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

Abstract

Relaxin, insulin-like peptide 3 (INSL3), relaxin-3, and INSL5 are the cognate ligands for the relaxin family peptide (RXFP) receptors 1-4, respectively. RXFP1 activates pleiotropic signaling pathways including the signalosome protein complex that facilitates high-sensitivity signaling; coupling to Gα(s), Gα(i), and Gα(o) proteins; interaction with glucocorticoid receptors; and the formation of hetero-oligomers with distinctive pharmacological properties. In addition to relaxin-related ligands, RXFP1 is activated by Clq-tumor necrosis factor-related protein 8 and by small-molecular-weight agonists, such as ML290 [2-isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenylcarbamoyl)phenyl)benzamide], that act allosterically. RXFP2 activates only the Gα(s)- and Gα(o)-coupled pathways. Relaxin-3 is primarily a neuropeptide, and its cognate receptor RXFP3 is a target for the treatment of depression, anxiety, and autism. A variety of peptide agonists, antagonists, biased agonists, and an allosteric modulator target RXFP3. Both RXFP3 and the related RXFP4 couple to Gα(i)/Gα(o) proteins. INSL5 has the properties of an incretin; it is secreted from the gut and is orexigenic. The expression of RXFP4 in gut, adipose tissue, and β-islets together with compromised glucose tolerance in INSL5 or RXFP4 knockout mice suggests a metabolic role. This review focuses on the many advances in our understanding of RXFP receptors in the last 5 years, their signal transduction mechanisms, the development of novel compounds that target RXFP1-4, the challenges facing the field, and current prospects for new therapeutics.

Fig. 1.

Functional domains of RXFP1. The N-terminal region of RXFP1 consists of an LDLa module that is essential for signaling promoted by relaxin but not the allosteric modulator ML290. Residues L29, Y31, and K39 are important for receptor activation by relaxin. The LDLa module is connected to the leucine-rich repeat region that contains the primary high-affinity binding site for relaxin. The relaxin B-chain residues R13 and R17 are believed to bind to E277 and D279 in LRR8 and D231 and E233 in LRR6, whereas I20 is thought to interact with W180 and I182 in LRR4 and L204 and V206 in LRR5. The ECL2 region contains the secondary low-affinity binding site for relaxin, whereas ECL3 contains two residues, G659 and T660, that are essential for activation of RXFP1 by ML290. Helix 8 contains binding motifs that are essential for interaction with AKAP79 that is required for signalosome formation. The distal region of ICL3 is required for coupling to Gα_s and the receptor containing D637 displays constitutive activity. In the C-terminal tail region S704 is required for β-arrestin binding and signalosome formation, whereas the final 10 residues and in particular R752 are required for coupling of RXFP1 to Gα_i3.

Fig. 2.

Functional domains of RXFP3. The first 33 residues from the N terminus do not appear to be involved in binding of relaxin-3. R12 and R16 in the relaxin-3 B-chain interact with RXFP3 E244 and D145, and relaxin-3 R26 can potentially form a salt bridge with RXFP3 E141. Transmembrane regions also determine ligand affinity and specificity, although precise residues have yet to be identified. There is evidence for transactivation of EGFR in the ERK1/2 response in those cells that express the EGFR.

Fig. 3.

Structure-activity relationships for the relaxin family peptides relaxin, relaxin-3, INSL3, and INSL5. All four peptides share common structural features including the two intrachain disulfide bonds and the interchain disulfide bond. However, the interaction sites between the peptides and their cognate receptors show distinct characteristics. Binding of relaxin to RXFP1 involves the RxxxRxxI/V motif in the B-chain, whereas alterations in A-chain length influence activity at RXFP1 and RXFP2. Binding of INSL3 to RXFP2 involves a similar motif but displaced one turn along the α-helix of the B-chain. N terminus truncation of either the A- or the B-chain reduces efficacy but not affinity and has been used as a strategy for producing antagonists. Disruption of intrachain disulfide bonds in INSL3 reduces binding to a minor extent but destroys agonist activity (C10S/C15S or C10del/C15del). Binding of relaxin-3 to RXFP3 involves predominantly the B-chain, and reduction of the A-chain removes activity at RXFP1 without affecting activity at RXFP3. Relaxin-3 R8, R16, I5, and F20 are required for binding to RXFP3 and RXFP4, with R12 required for RXFP3 but not RXFP4. Relaxin-3 R26 and W27 are required for activation of RXFP3 and RXFP4. Truncation of the B-chain and G23R produces an antagonist, and truncation of the A-chain retains high-affinity agonist activity. Binding of INSL5 to RXFP4 shows a number of differences from the relaxin-3/RXFP3 interaction. Unlike relaxin-3, the B-chain of INSL5 is inactive and minimized analogs generally show reduced affinity and efficacy. Many of the agonist and antagonist peptides active at RXFP3 are also active at RXFP4.

Fig. 4.

Signal transduction pathways activated by RXFP1. In the resting state at least some RXFP1 receptors are bound to AKAP79 and adenylyl cyclase in the signalosome complex. Low, subpicomolar concentrations of relaxin activate the signalosome to produce tightly controlled cAMP signals. When the relaxin concentration rises to nanomolar the signalosome dissociates and RXFP1 activates canonical pathways to increase cAMP, ERK1/2, and NO and to activate NFκB. In pathologic conditions, the increased expression of the AT₂R promotes formation of RXFP1/AT₂R heterodimers that are essential for the antifibrotic actions of relaxin. The allosteric agonist ML290 interacts with RXFP1 to produce a different signaling pattern to relaxin.

Fig. 5.

Signal transduction pathways activated by RXFP3. The cognate ligand relaxin-3 activates RXFP3 that couples to Gα_i/o proteins to inhibit adenylyl cyclase and activate ERK1/2 and p38MAPK phosphorylation to promote AP1 transcription. Stimulation of RXFP3 by relaxin activates only ERK1/2 and AP1 transcription. There is an allosteric site on RXFP3 that when occupied by a positive allosteric modulator 135PAM1 sensitizes responses to relaxin-3 amide. In tissues where they coexist, activation of RXFP3 may cause transactivation of the EGFR.