the related INSL3 receptor, RXFP2, where it binds in a fashion referred to as a hybrid mode, resembling a binding mode somewhere between relaxin-RXFP1 and INSL3-RXFP2 (Bruell et al., 2017). However, RXFP2 lacks the helical stretch of linker thought to bind relaxin and thus has a lower affinity for relaxin.…”

“…Kinetic experiments revealed complexities that were incompatible with a single-step binding mechanism, and dissociation experiments indicated the existence of at least two relaxin-bound states of RXFP1. To test the hypothesis that the two binding states are associated with the binding sites in the LRR and linker domains, we tested binding to RXFP2, which does not contain the linker-binding site (Bruell et al., 2017), and demonstrated a single-step binding mechanism. In addition, we created chimeric receptors whereby we inserted RXFP1 linker residues into RXFP2 and observed an improvement in both binding and signaling capacity in response to relaxin, as well as a biphasic dissociation profile more similar to that of RXFP1 than of wild-type RXFP2.…”

Diverse functions of insulin-like 3 peptide

“…Moreover, a RXFP2 splice variant expressed in human uterine tissue that lacks the LDLa module had intact ligand binding affinity but an impaired cAMP signaling when tested in HEK293T-RXFP2 cells (Scott et al 2006). Recent studies have shown that upon receptor binding to INSL3, the RXFP2 linker region located between the LDLa and LRRs may help to direct the LDLa domain to interact with the TM and activate the receptor together with the INSL3 A-chain (Bruell et al 2017).…”